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Human rotavirus (HRV) is a major cause of childhood diarrhea in developing countries where widespread malnutrition contributes to the decreased oral vaccine efficacy and increased prevalence of other enteric infections, which are major concerns for global health. Neonatal gnotobiotic (Gn) piglets closely resemble human infants in their anatomy, physiology, and outbred status, providing a unique model to investigate malnutrition, supplementations, and HRV infection. To understand the molecular signatures associated with immune enhancement and reduced diarrheal severity by Escherichia coli Nissle 1917 (EcN) and tryptophan (TRP), immunological responses and global nontargeted metabolomics and lipidomics approaches were investigated on the plasma and fecal contents of malnourished pigs transplanted with human infant fecal microbiota and infected with virulent (Vir) HRV. Overall, EcN + TRP combined (rather than individual supplement action) promoted greater and balanced immunoregulatory/immunostimulatory responses associated with greater protection against HRV infection and disease in malnourished humanized piglets. Moreover, EcN + TRP treatment upregulated the production of several metabolites with immunoregulatory/immunostimulatory properties: amino acids (

The disease-producing capacity of the opportunistic pathogen Enterococcus faecalis is enhanced by the ability of the bacterium to evade killing by antimicrobial agents. Survival of E. faecalis in the presence of the human antimicrobial enzyme lysozyme is mediated in part by the site 2 metalloprotease Eep; however, a complete model of enterococcal lysozyme resistance has not been elucidated. To better understand the molecular basis for lysozyme resistance in E. faecalis, we analyzed Δeep suppressor mutants that acquire resistance to lysozyme through mutation of the gene OG1RF_11713, a predicted teichoic acid biosynthesis-encoding gene located within the variable region of the enterococcal polysaccharide antigen (epa) locus. Sequence comparisons revealed that OG1RF_11713 is most similar to the cytidine-5′-diphosphate (CDP)-glycerol:poly-(glycerolphosphate)glycerophosphotransferase TagF from Staphylococcus epidermidis. Inactivation of OG1RF_11713 in both the wild-type and Δeep genetic backgrounds was sufficient to increase the resistance of E. faecalis OG1RF to lysozyme. Minimal amounts of N-acetylgalactosamine were detectable in cell wall carbohydrate extracts of OG1RF_11713 deletion mutants, and this was associated with a reduction in negative cell surface charge. Targeted disruption of OG1RF_11713 was also associated with increased susceptibility to the antibiotic polymyxin B and membrane-targeting detergents and decreased susceptibility to the lantibiotic nisin. This work implicates OG1RF_11713 as a major determinant of cell envelope integrity and provides further validation that lysozyme resistance is intrinsically linked to the modification of enterococcal cell wall polysaccharides. IMPORTANCE Enterococcus faecalis is a leading cause of health-care-associated infections for which there are limited treatment options. E. faecalis is resistant to several antibiotics and to high concentrations of the human antimicrobial enzyme lysozyme. The molecular mechanisms that mediate lysozyme resistance in E. faecalis are complex and remain incompletely characterized. This work demonstrates that a gene located within the variable region of the enterococcal polysaccharide antigen locus of E. faecalis strain OG1RF (OG1RF_11713), which is predicted to encode a component of the teichoic acid biosynthesis machinery, is part of the lysozyme resistance circuitry and is important for enterococcal cell wall integrity. These findings suggest that OG1RF_11713 is a potential target for new therapeutic strategies to combat enterococcal infections.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has emerged to cause pandemic respiratory disease in the past 2 years, leading to significant worldwide morbidity and mortality. At the beginning of the pandemic, only nonspecific treatments were available, but recently two oral antivirals have received emergency use authorization from the U.S. Food and Drug Administration for the treatment of mild to moderate coronavirus disease (COVID-19). Molnupiravir targets the viral polymerase and causes lethal mutations within the virus during replication. Nirmatrelvir targets SARS-CoV-2's main protease, and it is combined with ritonavir to delay its metabolism and allow nirmatrelvir to inhibit proteolytic cleavage of viral polyproteins during replication, preventing efficient virus production. Both drugs inhibit

Vif is a lentiviral accessory protein that counteracts the antiviral activity of cellular APOBEC3 (A3) cytidine deaminases in infected cells. The exact contribution of each member of the A3 family for the restriction of HIV-2 is still unclear. Thus, the aim of this work was to identify the A3s with anti-HIV-2 activity and compare their restriction potential for HIV-2 and HIV-1. We found that A3G is a strong restriction factor of both types of viruses and A3C restricts neither HIV-1 nor HIV-2. Importantly, A3B exhibited potent antiviral activity against HIV-2, but its effect was negligible against HIV-1. Whereas A3B is packaged with similar efficiency into both viruses in the absence of Vif, HIV-2 and HIV-1 differ in their sensitivity to A3B. HIV-2 Vif targets A3B by reducing its cellular levels and inhibiting its packaging into virions, whereas HIV-1 Vif did not evolve to antagonize A3B. Our observations support the hypothesis that during wild-type HIV-1 and HIV-2 infections, both viruses are able to replicate in host cells expressing A3B but using different mechanisms, probably resulting from a Vif functional adaptation over evolutionary time. Our findings provide new insights into the differences between Vif protein and their cellular partners in the two human viruses. Of note, A3B is highly expressed in some cancer cells and may cause deamination-induced mutations in these cancers. Thus, A3B may represent an important therapeutic target. As such, the ability of HIV-2 Vif to induce A3B degradation could be an effective tool for cancer therapy. IMPORTANCE Primate lentiviruses encode a series of accessory genes that facilitate virus adaptation to its host. Among those, the vif-encoded protein functions primarily by targeting the APOBEC3 (A3) family of cytidine deaminases. All lentiviral Vif proteins have the ability to antagonize A3G; however, antagonizing other members of the A3 family is variable. Here, we report that HIV-2 Vif, unlike HIV-1 Vif, can induce degradation of A3B. Consequently, HIV-2 Vif but not HIV-1 Vif can inhibit the packaging of A3B. Interestingly, while A3B is packaged efficiently into the core of both HIV-1 and HIV-2 virions in the absence of Vif, it only affects the infectivity of HIV-2 particles. Thus, HIV-1 and HIV-2 have evolved two distinct mechanisms to antagonize the antiviral activity of A3B. Aside from its antiviral activity, A3B has been associated with mutations in some cancers. Degradation of A3B by HIV-2 Vif may be useful for cancer therapies.

APOBEC3 proteins APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H (A3H) are host restriction factors that inhibit HIV-1 through DNA cytidine deaminase-dependent and -independent mechanisms and have either one (A3H) or two (A3F and A3G) zinc-binding domains. A3H antiviral activity encompasses multiple molecular functions, all of which depend on recognition of RNA or DNA. A3H crystal structures revealed an unusual interaction with RNA wherein an RNA duplex mediates dimerization of two A3H proteins. In this study, we sought to determine the importance of RNA-binding amino acids in the antiviral and biochemical properties of A3H. We show that the wild-type A3H-RNA interaction is essential for A3H antiviral activity and for two deaminase-independent processes: encapsidation into viral particles and inhibition of reverse transcription. Furthermore, an extensive mutagenesis campaign revealed distinct roles for two groups of amino acids at the RNA binding interface. C-terminal helix residues exclusively bind RNA, and loop 1 residues play a dual role in recognition of DNA substrates and in RNA binding. Weakening the interface between A3H and RNA allows DNA substrates to bind with greater affinity and enhances deamination rates, suggesting that RNA binding must be disrupted to accommodate DNA. Intriguingly, we demonstrate that A3H can deaminate overhanging DNA strands of RNA/DNA heteroduplexes, which are early intermediates during reverse transcription and may represent natural A3H substrates. Overall, we present a mechanistic model of A3H restriction and a step-by-step elucidation of the roles of RNA-binding residues in A3H activity, particle incorporation, inhibition of reverse transcriptase inhibition, and DNA cytidine deamination. IMPORTANCE APOBEC3 proteins are host factors that protect the integrity of the host genome by inhibiting retroelements as well as retroviruses, such as HIV-1. To do this, the APOBEC3H protein has evolved unique interactions with structured RNAs. Here, we studied the importance of these interactions in driving antiviral activity of APOBEC3H. Our results provide a clear picture of how RNA binding drives the ability of APOBEC3H to infiltrate new viruses and prevent synthesis of viral DNA. We also explore how RNA binding by APOBEC3H influences recognition and deamination of viral DNA and describe two possible routes by which APOBEC3H might hypermutate the HIV-1 genome. These results highlight how one protein can sense many nucleic acid species for a variety of antiviral activities.

