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Although antiretroviral therapy can suppress HIV-1 replication effectively, virus reservoirs persist in infected individuals and virus replication rapidly rebounds if therapy is interrupted. Currently, there is a need for therapeutic approaches that eliminate, reduce, or control persistent viral reservoirs if a cure is to be realized. This work focuses on the preclinical development of novel, small-molecule inhibitors of the HIV-1 Vif protein. Vif inhibitors represent a new class of antiretroviral drugs that may expand treatment options to more effectively suppress virus replication or to drive HIV-1 reservoirs to a nonfunctional state by harnessing the activity of the DNA-editing cytidine deaminase A3G, a potent, intrinsic restriction factor expressed in macrophage and CD4+ T cells. In this study, we derived inhibitor escape variants to characterize the mechanism by which these novel agents inhibit virus replication and to provide evidence for target validation., The HIV-1 accessory protein Vif, which counteracts the antiviral action of the DNA-editing cytidine deaminase APOBEC3G (A3G), is an attractive and yet unexploited therapeutic target. Vif reduces the virion incorporation of A3G by targeting the restriction factor for proteasomal degradation in the virus-producing cell. Compounds that inhibit Vif-mediated degradation of A3G in cells targeted by HIV-1 would represent a novel antiviral therapeutic. We previously described small molecules with activity consistent with Vif antagonism. In this study, we derived inhibitor escape HIV-1 variants to characterize the mechanism by which these novel agents inhibit virus replication. Here we show that resistance to these agents is dependent on an amino acid substitution in Vif (V142I) and on a point mutation that likely upregulates transcription by modifying the lymphocyte enhancing factor 1 (LEF-1) binding site. Molecular modeling demonstrated a docking site in the Vif-Elongin C complex that is disrupted by these inhibitors. This docking site is lost when Vif acquires the V142I mutation that leads to inhibitor resistance. Competitive fitness experiments indicated that the V142I Vif and LEF-1 binding site mutations created a virus that is better adapted to growing in the presence of A3G than the wild-type virus.

Enterovirus 71 (EV71), a single-stranded positive-sense RNA virus, is the causative agent of hand, foot, and mouth disease (HFMD), for which no effective antiviral therapy is currently available. Apolipoprotein B messenger RNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) is a cytidine deaminase that inhibits the replication of several viruses, such as human immunodeficiency virus-1, hepatitis B virus and hepatitis C virus. In our efforts toward understanding the antiviral spectrum and mechanism of A3G, we found that ectopic expression of A3G inhibited EV71 replication, whereas knockdown of endogenous A3G expression promoted EV71 replication. Moreover, inhibition of EV71 replication by IMB-Z, a N-phenylbenzamide derivative, is associated with increased levels of intracellular A3G, but reducing the level of A3G by RNA interference diminished the antiviral activity of IMB-Z. Mechanistically, we obtained evidence suggesting that the cytidine deaminase activity is not required for A3G inhibition of EV71 replication. Instead, we demonstrated that A3G can interact with viral 3D RNA-dependent RNA polymerase (RdRp) and viral RNA and be packaged into progeny virions to reduce its infectivity. Taken together, our results indicate that A3G is a cellular restriction factor of EV71 and mediator of the antiviral activity of IMB-Z. Pharmacological induction and/or stabilization of A3G is a potential therapeutic approach to treat diseases caused by EV71 infection, such as HFMD.

Background The apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) genes A3D, A3F, A3G and A3H have all been implicated in the restriction of human immunodeficiency virus type 1 (HIV-1) replication. Polymorphisms in these genes are likely to impact viral replication and fitness, contributing to viral diversity. Currently, only a few studies indicate that polymorphisms in the A3 genes may be correlated with infection risk and disease progression. Methods To characterize polymorphisms in the coding regions of these APOBEC3 genes in an HIV-1 infected population from the Limpopo Province of South Africa, APOBEC3 gene fragments were amplified from genomic DNA of 192 HIV-1 infected subjects and sequenced on an Illumina MiSeq platform. SNPs were confirmed and compared to SNPs in other populations reported in the 1000 Genome Phase III and HapMap databases, as well as in the ExAC exome database. Hardy-Weinberg Equilibrium was calculated and haplotypes were inferred using the LDlink 3.0 web tool. Linkage Disequilibrium (LD) for these SNPS were calculated in the total 1000 genome and AFR populations using the same tool. Results Known variants compared to the GRCh37 consensus genome sequence were detected at relatively high frequencies (> 5%) in all of the APOBEC3 genes. A3H showed the most variation, with several of the variants present in both alleles in almost all of the patients. Several minor allele variants (

APOBEC3G (A3G) is a host enzyme that mutates the genomes of retroviruses like HIV. Since A3G is expressed pre-infection, it has classically been considered an agent of innate immunity. We and others previously showed that the impact of A3G-induced mutations on the HIV genome extends to adaptive immunity also, by generating cytotoxic T cell (CTL) escape mutations. Accordingly, HIV genomic sequences encoding CTL epitopes often contain A3G-mutable “hotspot” sequence motifs, presumably to channel A3G action toward CTL escape. Here, we studied the depths and consequences of this apparent viral genome co-evolution with A3G. We identified all potential CTL epitopes in Gag, Pol, Env, and Nef restricted to several HLA class I alleles. We simulated A3G-induced mutations within CTL epitope-encoding sequences, and flanking regions. From the immune recognition perspective, we analyzed how A3G-driven mutations are predicted to impact CTL-epitope generation through modulating proteasomal processing and HLA class I binding. We found that A3G mutations were most often predicted to result in diminishing/abolishing HLA-binding affinity of peptide epitopes. From the viral genome evolution perspective, we evaluated enrichment of A3G hotspots at sequences encoding CTL epitopes and included control sequences in which the HIV genome was randomly shuffled. We found that sequences encoding immunogenic epitopes exhibited a selective enrichment of A3G hotspots, which were strongly biased to translate to non-synonymous amino acid substitutions. When superimposed on the known mutational gradient across the entire length of the HIV genome, we observed a gradient of A3G hotspot enrichment, and an HLA-specific pattern of the potential of A3G hotspots to lead to CTL escape mutations. These data illuminate the depths and extent of the co-evolution of the viral genome to subvert the host mutator A3G.

The APOBEC3 family of deoxycytidine deaminases has the ability to restrict HIV-1 through deamination-dependent and deamination-independent mechanisms. Although the generation of mutations through deamination of cytosine to uracil in single-stranded HIV-1 (-) DNA is the dominant mechanism of restriction, the deaminase-independent mechanism additionally contributes. Previous observations indicate that APOBEC3 enzymes competitively bind the RNA template or reverse transcriptase (RT) and act as a roadblock to DNA polymerization. Here we studied how the deamination-independent inhibition of HIV-1 RT by APOBEC3C S188I, APOBEC3F, APOBEC3G, and APOBEC3H affected RT template switching. We found that APOBEC3F could promote template switching of RT, and this was dependent on the high affinity with which it bound nucleic acids, suggesting than an APOBEC3 "road-block" can force template switching. Our data demonstrate that the deamination-independent functions of APOBEC3 enzymes extend beyond only disrupting RT DNA polymerization. Since alterations to the RT template switching frequency can result in insertions or deletions, our data support a model in which APOBEC3 enzymes use multiple mechanisms to increase the probability of generating a mutated and nonfunctional virus in