Cytidine deaminases (CDAs) catalyze the hydrolytic deamination of cytidine and deoxycytidine in the pyrimidine salvage pathway. In kinetoplastids, pyrimidine metabolism has been extensively studied as a source of potential drug targets, given the fact that many of the enzymes of the pathway are essential. Thymidylate (dTMP) synthesis in Trypanosoma brucei exhibits unique characteristics. Thus, it has been suggested that the production of dUMP, the substrate for dTMP formation, is solely dependent on cytidine deaminase and thymidine kinase. Here we characterize recombinant T. brucei CDA (TbCDA) and present evidence that indeed the alternative route for dUMP formation via deoxyuridine 5′-triphosphate nucleotidohydrolase does not have a prominent role in de novo dTMP formation. Furthermore, we provide a scheme for the compartmentalization of dTMP biosynthesis, taking into account the observation that CDA is located in the mitochondrion, together with available information on the intracellular localization of other enzymes involved in the dTTP biosynthetic pathway., Cytidine deaminase (CDA) is a pyrimidine salvage enzyme that catalyzes cytidine and deoxycytidine hydrolytic deamination to yield uridine and deoxyuridine. Here we report the biochemical characterization of Trypanosoma brucei CDA as an enzyme within the tetrameric class of the CDA family that efficiently deaminates cytidine, deoxycytidine, and the nucleoside analogue 5-methyl-2′-deoxycytidine. In line with previous studies, we show that RNA interference (RNAi)-mediated CDA depletion impairs T. brucei proliferation when grown in pyrimidine-deficient medium, while supplementation with thymidine or deoxyuridine restores growth, further underscoring the role of this enzyme in providing deoxyuridine for dUMP formation via thymidine kinase, the substrate required for de novo thymidylate biosynthesis. This observation contrasts with the existence in T. brucei of a dimeric deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase), an essential enzyme that can produce dUMP via the hydrolysis of dUTP/dUDP. Thus, T. brucei dUTPase-null mutants are thymidine auxotrophs, suggesting that dUTPase might have a role in providing dUMP for thymidylate biosynthesis. We show that overexpression of human dCMP deaminase (DCTD), an enzyme that provides directly dUMP through dCMP deamination, does not reverse the lethal phenotype of dUTPase knockout cells, which further supports the notion that in T. brucei, CDA is uniquely involved in providing dUMP, while the main role of dUTPase would be the withdrawal of the excess of dUTP to avoid its incorporation into DNA. Furthermore, we report the mitochondrial localization of CDA, highlighting the importance of this organelle in pyrimidine metabolism. IMPORTANCE Cytidine deaminases (CDAs) catalyze the hydrolytic deamination of cytidine and deoxycytidine in the pyrimidine salvage pathway. In kinetoplastids, pyrimidine metabolism has been extensively studied as a source of potential drug targets, given the fact that many of the enzymes of the pathway are essential. Thymidylate (dTMP) synthesis in Trypanosoma brucei exhibits unique characteristics. Thus, it has been suggested that the production of dUMP, the substrate for dTMP formation, is solely dependent on cytidine deaminase and thymidine kinase. Here we characterize recombinant T. brucei CDA (TbCDA) and present evidence that indeed the alternative route for dUMP formation via deoxyuridine 5′-triphosphate nucleotidohydrolase does not have a prominent role in de novo dTMP formation. Furthermore, we provide a scheme for the compartmentalization of dTMP biosynthesis, taking into account the observation that CDA is located in the mitochondrion, together with available information on the intracellular localization of other enzymes involved in the dTTP biosynthetic pathway.

Prolonged norovirus shedding may occur in certain patients, such as organ transplant recipients. We established a mouse model for persistent norovirus infection (using the mouse norovirus MNV.CR6 strain). The nucleoside viral polymerase inhibitor 2′- C -methylcytidine (2CMC), but not favipiravir (T-705), reduced viral shedding to undetectable levels. Viral rebound was observed after stopping treatment, which was again effectively controlled by treatment with 2CMC. No drug-resistant variants emerged.

Morbidity and mortality resulting from influenza-like disease are a threat, especially for older adults. To improve case management, next-generation broad-spectrum antiviral therapeutics that are efficacious against major drivers of influenza-like disease, including influenza viruses and respiratory syncytial virus (RSV), are urgently needed. Using a dual-pathogen high-throughput screening protocol for influenza A virus (IAV) and RSV inhibitors, we have identified N4-hydroxycytidine (NHC) as a potent inhibitor of RSV, influenza B viruses, and IAVs of human, avian, and swine origins. Biochemical in vitro polymerase assays and viral RNA sequencing revealed that the ribonucleotide analog is incorporated into nascent viral RNAs in place of cytidine, increasing the frequency of viral mutagenesis. Viral passaging in cell culture in the presence of an inhibitor did not induce robust resistance. Pharmacokinetic profiling demonstrated dose-dependent oral bioavailability of 36 to 56%, sustained levels of the active 5'-triphosphate anabolite in primary human airway cells and mouse lung tissue, and good tolerability after extended dosing at 800 mg/kg of body weight/day. The compound was orally efficacious against RSV and both seasonal and highly pathogenic avian IAVs in mouse models, reducing lung virus loads and alleviating disease biomarkers. Oral dosing reduced IAV burdens in a guinea pig transmission model and suppressed virus spread to uninfected contact animals through direct transmission. Based on its broad-spectrum efficacy and pharmacokinetic properties, NHC is a promising candidate for future clinical development as a treatment option for influenza-like diseases.

Norovirus (NoV) is a positive-sense single-stranded RNA virus that causes acute gastroenteritis and is responsible for 200,000 deaths per year worldwide. No effective vaccine or treatment is available. Recent studies have shown that the nucleoside analogs favipiravir (T-705) and 2′- C -methyl-cytidine (2CM-C) inhibit NoV replication in vitro and in animal models, but their precise mechanism of action is unknown. We evaluated the molecular interactions between nucleoside triphosphates and NoV RNA-dependent RNA polymerase (NoVpol), the enzyme responsible for replication and transcription of NoV genomic RNA. We found that T-705 ribonucleoside triphosphate (RTP) and 2CM-C triphosphate (2CM-CTP) equally inhibited human and mouse NoVpol activities at concentrations resulting in 50% of maximum inhibition (IC 50 s) in the low micromolar range. 2CM-CTP inhibited the viral polymerases by competing directly with natural CTP during primer elongation, whereas T-705 RTP competed mostly with ATP and GTP at the initiation and elongation steps. Incorporation of 2CM-CTP into viral RNA blocked subsequent RNA synthesis, whereas T-705 RTP did not cause immediate chain termination of NoVpol. 2CM-CTP and T-705 RTP displayed low levels of enzyme selectivity, as they were both recognized as substrates by human mitochondrial RNA polymerase. The level of discrimination by the human enzyme was increased with a novel analog of T-705 RTP containing a 2′- C -methyl substitution. Collectively, our data suggest that 2CM-C inhibits replication of NoV by acting as a classic chain terminator, while T-705 may inhibit the virus by multiple mechanisms of action. Understanding the precise mechanism of action of anti-NoV compounds could provide a rational basis for optimizing their inhibition potencies and selectivities.

The APOBEC3 (A3) enzymes, A3G and A3F, are coordinately expressed in CD4 + T cells and can become coencapsidated into HIV-1 virions, primarily in the absence of the viral infectivity factor (Vif). A3F and A3G are deoxycytidine deaminases that inhibit HIV-1 replication by inducing guanine-to-adenine hypermutation through deamination of cytosine to form uracil in minus-strand DNA. The effect of the simultaneous presence of both A3G and A3F on HIV-1 restriction ability is not clear. Here, we used a single-cycle infectivity assay and biochemical analyses to determine if coencapsidated A3G and A3F differ in their restriction capacity from A3G or A3F alone. Proviral DNA sequencing demonstrated that compared to each A3 enzyme alone, A3G and A3F, when combined, had a coordinate effect on hypermutation. Using size exclusion chromatography, rotational anisotropy, and in vitro deamination assays, we demonstrate that A3F promotes A3G deamination activity by forming an A3F/G hetero-oligomer in the absence of RNA which is more efficient at deaminating cytosines. Further, A3F caused the accumulation of shorter reverse transcripts due to decreasing reverse transcriptase efficiency, which would leave single-stranded minus-strand DNA exposed for longer periods of time, enabling more deamination events to occur. Although A3G and A3F are known to function alongside each other, these data provide evidence for an A3F/G hetero-oligomeric A3 with unique properties compared to each individual counterpart. IMPORTANCE The APOBEC3 enzymes APOBEC3F and APOBEC3G act as a barrier to HIV-1 replication in the absence of the HIV-1 Vif protein. After APOBEC3 enzymes are encapsidated into virions, they deaminate cytosines in minus-strand DNA, which forms promutagenic uracils that induce transition mutations or proviral DNA degradation. Even in the presence of Vif, footprints of APOBEC3-catalyzed deaminations are found, demonstrating that APOBEC3s still have discernible activity against HIV-1 in infected individuals. We undertook a study to better understand the activity of coexpressed APOBEC3F and APOBEC3G. The data demonstrate that an APOBEC3F/APOBEC3G hetero-oligomer can form that has unique properties compared to each APOBEC3 alone. This hetero-oligomer has increased efficiency of virus hypermutation, raising the idea that we still may not fully realize the antiviral mechanisms of endogenous APOBEC3 enzymes. Hetero-oligomerization may be a mechanism to increase their antiviral activity in the presence of Vif.

No antiviral therapies are available for the tick-borne flaviviruses associated with hemorrhagic fevers: Kyasanur Forest disease virus (KFDV), both classical and the Alkhurma hemorrhagic fever virus (AHFV) subtype, and Omsk hemorrhagic fever virus (OHFV). We tested compounds reported to have antiviral activity against members of the Flaviviridae family for their ability to inhibit AHFV replication. 6-Azauridine (6-azaU), 2′-C-methylcytidine (2′-CMC), and interferon alpha 2a (IFN-α2a) inhibited the replication of AHFV and also KFDV, OHFV, and Powassan virus. The combination of IFN-α2a and 2′-CMC exerted an additive antiviral effect on AHFV, and the combination of IFN-α2a and 6-azaU was moderately synergistic. The combination of 2′-CMC and 6-azaU was complex, being strongly synergistic but with a moderate level of antagonism. The antiviral activity of 6-azaU was reduced by the addition of cytidine but not guanosine, suggesting that it acted by inhibiting pyrimidine biosynthesis. To investigate the mechanism of action of 2′-CMC, AHFV variants with reduced susceptibility to 2′-CMC were selected. We used a replicon system to assess the substitutions present in the selected AHFV population. A double NS5 mutant, S603T/C666S, and a triple mutant, S603T/C666S/M644V, were more resistant to 2′-CMC than the wild-type replicon. The S603T/C666S mutant had a reduced level of replication which was increased when M644V was also present, although the replication of this triple mutant was still below that of the wild type. The S603 and C666 residues were predicted to lie in the active site of the AHFV NS5 polymerase, implicating the catalytic center of the enzyme as the binding site for 2′-CMC.

Mericitabine (RG7128) is the prodrug of a highly selective cytidine nucleoside analog inhibitor (RO5855) of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. This study evaluated the effects of combining RO5855 and ribavirin on HCV replication in the HCV subgenomic replicon by using two drug-drug interaction models. The effects of RO5855 and ribavirin on the intracellular metabolism of each compound, on interferon-stimulated gene (ISG) expression, and on the viability of hepatocyte-derived cells were also investigated. RO5855 and ribavirin had additive inhibitory activities against HCV subgenomic replicon replication in drug-drug interaction analyses. RO5855 did not affect the uptake or phosphorylation of ribavirin in primary human hepatocytes, human peripheral blood mononuclear cells, or genotype 1b (G1b) replicon cells. Similarly, ribavirin did not affect the concentrations of intracellular species derived from RO5855 in primary human hepatocytes or the formation of the triphosphorylated metabolites of RO5855. Ribavirin at concentrations of >40 μM significantly reduced the viability of primary hepatocytes but not of Huh7, the G1b replicon, or interferon-cured Huh7 cells. RO5855 alone or with ribavirin did not significantly alter the viability of Huh7 or G1b replicon cells, and it did not significantly affect the viability of primary hepatocytes when it was administered alone. The viability of primary hepatocytes was reduced when they were incubated with RO5855 and ribavirin, similar to the effects of ribavirin alone. RO5855 alone or with ribavirin had no effect on ISG mRNA levels in any of the cells tested. In conclusion, RO5855 did not show any unfavorable interactions with ribavirin in human hepatocytes or an HCV subgenomic replicon system.

The host deploys a subset of immune responses to expel helminths, which differs depending on the nature of the helminth. Strongyloides venezuelensis , a counterpart of the human pathogen S. stercoralis , naturally infects rodents and has been used as an experimental model. Here we show that induction of immunoglobulin G (IgG) and IgE is a prerequisite for rapid expulsion of S. venezuelensis during a primary infection. Activation-induced cytidine deaminase-deficient (AID −/− ) mice, which lack the ability to switch IgM to other isotypes, normally developed T-helper 2 (Th2) cells and intestinal mastocytosis after infection with S. venezuelensis . Although AID −/− mice expelled Nippostrongylus brasiliensis normally, they required a much longer period to expel S. venezuelensis than wild-type (WT) mice. Adoptive transfers of immune sera from S. venezuelensis -infected but not N. brasiliensis -infected mice restored the ability of AID −/− mice to promptly expel S. venezuelensis . Immune serum-derived IgG and IgE induced worm expulsion via Fc γ receptor III (FcγRIII) and Fc ε receptor I (FcεRI), respectively, and a mixture of IgG and IgE showed collaborative effects. Whereas FcγRIII −/− mice or FcεRIα −/− mice normally could expel S. venezuelensis , FcγRIII −/− mice, when their IgE was neutralized by anti-IgE, or FcεRIα −/− mice, when their IgG binding to FcγRIII was blocked by anti-FcγRIII, showed a markedly reduced ability to expel S. venezuelensis . These data reveal that IgG and IgE play redundant roles but act in concert to accelerate S. venezuelensis expulsion. Mast cell-deficient mice, even those equipped with immune serum-derived IgG or IgE, failed to expel S. venezuelensis promptly, suggesting that mast cells are cellular targets of IgG and IgE.

The Vif protein of human immunodeficiency virus type 1 (HIV-1) promotes viral replication by downregulation of the cell-encoded, antiviral APOBEC3 proteins. These proteins exert their suppressive effects through the inhibition of viral reverse transcription as well as the induction of cytidine deamination within nascent viral cDNA. Importantly, these two effects have not been characterized in detail in human CD4 + T cells, leading to controversies over their possible contributions to viral inhibition in the natural cell targets of HIV-1 replication. Here we use wild-type and Vif-deficient viruses derived from the CD4 + T cells of multiple donors to examine the consequences of APOBEC3 protein function at natural levels of expression. We demonstrate that APOBEC3 proteins impart a profound deficiency to reverse transcription from the initial stages of cDNA synthesis, as well as excessive cytidine deamination (hypermutation) of the DNAs that are synthesized. Experiments using viruses from transfected cells and a novel method for mapping the 3′ termini of cDNAs indicate that the inhibition of reverse transcription is not limited to a few specific sites, arguing that APOBEC3 proteins impede enzymatic processivity. Detailed analyses of mutation spectra in viral cDNA strongly imply that one particular APOBEC3 protein, APOBEC3G, provides the bulk of the antiviral phenotype in CD4 + T cells, with the effects of APOBEC3F and APOBEC3D being less significant. Taken together, we conclude that the dual mechanisms of action of APOBEC3 proteins combine to deliver more effective restriction of HIV-1 than either function would by itself.

The complete genome of porcine circovirus type 2 (PCV2) strain YiY-3-2-H5 contains a cytidine insertion at position 962 in open reading frame 1. This insertion causes a reading frameshift of the rep gene, and thereafter a premature stop codon is present at the 3′ terminal end of this gene.

We used classical mutagens in Gram-negative Escherichia coli to study synergies with different classes of antibiotics, test models of antibiotic mechanisms of action, and examine the basis of synergy. We used 4-nitroquinoline 1-oxide (4NQO), zebularine (ZEB), 5-azacytidine (5AZ), 2-aminopurine (2AP), and 5-bromodeoxyuridine (5BrdU) as mutagens (with bactericidal potency of 4NQOZEB5AZ2AP5BrdU) and vancomycin (VAN), ciprofloxacin (CPR), trimethoprim (TMP), gentamicin (GEN), tetracycline (TET), erythromycin (ERY), and chloramphenicol (CHL) as antibiotics. We detected the strongest synergies with 4NQO, an agent that oxidizes guanines and ultimately results in double-strand breaks when paired with the bactericidal antibiotics VAN, TMP, CPR, and GEN, but no synergies with the bacteriostatic antibiotics TET, ERY, and CHL. Each of the other mutagens displays synergies with the bactericidal antibiotics to various degrees that reflect their potencies, as well as with some of the other mutagens. The results support recent models showing that bactericidal antibiotics kill bacteria principally by ultimately generating more double-strand breaks than can be repaired. We discuss the synergies seen here and elsewhere as representing dose effects of not the proximal target damage but rather the ultimate resulting double-strand breaks. We also used the results of pairwise tests to place the classic mutagens into functional antibacterial categories within a previously defined drug interaction network.

The human APOBEC3 family consists of seven cytidine deaminases (A3A to A3H), some of which display potent antiretroviral activity against HIV-1 and other retroviruses. Studies that analyzed the effect of A3G on human T-lymphotropic virus type 1 (HTLV-1) infectivity resulted in conflicting findings, and our knowledge of HTLV-1 restriction by other A3 proteins remains limited. Since HTLV-1, much like HIV, targets CD4 + T cells, we hypothesized that A3 proteins other than A3G restrict HTLV-1. All seven human A3 proteins were tested in HTLV-1 reporter and HIV-1 infectivity assays. We show that A3A, A3B, and A3H haplotype 2 (A3H hapII) acted as potent inhibitors of HTLV-1. Wild-type HIV-1, in contrast, was restricted by A3B and A3H hapII, but not by A3A. Catalytic site mutants of A3A, A3B, and A3H hapII showed that A3A and A3B restriction of HTLV-1 required deaminase activity. However, A3H hapII acted in a deaminase-independent manner when restricting HTLV-1, while requiring deaminase activity for HIV-1 restriction. We also analyzed A3 editing of HTLV-1 in five T-cell lines obtained from HTLV-1-infected patients. These cell lines contained extensively edited HTLV-1 sequences with G-to-A mutations in dinucleotide contexts suggestive of APOBEC3 mutagenesis. Comparison of the A3-induced mutations from reporter cells and the patient-derived cell lines indicate that A3G but also other A3 members, possibly A3A and A3B, affect HTLV-1 in vivo . Taken together, our data indicate that HTLV-1 is a likely target for multiple A3 proteins.

The APOBEC3 family comprises seven cytidine deaminases (APOBEC3A [A3A] to A3H), which are expressed to various degrees in HIV-1 susceptible cells. The HIV-1 Vif protein counteracts APOBEC3 restriction by mediating its degradation by the proteasome. We hypothesized that Vif proteins from various HIV-1 subtypes differ in their abilities to counteract different APOBEC3 proteins. Seventeen Vif alleles from seven HIV-1 subtypes were tested for their abilities to degrade and counteract A3G, A3F, and A3H haplotype II (hapII). We show that most Vif alleles neutralize A3G and A3F efficiently but display differences with respect to the inhibition of A3H hapII. The majority of non-subtype B Vif alleles tested presented some activity against A3H hapII, with two subtype F Vif variants being highly effective in counteracting A3H hapII. The residues required for activity were mapped to two residues in the amino-terminal region of Vif (positions 39F and 48H). Coimmunoprecipitations showed that these two amino acids were necessary for association of Vif with A3H hapII. These findings suggest that the A3H hapII binding site in Vif is distinct from the regions important for A3G and A3F recognition and that it requires specific amino acids at positions 39 and 48. The differential Vif activity spectra, especially against A3H hapII, suggest adaptation to APOBEC3 repertoires representative of different human ancestries. Phenotypic assessment of anti-APOBEC3 activity of Vif variants against several cytidine deaminases will help reveal the requirement for successful replication in vivo and ultimately point to interventions targeting the Vif-APOBEC3 interface.

Chronic infection with hepatitis C virus (HCV) is a major global health burden and is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma. Current therapy for HCV infection has limited efficacy, particularly against genotype 1 virus, and is hampered by a range of adverse effects. Therefore, there is a clear unmet medical need for efficacious and safe direct antiviral drugs for use in combination with current treatments to increase cure rates and shorten treatment times. The broad genotypic coverage achievable with nucleosides or nucleotides and the high genetic barrier to resistance of these compounds observed in vitro and in vivo suggest that this class of inhibitors could be a valuable component of future therapeutic regimens. Here, we report the in vitro inhibitory activity and mode of action of 2-deoxy-2-spirocyclopropylcytidine (TMC647078), a novel and potent nucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase that causes chain termination of the nascent HCV RNA chain. In vitro combination studies with a protease inhibitor resulted in additive efficacy in the suppression of HCV RNA replication, highlighting the potential for the combination of these two classes in the treatment of chronic HCV infection. No cytotoxic effects were observed in various cell lines. Biochemical studies indicated that TMC647078 is phosphorylated mainly by deoxycytidine kinase (dCK) without inhibiting the phosphorylation of the natural substrate, and high levels of triphosphate were observed in Huh7 cells and in primary hepatocytes in vitro. TMC647078 is a potent novel nucleoside inhibitor of HCV replication with a promising in vitro virology and biology profile. Infection with hepatitis C virus (HCV), the causative agent of hepatitis C, is an important global health burden, with an estimated 120 to 170 million persons chronically infected (7, 11). Chronic HCV infection can lead to liver cirrhosis and hepatocellular carcinoma and is the leading cause of liver transplantation (10). The virus is transmitted mainly via bloodblood contact, and spontaneous virus clearance has been estimated to be achieved for 26% of infected subjects (29). HCV is a member of the Flaviviridae family of viruses in the genus Hepacivirus, comprising at least six major genotypes and multiple subtypes. The 9.6-kb positive-sense, single-stranded RNA genome of HCV encodes four structural proteins (the core protein, the envelope glycoproteins E1 and E2, and p7) and six nonstructural proteins responsible for viral replication (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). The nonstructural protein NS5B is an RNA-dependent RNA polymerase (RdRp) responsible for the amplification of the HCV RNA and the assembly of the replicase complex at the endoplasmic reticulum membrane (3, 25).

7SL RNA promotes the formation of the signal recognition particle that targets secretory and membrane proteins to the endoplasmic reticulum. 7SL RNA is also selectively packaged by many retroviruses, including HIV-1. Here, we demonstrate that 7SL RNA is an integral component of the viral ribonucleoprotein (RNP) complex containing Gag, viral genomic RNA, and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathbf{tRNA}_{3}^{Lys}\) \end{document} . Only the potent anti-HIV-1 cytidine deaminases can bind to 7SL RNA and target to HIV-1 RNP. A conserved motif in the amino-terminal region of A3G is important for 7SL RNA interaction. The weak anti-HIV-1 A3C did not interact with 7SL RNA and failed to target to viral RNPs, despite efficient virion packaging. However, a chimeric construct of A3C plus the 7SL-binding amino terminus of A3G did target to viral RNPs and showed enhanced anti-HIV-1 activity. 7SL RNA binding is a conserved feature of human anti-HIV-1 cytidine deaminases. Thus, potent anti-HIV-1 cytidine deaminases have evolved to possess a unique RNA-binding ability for precise HIV-1 targeting and viral inhibition.

Human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G, hereinafter referred to as A3G) is an innate virus restriction factor that inhibits human immunodeficiency virus type 1 (HIV-1) replication and induces excessive deamination of cytidine residues in nascent reverse transcripts. To test the hypothesis that this enzyme can also help generate viral sequence diversification and the evolution of beneficial viral variants, we have examined the impact of A3G on the acquisition of (−)2′,3′-dideoxy-3′-thiacytidine (3TC) resistance in vitro . That characteristic resistance mutations are rapidly fixed in the presence of A3G and 3TC suggests that A3G-mediated editing can be an important source of genetic variation on which natural selection acts to shape the structure of HIV-1 populations.

ß- l -2′,3′-Didehydro-2′,3′-dideoxy-N 4 -hydroxycytidine ( l -Hyd4C) was demonstrated to be an effective and highly selective inhibitor of hepatitis B virus (HBV) replication in HepG2.2.15 cells (50% effective dose [ED 50 ] = 0.03 μM; 50% cytotoxic dose [CD 50 ] = 2,500 μM). In the present study, we investigated the intracellular pharmacology of tritiated l -Hyd4C in HepG2 cells. l -[ 3 H]Hyd4C was shown to be phosphorylated extensively and rapidly to the 5′-mono-, 5′-di-, and 5′-triphosphate derivatives. Other metabolites deriving from a reduction or removal of the NHOH group of l -Hyd4C could not be detected, although both reactions were described as the primary catabolic pathways of the stereoisomer ß- d -N 4 -hydroxycytidine in HepG2 cells. Also, the formation of liponucleotide metabolites, such as the 5′-diphosphocholine derivative of l -Hyd4C, as described for some l -deoxycytidine analogues, seems to be unlikely. After incubation of HepG2 cells with 10 μM l -[ 3 H]Hyd4C for 24 h, the 5′-triphosphate accumulated to 19.4 ± 2.7 pmol/10 6 cells. The predominant peak belonged to 5-diphosphate, with 43.5 ± 4.3 pmol/10 6 cells. The intracellular half-life of the 5′-triphosphate was estimated to be 29.7 h. This extended half-life probably reflects a generally low affinity of 5′-phosphorylated l -deoxycytidine derivatives for phosphate-degrading enzymes but may additionally be caused by an efficient rephosphorylation of the 5′-diphosphate during a drug-free incubation. The high 5′-triphosphate level and its extended half-life in HepG2 cells are consistent with the potent antiviral activity of l -Hyd4C.

We developed a high-throughput quantitative capillary assay and demonstrated that Pseudomonas putida strains F1 and PRS2000 were attracted to cytosine, but not thymine or uracil. In contrast, Pseudomonas aeruginosa PAO1 was not chemotactic to any pyrimidines. Chemotaxis assays with a mutant strain of F1 in which the putative methyl-accepting chemotaxis protein-encoding gene Pput_0623 was deleted revealed that this gene (designated mcpC ) encodes a chemoreceptor for positive chemotaxis to cytosine. P. putida F1 also responded weakly to cytidine, uridine, and thymidine, but these responses were not mediated by mcpC . Complementation of the F1 Δ mcpC mutant XLF004 with the wild-type gene restored chemotaxis to cytosine. In addition, introduction of this gene into P. aeruginosa PAO1 conferred the ability to respond to cytosine. To our knowledge, this is the first report of a chemoreceptor for cytosine.

The APOBEC3 cytidine deaminases are potent antiviral factors that restrict the replication of human immunodeficiency virus type 1 (HIV-1). In HIV-1-infected CD4 + T cells, the viral accessory protein Vif binds to APOBEC3G (A3G), APOBEC3F (A3F), and APOBEC3C (A3C) and targets these proteins for polyubiquitination by forming an E3 ubiquitin ligase with cullin 5. Previous studies identified regions of HIV-1 Vif, 40 YRHHY 44 and 12 QVDRMR 17 , which are important for interaction with A3G and A3F, respectively, and showed that Vif residues 54 to 71 are sufficient for A3G binding. Here, we identify 69 YXXL 72 as a novel conserved motif in HIV-1 Vif that mediates binding to human A3G and its subsequent degradation. Studies on other APOBEC3 proteins revealed that Tyr69 and Leu72 are important for the degradation of A3F and A3C as well. Similar to A3F, A3C regulation is also mediated by Vif residues 12 QVDRMR 17 . Simian immunodeficiency virus (SIV) Vif was shown to bind and degrade African green monkey A3G (agmA3G) and, unexpectedly, human A3C. The YXXL motif of SIVagm Vif was important for the inactivation of agmA3G and human A3C. Unlike HIV-1 Vif, however, SIVagm Vif does not require Tyr40 and His43 for agmA3G degradation. Tyr69 in the YXXL motif was critical for binding of recombinant glutathione S -transferase-Vif(1-94) to A3G in vitro. These results suggest that the YXXL motif in Vif is a potential target for small-molecule inhibitors to block Vif interaction with A3G, A3F, and A3C, and thereby protect cells against HIV-1 infection.

Streptococcus pneumoniae has unusually complex cell wall teichoic acid and lipoteichoic acid, both of which contain a ribitol phosphate moiety. The lic region of the pneumococcal genome contains genes for the uptake and activation of choline, the attachment of phosphorylcholine to teichoic acid precursors, and the transport of these precursors across the cytoplasmic membrane. The role of two other, so far uncharacterized, genes, spr1148 and spr1149, in the lic region was determined. TarJ (spr1148) encodes an NADPH-dependent alcohol dehydrogenase for the synthesis of ribitol 5-phosphate from ribulose 5-phosphate. TarI (spr1149) encodes a cytidylyl transferase for the synthesis of cytidine 5′-diphosphate (CDP)-ribitol from ribitol 5-phosphate and cytidine 5′-triphosphate. We also present the crystal structure of TarI with and without bound CDP, and the structures present a rationale for the substrate specificity of this key enzyme. No transformants were obtained with insertion plasmids designed to interrupt the tarIJ genes, indicating that their function could be essential for cell growth. CDP-activated ribitol is a precursor for the synthesis of pneumococcal teichoic acids and some of the capsular polysaccharides. Thus, all eight genes in the lic region have a role in teichoic acid synthesis.

Human APOBEC3H belongs to the APOBEC3 family of cytidine deaminases that potently inhibit exogenous and endogenous retroviruses. The impact of single nucleotide polymorphisms (SNP) and alternative splicing on the antiretroviral activity of human APOBEC3H is currently unknown. In this study, we show that APOBEC3H transcripts derived from human peripheral blood mononuclear cells are polymorphic in sequence and subject to alternative splicing. We found that APOBEC3H variants encoding a SNP cluster (G105R, K121D and E178D, hapII-RDD) restricted human immunodeficiency virus type 1 (HIV-1) more efficiently than wild-type APOBEC3H (hapI-GKE). All APOBEC3H variants tested were resistant to HIV-1 Vif, the viral protein that efficiently counteracts APOBEC3G/3F activity. Alternative splicing of APOBEC3H was common and resulted in variants with distinct C-terminal regions and variable antiretroviral activities. Splice variants of hapI-GKE displayed a wide range of antiviral activities, whereas similar splicing events in hapII-RDD resulted in proteins that uniformly and efficiently restricted viral infectivity (>20-fold). Site-directed mutagenesis identified G105R in hapI-GKE and D121K in hapII-RDD as critical substitutions leading to an average additional 10-fold increase in antiviral activity. APOBEC3H variants were catalytically active and, similarly to APOBEC3F, favored a GA dinucleotide context. HIV-1 mutagenesis as a mode of action for APOBEC3H is suggested by the decrease of restriction observed with a cytidine deaminase domain mutant and the inverse correlation between G-to-A mutations and infectivity. Thus, the anti-HIV activity of APOBEC3H seems to be regulated by a combination of genomic variation and alternative splicing. Since prevalence of hapII-RDD is high in populations of African descent, these findings raise the possibility that some individuals may harbor effective as well as HIV-1 Vif-resistant intracellular antiviral defense mechanisms.

PSI-6130 (β- d -2′-deoxy-2′-fluoro-2′- C -methylcytidine) is a selective inhibitor of hepatitis C virus (HCV) replication that targets the NS5B polymerase. R7128, the prodrug of PSI-6130, has shown antiviral efficacy in patients chronically infected with HCV genotype 1a (GT-1a) and GT-1b. We observed that the compound exhibited potent in vitro activity against laboratory-optimized HCV replicons as well as against a panel of replicons containing NS5B HCV polymerases derived from GT-1a and GT-1b clinical isolates. We used the HCV replicon cell system to examine the emergence of variants with reduced sensitivity to PSI-6130. Short-term treatment of cells harboring the HCV subgenomic replicon with PSI-6130 cleared the replicon without generating resistant variants. Long-term culture of the cells under the compound selection generated the S282T substitution in a complex pattern with other amino acid substitutions in the NS5B polymerase. The presence of the coselected substitutions did not increase the moderate three- to sixfold loss of sensitivity to PSI-6130 mediated by the S282T substitution; however, their presence enhanced the replication capacity compared to the replication levels seen with the S282T substitution alone. We also observed a lack of cross-resistance between PSI-6130 and R1479 and demonstrated that long-term culture selection with PSI-6130 in replicon cells harboring preexisting mutations resistant to R1479 (S96T/N142T) results in the emergence of the S282T substitution and the reversion of S96T to wild-type serine. In conclusion, PSI-6130 presents a high barrier to resistance selection in vitro, selects for variants exhibiting only low-level resistance, and lacks cross-resistance with R1479, supporting the continued development of the prodrug R7128 as a therapeutic agent for the treatment of HCV infection.

Specific inhibitors of hepatitis C virus (HCV) replication that target the NS3/4A protease (e.g., VX-950) or the NS5B polymerase (e.g., R1479/R1626, PSI-6130/R7128, NM107/NM283, and HCV-796) have advanced into clinical development. Treatment of patients with VX-950 or HCV-796 rapidly selected for drug-resistant variants after a 14-day monotherapy treatment period. However, no viral resistance was identified after monotherapy with R1626 (prodrug of R1479) or NM283 (prodrug of NM107) after 14 days of monotherapy. Based upon the rapid selection of resistance to the protease and nonnucleoside inhibitors during clinical trials and the lack of selection of resistance to the nucleoside inhibitors, we used the replicon system to determine whether nucleoside inhibitors demonstrate a higher genetic barrier to resistance than protease and nonnucleoside inhibitors. Treatment of replicon cells with nucleoside inhibitors at 10 and 15 times the 50% effective concentration resulted in clearance of the replicon, while treatment with a nonnucleoside or protease inhibitor selected resistant colonies. In combination, the presence of a nucleoside inhibitor reduced the frequency of colonies resistant to the other classes of inhibitors. These results indicate that the HCV replicon presents a higher barrier to the selection of resistance to nucleoside inhibitors than to nonnucleoside or protease inhibitors. Furthermore, the combination of a nonnucleoside or protease inhibitor with a nucleoside polymerase inhibitor could have a clear clinical benefit through the delay of resistance emergence.

Apobec proteins are a family of cellular cytidine deaminases, among which several members have been shown to have potent antiviral properties. This antiviral activity is associated with the ability to cause hypermutation of retroviral cDNA. However, recent research has indicated that Apobec proteins are also able to inhibit retroviruses by other mechanisms that are independent of their deaminase activity. We have compared the antiviral activities of human and murine Apobec3 (A3) proteins, and we have found that, consistent with previous reports, human immunodeficiency virus (HIV) is able to resist human A3G but is sensitive to murine A3, whereas murine leukemia virus (MLV) is relatively resistant to murine A3 (mA3) but sensitive to human A3G. In contrast to previous studies, we observed that mA3 is packaged efficiently into MLV particles. The C-terminal cytidine deaminase domain (CDD2) is required for packaging of mA3 into MLV particles, and packaging did not depend on the MLV viral RNA. However, mA3 packed into MLV particles failed to cause hypermutation of viral DNA, indicating that its deaminase activity is blocked or inhibited. hA3G also caused significantly less hypermutation of MLV than of HIV DNA. Both mA3 and the splice variant mA3Δ5 exhibited some residual antiviral activity against MLV and caused a reduction in the ability of MLV particles to generate reverse transcription products. These results suggest that MLV has evolved specific mechanisms to block the ability of Apobec proteins to mediate deaminase-dependent hypermutation.

Interferons (IFNs) play a major role in the control of hepatitis B virus (HBV), whether as endogenous cytokines limiting the spread of the virus during the acute phase of the infection or as drugs for the treatment of its chronic phase. However, the mechanism by which IFNs inhibit HBV replication has so far remained elusive. Here, we show that type I and II IFN treatment of human hepatocytes induces the production of APOBEC3G (A3G) and, to a lesser extent, that of APOBEC3F (A3F) and APOBEC3B (A3B) but not that of two other cytidine deaminases also endowed with anti-HBV activity, activation-induced cytidine deaminase (AID), and APOBEC1. Most importantly, we reveal that blocking A3B, A3F, and A3G by combining RNA interference and the virion infectivity factor (Vif) protein of human immunodeficiency virus does not abrogate the inhibitory effect of IFNs on HBV. We conclude that these cytidine deaminases are not essential effectors of IFN in its action against this pathogen.

Novel N 4 -hydroxy- and 5-methyl-modified β- l -deoxycytidine analogues were synthesized and evaluated as anti-hepatitis B virus (HBV) agents. Their in vitro efficiencies were investigated in HepG2.2.15 cells stably transfected with HBV. β- l -2′,3′-Didehydro-2′,3′-dideoxy-N 4 -hydroxycytidine (β- l -Hyd4C) was most effective in reducing secreted HBV DNA (50% effective concentration [EC 50 ], 0.03 μM), followed by β- l -2′,3′-dideoxy-3′-thia-N 4 -hydroxycytidine (EC 50 , 0.51 μM), β- l -2′,3′-dideoxy-N 4 -hydroxycytidine (EC 50 , 0.55 μM), and β- l -5-methyl-2′-deoxycytidine (EC 50 , 0.9 μM). The inhibition of the presumed target, the HBV DNA polymerase, by the triphosphates of some of the β- l -cytidine derivatives was also assessed. In accordance with the cell culture data, β- l -Hyd4C triphosphate was the most active inhibitor, with a 50% inhibitory concentration of 0.21 μM. The cytotoxicities of some of the 4-NHOH-modified β- l -nucleosides were dramatically lower than those of the corresponding cytidine analogues with the unmodified 4-NH 2 group. The 50% cytotoxic concentrations for β- l -Hyd4C in HepG2 and HL-60 cells were 2,500 μM and 3,500 μM, respectively. In summary, our results demonstrate that at least β- l -Hyd4C can be recommended as a highly efficient and extremely selective inhibitor of HBV replication for further investigations.

The HIV-1 Vif protein inactivates the cellular antiviral cytidine deaminase APOBEC3F (A3F) in virus-infected cells by specifically targeting it for proteasomal degradation. Several studies identified Vif sequence motifs involved in A3F interaction, whereas a Vif-binding A3F interface was proposed based on our analysis of highly similar APOBEC3C (A3C). However, the structural mechanism of specific Vif-A3F recognition is still poorly understood. Here we report structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Alanine-scanning analysis of Vif revealed that six residues located within the conserved Vif F1-, F2-, and F3-box motifs are essential for both A3C and A3F degradation, and an additional four residues are uniquely required for A3F degradation. Modeling of the Vif structure on an HIV-1 Vif crystal structure revealed that three discontinuous flexible loops of Vif F1-, F2-, and F3-box motifs sterically cluster to form a flexible A3F interaction interface, which represents hydrophobic and positively charged surfaces. We found that the basic Vif interface patch (R17, E171, and R173) involved in the interactions with A3C and A3F differs. Furthermore, our crystal structure determination and extensive mutational analysis of the A3F C-terminal domain demonstrated that the A3F interface includes a unique acidic stretch (L291, A292, R293, and E324) crucial for Vif interaction, suggesting additional electrostatic complementarity to the Vif interface compared with the A3C interface. Taken together, these findings provide structural insights into the A3F-Vif interaction mechanism, which will provide an important basis for development of novel anti-HIV-1 drugs using cellular cytidine deaminases. IMPORTANCE HIV-1 Vif targets cellular antiviral APOBEC3F (A3F) enzyme for degradation. However, the details on the structural mechanism for specific A3F recognition remain unclear. This study reports structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Three discontinuous sequence motifs of Vif, F1, F2, and F3 boxes, assemble to form an A3F interaction interface. In addition, we determined a crystal structure of the wild-type A3F C-terminal domain responsible for the Vif interaction. These results demonstrated that both electrostatic and hydrophobic interactions are the key force driving Vif-A3F binding and that the Vif-A3F interfaces are larger than the Vif-A3C interfaces. These findings will allow us to determine the configurations of the Vif-A3F complex and to construct a structural model of the complex, which will provide an important basis for inhibitor development.

Streptococcus mutans has a significant number of transporters of the ATP-binding cassette (ABC) superfamily. Members of this superfamily are involved in the translocation of a diverse range of molecules across membranes. However, the functions of many of these members remain unknown. We have investigated the role of the single S. mutans representative of the second subfamily of carbohydrate uptake transporters (CUT2) of the ABC superfamily. The genetic context of genes encoding this transporter indicates that it may have a role in ribonucleoside scavenging. Inactivation of rnsA (ATPase) or rnsB (solute binding protein) resulted in strains resistant to 5-fluorocytidine and 5-fluorouridine (toxic ribonucleoside analogues). As other ribonucleosides including cytidine, uridine, adenosine, 2-deoxyuridine, and 2-deoxycytidine protected S. mutans from 5-fluorocytidine and 5-fluorouridine toxicity, it is likely that this transporter is involved in the uptake of these molecules. Indeed, the rnsA and rnsB mutants were unable to transport [2- 14 C]cytidine or [2- 14 C]uridine and had significantly reduced [8- 14 C]adenosine uptake rates. Characterization of this transporter in wild-type S. mutans indicates that it is a high-affinity ( K m = 1 to 2 μM) transporter of cytidine, uridine, and adenosine. The inhibition of [ 14 C]cytidine uptake by a range of structurally related molecules indicates that the CUT2 transporter is involved in the uptake of most ribonucleosides, including 2-deoxyribonucleosides, but not ribose or nucleobases. The characterization of this permease has directly shown for the first time that an ABC transporter is involved in the uptake of ribonucleosides and extends the range of substrates known to be transported by members of the ABC transporter superfamily.

Earlier, our laboratory reported that purified glutathione S -transferase-virion host shutoff (GST- vhs ) protein exhibited endoribonucleolytic activity in in vitro assays using as substrates in vitro-transcribed regions of IEX-1 mRNA. Here, we report that studies of the cleavage patterns of synthetic RNA oligonucleotides defined the activity of GST- vhs as being similar to that of RNase A. Thus, GST- vhs cleaved the RNA at the 3′ end of single-stranded cytidine and uridine residues. Since the GST-m vhs nuclease-defective mutant protein failed to cleave the synthetic RNAs, the results unambiguously attribute the activity to vhs .

The human cytidine deaminases APOBEC3G (hA3G) and APOBEC3F (hA3F) are intracellular antiretroviral factors that can hypermutate nascent reverse transcripts and inhibit the replication of human immunodeficiency virus type 1 (HIV-1). Both enzymes have two cytidine deaminase motifs, although only the C-terminal motif is catalytic. Current models of APOBEC protein function imply editing is the principal mechanism of antiviral activity. In particular, hA3G is a more potent inhibitor of HIV-1 infectivity than hA3F and also induces a greater frequency of mutations in HIV-1 cDNA. We used hA3G/hA3F chimeric proteins to investigate whether cytidine deaminase potential reflects antiviral potency. We show here that the origin of the C-terminal deaminase motif is sufficient to determine the degree of mutation induced in a bacterial assay that measures mutations in chromosomal DNA. In contrast, this was not the case in the context of HIV-1 infection where the N-terminal deaminase motif also modulated the editing capabilities of the chimeras. Surprisingly, although three of the chimeric proteins induced levels of mutation that approximated those of parental hA3F, they displayed lower levels of antiviral activity. Most importantly, real-time PCR experiments revealed that the quantity of reverse transcripts detected in target cells, rather than the mutational burden carried by such DNAs, corresponded closely with viral infectivity. In other words, the antiviral phenotype of APOBEC proteins correlates with their ability to prevent the accumulation of reverse transcripts and not with the induction of hypermutation.

The APOBEC3 genes encode cytidine deaminases that act as components of an intrinsic immune defense that have potent activity against a variety of retroelements. This family of genes has undergone a rapid expansion from one or two genes in nonprimate mammals to at least seven members in primates. Here we describe the evolution and function of an uncharacterized antiviral effector, APOBEC3H , which represents the most evolutionarily divergent APOBEC3 gene found in primates. We found that APOBEC3H has undergone significant adaptive evolution in primates. Consistent with our previous findings implicating adaptively evolving APOBEC3 genes as antiviral effectors, APOBEC3H from Old World monkeys (OWMs) has efficient antiviral activity against primate lentiviruses, is sensitive to inactivation by the simian immunodeficiency virus Vif protein, and is capable of hypermutating retroviral genomes. In contrast, human APOBEC3H is inherently poorly expressed in primate cells and is ineffective at inhibiting retroviral replication. Both OWM and human APOBEC3H proteins can be expressed in bacteria, where they display significant DNA mutator activity. Thus, humans have retained an APOBEC3H gene that encodes a functional, but poorly expressed, cytidine deaminase with no apparent antiviral activity. The consequences of the lack of antiviral activity of human APOBEC3H are likely to be relevant to the current-day abilities of humans to combat retroviral challenges.

β- d - N 4 -Hydroxycytidine (NHC) was found to have selective anti-hepatitis C virus (HCV) activity in the HCV replicon system (clone A). The intracellular metabolism of tritiated NHC was investigated in the HCV replicon system, Huh-7 cells, HepG2 cells, and primary human hepatocytes. Incubation of cells with 10 μM radiolabeled NHC demonstrated extensive and rapid phosphorylation in all liver cells. Besides the 5′-mono, -di-, and -triphosphate metabolites of NHC, other metabolites were characterized. These included cytidine and uridine mono-, di-, and triphosphates. UTP was the predominant early metabolite in Huh-7 cells and primary human hepatocytes, suggesting deamination of NHC as the primary catabolic pathway. The intracellular half-lives of radiolabeled NHC-triphosphate and of CTP and UTP derived from NHC incubation in Huh-7 cells were calculated to be 3.0 ± 1.3, 10.4 ± 3.3, and 13.2 ± 3.5 h (means ± standard deviations), respectively. Studies using monkey and human whole blood demonstrated more-rapid deamination and oxidation in monkey cells than in human cells, suggesting that NHC may not persist long enough in plasma to be delivered to liver cells.

A method for measuring internal nucleoside triphosphate pools of lactococci was optimized and validated. This method is based on extraction of 33 P-labeled nucleotides with formic acid and evaluation by two-dimensional chromatography with a phosphate buffer system for the first dimension and with an H 3 BO 3 -LiOH buffer for separation in the second dimension. We report here the sizes of the ribo- and deoxyribonucleotide pools in laboratory strain MG1363 during growth in a defined medium. We found that purine- and pyrimidine-requiring strains may be used to establish physiological conditions in batch fermentations with altered nucleotide pools and growth rates by addition of nucleosides in different combinations. Addition of cytidine together with inosine to a purine-requiring strain leads to a reduction in the internal purine nucleotide pools and a decreased growth rate. This effect was not seen if cytidine was replaced by uridine. A similar effect was observed if cytidine and inosine were added to a pyrimidine-requiring strain; the UTP pool size was significantly decreased, and the growth rate was reduced. To explain the observed inhibition, the nucleoside transport systems in Lactococcus lactis were investigated by measuring the uptake of radioactively labeled nucleosides. The K m for for inosine, cytidine, and uridine was determined to be in the micromolar range. Furthermore, it was found that cytidine and inosine are competitive inhibitors of each other, whereas no competition was found between uridine and either cytidine or inosine. These findings suggest that there are two different high-affinity nucleoside transporters, one system responsible for uridine uptake and another system responsible for the uptake of all purine nucleosides and cytidine.

A base-modified nucleoside analogue, β- d -N 4 -hydroxycytidine (NHC), was found to have antipestivirus and antihepacivirus activities. This compound inhibited the production of cytopathic bovine viral diarrhea virus (BVDV) RNA in a dose-dependant manner with a 90% effective concentration (EC 90 ) of 5.4 μM, an observation that was confirmed by virus yield assays (EC 90 = 2 μM). When tested for hepatitis C virus (HCV) replicon RNA reduction in Huh7 cells, NHC had an EC 90 of 5 μM on day 4. The HCV RNA reduction was incubation time and nucleoside concentration dependent. The in vitro antiviral effect of NHC was additive with recombinant alpha interferon-2a and could be prevented by the addition of exogenous cytidine and uridine but not of other natural ribo- or 2′-deoxynucleosides. When HCV RNA replicon cells were cultured in the presence of increasing concentrations of NHC (up to 40 μM) for up to 45 cell passages, no resistant replicon was selected. Similarly, resistant BVDV could not be selected after 20 passages. NHC was phosphorylated to the triphosphate form in Huh7 cells, but in cell-free HCV NS5B assays, synthetic NHC-triphosphate (NHC-TP) did not inhibit the polymerization reaction. Instead, NHC-TP appeared to serve as a weak alternative substrate for the viral polymerase, thereby changing the mobility of the product in polyacrylamide electrophoresis gels. We speculate that incorporated nucleoside analogues with the capacity of changing the thermodynamics of regulatory secondary structures (with or without introducing mutations) may represent an important class of new antiviral agents for the treatment of RNA virus infections, especially HCV.

Escherichia coli tRNA contains four naturally occurring nucleosides modified with sulfur. Cysteine is the intracellular sulfur source for each of these modified bases. We previously found that the iscS gene, a member of the nifS cysteine desulfurase gene family, is required for 4-thiouridine biosynthesis in E. coli . Since IscS does not bind tRNA, its role is the mobilization and distribution of sulfur to enzymes that catalyze the sulfur insertion steps. In addition to iscS , E. coli contains two other nifS homologs, csdA and csdB , each of which has cysteine desulfurase activity and could potentially donate sulfur for thionucleoside biosynthesis. Double csdA csdB and iscS csdA mutants were prepared or obtained, and all mutants were analyzed for thionucleoside content. It was found that unfractionated tRNA isolated from the iscS mutant strain contained 35 S]cysteine showed that only a small fraction of 2-thiocytidine was present, while the other thionucleosides were absent when cells were isolated during log phase. As expected, digests from the iscS mutant strain contained 6- N -dimethylallyl adenosine (i 6 A) in place of 6- N -dimethylallyl-2-methylthioadenosine and 5-methylaminomethyl uridine (mnm 5 U) instead of 5-methylaminomethyl-2-thiouridine. Prolonged growth of the iscS and iscS csdA mutant strains revealed a gradual increase in levels of 2-thiocytidine and 6- N -dimethylallyl-2-methylthioadenosine with extended incubation (>24 h), while the thiouridines remained absent. This may be due to a residual level of Fe-S cluster biosynthesis in iscS deletion strains. An overall scheme for thionucleoside biosynthesis in E. coli is discussed.

The antiviral efficacies and cytotoxicities of 2′,3′- and 4′-substituted 2′,3′-didehydro-2′,3′-dideoxycytidine analogs were evaluated. All compounds were tested (i) against a wild-type human immunodeficiency virus type 1 (HIV-1) isolate (strain xxBRU) and lamivudine-resistant HIV-1 isolates, (ii) for their abilities to inhibit hepatitis B virus (HBV) production in the inducible HepAD38 cell line, and (iii) for their abilities to inhibit bovine viral diarrhea virus (BVDV) production in acutely infected Madin-Darby bovine kidney cells. Some compounds demonstrated potent antiviral activities against the wild-type HIV-1 strain (range of 90% effective concentrations [EC 90 s], 0.14 to 5.2 μM), but marked increases in EC 90 s were noted when the compounds were tested against the lamivudine-resistant HIV-1 strain (range of EC 90 s, 53 to >100 μM). The β- l -enantiomers of both classes of compounds were more potent than the corresponding β- d -enantiomers. None of the compounds showed antiviral activity in the assay that determined their abilities to inhibit BVDV, while two compounds inhibited HBV production in HepAD38 cells (EC 90 , 0.25 μM). The compounds were essentially noncytotoxic in human peripheral blood mononuclear cells and HepG2 cells. No effect on mitochondrial DNA levels was observed after a 7-day incubation with the nucleoside analogs at 10 μM. These studies demonstrate that (i) modification of the sugar ring of cytosine nucleoside analogs with a 4′-thia instead of an oxygen results in compounds with the ability to potently inhibit wild-type HIV-1 but with reduced potency against lamivudine-resistant virus and (ii) the antiviral activity of β- d -2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine against wild-type HIV-1 (EC 90 , 0.08 μM) and lamivudine-resistant HIV-1 (EC 90 = 0.15 μM) is markedly reduced by introduction of a 3′-fluorine in the sugar (EC 90 s of compound 2a, 37.5 and 494 μM, respectively).

Highly active antiretroviral therapy (HAART) is the standard treatment for infection with the human immunodeficiency virus (HIV). HAART regimens consist of protease inhibitors or nonnucleoside reverse transcriptase inhibitors combined with two or more nucleoside reverse transcriptase inhibitors (NRTIs). DPC 817, 2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine (PSI 5582 D-D4FC) is a potent inhibitor of HIV type 1 replication in vitro. Importantly, DPC 817 retains activity against isolates harboring mutations in the reverse transcriptase gene that confer resistance to lamivudine (3TC) and zidovudine (AZT), which are frequent components of initial HAART regimens. DPC 817 combines this favorable resistance profile with rapid uptake and conversion to the active metabolite DPC 817-triphosphate, which has an intracellular half-life of 13 to 17 h. Pharmacokinetics in the rhesus monkey suggest low clearance of parent DPC 817 and a plasma half-life longer than that of either AZT or 3TC. Together, these properties suggest that DPC 817 may be useful as a component of HAART regimens in individuals with resistance to older NRTI agents.

Members of the APOBEC3 family of cytidine deaminases vary in their proportions of a virion-incorporated enzyme that is localized to mature retrovirus cores. We reported previously that APOBEC3F (A3F) was highly localized into mature human immunodeficiency virus type 1 (HIV-1) cores and identified that L306 in the C-terminal cytidine deaminase (CD) domain contributed to its core localization (C. Song, L. Sutton, M. Johnson, R. D'Aquila, J. Donahue, J Biol Chem 287: 16965–16974, 2012, http://dx.doi.org/10.1074/jbc.M111.310839 ). We have now determined an additional genetic determinant(s) for A3F localization to HIV-1 cores. We found that one pair of leucines in each of A3F's C-terminal and N-terminal CD domains jointly determined the degree of localization of A3F into HIV-1 virion cores. These are A3F L306/L368 (C-terminal domain) and A3F L122/L184 (N-terminal domain). Alterations to one of these specific leucine residues in either of the two A3F CD domains (A3F L368A, L122A, and L184A) decreased core localization and diminished HIV restriction without changing virion packaging. Furthermore, double mutants in these leucine residues in each of A3F's two CD domains (A3F L368A plus L184A or A3F L368A plus L122A) still were packaged into virions but completely lost core localization and anti-HIV activity. HIV virion core localization of A3F is genetically separable from its virion packaging, and anti-HIV activity requires some core localization. IMPORTANCE Specific leucine-leucine interactions are identified as necessary for A3F's core localization and anti-HIV activity but not for its packaging into virions. Understanding these signals may lead to novel strategies to enhance core localization that may augment effects of A3F against HIV and perhaps of other A3s against retroviruses, parvoviruses, and hepatitis B virus.

We show here that deoxycytidine deaminase (DCD)-deficient mutants of Escherichia coli are hypersensitive to killing by exogenous cytidine, adenosine, or guanosine, whereas wild-type cells are not. This hypersensitivity is reversed by exogenous thymidine. The mechanism likely involves the allosteric regulation of ribonucleotide reductase and severe limitations of the dTTP pools, resulting in thymineless death, the phenomenon of cell death due to thymidine starvation. We also report here that DCD-deficient mutants of E. coli are more sensitive to a series of different antibiotics, including vancomycin, and we show synergistic killing with the combination of vancomycin and cytidine. One possibility is that a very low, subinhibitory concentration of vancomycin enters Gram-negative cells and that this concentration is potentiated by chromosomal lesions resulting from the thymineless state. A second possibility is that the metabolic imbalance resulting from DCD deficiency affects the assembly of the outer membrane, which normally presents a barrier to drugs such as vancomycin. We consider these findings with regard to ideas of rendering Gram-negative bacteria sensitive to drugs such as vancomycin.

The B. subtilis pyrG gene, which encodes CTP synthetase, is located far from the pyrimidine biosynthetic operon on the chromosome and is independently regulated. The pyrG promoter and 5′ leader were fused to lacZ and integrated into the chromosomes of several B. subtilis strains having mutations in genes of pyrimidine biosynthesis and salvage. These mutations allowed the intracellular pools of cytidine and uridine nucleotides to be manipulated by the composition of the growth medium. These experiments indicated that pyrG expression is repressed by cytidine nucleotides but is largely independent of uridine nucleotides. The start of pyrG transcription was mapped by primer extension to a position 178 nucleotides upstream of the translation initiation codon. A factor-independent termination hairpin lying between the pyrG promoter and its coding region is essential for regulation of pyrG expression. Primer-extended transcripts were equally abundant in repressed and derepressed cells when the primer bound upstream of the terminator, but they were much less abundant in repressed cells when the primer bound downstream of the terminator. Furthermore, deletion of the terminator from pyrG-lacZ fusions integrated into the chromosome yielded elevated levels of expression that was not repressible by cytidine. We suggest that cytidine repression of pyrG expression is mediated by an antitermination mechanism in which antitermination by a putative trans -acting protein is reduced by elevated levels of cytidine nucleotides. Conservation of sequences and secondary structural elements in the pyrG 5′ leaders of several other gram-positive bacteria indicates that their pyrG genes are regulated by a similar mechanism.

A novel enzyme which catalyzes the oxidation of nucleosides to nucleoside-5(prm1)-carboxylic acids, forming hydrogen peroxide, was purified to homogeneity from Flavobacterium meningosepticum T-2799. The enzyme has a molecular weight of about 500,000, and four nonidentical subunits (molecular weights of 81,000, 69,000, 33,000, and 16,000) were detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On the basis of visible absorption spectra of the purified enzyme, the enzyme is concluded to be a hemoprotein. It also contains covalently bound flavin adenine dinucleotide. The various nucleosides, such as adenosine (K(infm) = 48 (mu)M), inosine (K(infm) = 66 (mu)M), guanosine (K(infm) = 21 (mu)M), thymidine (K(infm) = 50 (mu)M), uridine (K(infm) = 80 (mu)M), and cytidine (K(infm) = 50 (mu)M), were oxidized by the enzyme, but nucleotides, bases, and ribose were not.

The apolipoprotein B editing complex 3 (APOBEC3) family of proteins is a group of intrinsic antiviral factors active against a number of retroviral pathogens, including HIV in humans and mouse mammary tumor virus (MMTV) in mice. APOBEC3 restricts its viral targets through cytidine deamination of viral DNA during reverse transcription or via deaminase-independent means. Here, we used virions from the mammary tissue of MMTV-infected inbred wild-type mice with different allelic APOBEC3 variants (APOBEC3 BALB and APOBEC3 BL/6 ) and knockout mice to determine whether cytidine deamination was important for APOBEC3's anti-MMTV activity. First, using anti-murine APOBEC3 antiserum, we showed that both APOBEC3 allelic variants are packaged into the cores of milk-borne virions produced in vivo . Next, using an in vitro deamination assay, we determined that virion-packaged APOBEC3 retains its deamination activity and that allelic differences in APOBEC3 affect the sequence specificity. In spite of this in vitro activity, cytidine deamination by virion-packaged APOBEC3 of MMTV early reverse transcription DNA occurred only at low levels. Instead, the major means by which in vivo virion-packaged APOBEC3 restricted virus was through inhibition of early reverse transcription in both cell-free virions and in vitro infection assays. Moreover, the different wild-type alleles varied in their ability to inhibit this step. Our data suggest that while APOBEC3-mediated cytidine deamination of MMTV may occur, it is not the major means by which APOBEC3 restricts MMTV infection in vivo . This may reflect the long-term coexistence of MMTV and APOBEC3 in mice.

(-)-beta-L-2',3'-Dideoxycytidine (L-ddC) and (-)-beta-L-2',3'-dideoxy-5-fluorocytidine (L-FddC) have been reported to be potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) in vitro. In the present study, the 5'-triphosphates of L-ddC (L-ddCTP) and L-FddC (L-FddCTP) were demonstrated to competitively inhibit HIV-1 reverse transcriptase (RT), with inhibition constants (KiS) of 2 and 1.6 microM, respectively, when a poly(rI).oligo(dC)10-15 template primer was used; in comparison Ki values for beta-D-2',3'-dideoxycytidine 5'-triphosphate (D-ddCTP) and beta-D-2',3'-dideoxy-5-fluorocytidine 5'-triphosphate (D-FddCTP) were 1.1 and 1.4 microM, respectively. Use of the mutant RT at position 184 (substitution of methionine to valine [M184V]), which is associated with resistance to beta-L-2',3'-dideoxy-3'-thiacytidine (3TC) and beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC), resulted in significant increases (50- to 60-fold) in Ki values for L-ddCTP and L-FddCTP, whereas the elevation in Ki values for D-ddCTP and D-FddCTP was moderate (2-fold). L-ddCTP and L-FddCTP did not inhibit human DNA polymerases alpha and beta up to 100 microM. In contrast, D-ddCTP and D-FddCTP inhibited human DNA polymerase beta, with Ki values of 0.5 and 2.5 microM, respectively. By using sequencing analysis, L-ddCTP and L-FddCTP exhibited DNA chain-terminating activities toward the parental HIV-1 RT, whereas they were not a substrate for the mutant M184V HIV-1 RT.L-ddC and L-FddC did not inhibit the mitochondrial DNA content of human cells up to a concentration of 10 microM, whereas D-ddC and D-FddC decreased the mitochondrial DNA content by 90% at concentrations of 1 and 10 microM, respectively. All of these results suggest that further development of L-ddC, and L-FddC in particular, is warranted as a possible anti-HIV candidate.

In vivo, gonococci encounter a myriad of conditions not present in vitro. At some stages of infection and disease, gonococci may grow anaerobically, probably by using sodium nitrite as a terminal electron acceptor. Also, gonococci sialylate their lipooligosaccharide (LOS) in vivo, by using low concentrations of cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA) present in host tissue. This sialylation is responsible for the acquired resistance of gonococci to both normal and immune human serum. Given that gonococci grown in the absence of oxygen or in the presence of CMP-NANA probably more closely resemble gonococci grown inside a human host, we studied the serum resistance of gonococci cultivated under these conditions. In the absence of CMP-NANA, anaerobically grown (anaerobic) gonococci were somewhat less sensitive to serum killing than were aerobically grown (aerobic) gonococci. However, anaerobic gonococci grown with 6 micrograms of CMP-NANA per ml exhibited almost complete serum resistance, while aerobic gonococci required 16-fold-higher CMP-NANA concentrations to achieve significant serum resistance. Anaerobic gonococci incubated in CMP-NANA converted to serum resistance two to three times faster than did similarly treated aerobic gonococci and incorporated up to six times as much sialic acid into their LOS. Gonococci can express several different LOS molecules. Anaerobic gonococci expressed the LOS molecule that acts as an acceptor for sialic acid from CMP-NANA in greater quantity than aerobic gonococci did. Finally, Triton X-100 extracts of anaerobic gonococci contained about four times more sialyltransferase activity than did extracts of aerobic gonococci. Sialyltransferase activity in these extracts was not inhibited by oxygen or enhanced by anaerobiosis. These data indicate that anaerobic conditions lead to altered LOS biosynthesis and to induction of sialyltransferase activity in gonococci. In vivo, where decreased oxygen levels and relevant concentrations of CMP-NANA are found, gonococci could readily become resistant to killing by normal and immune human serum.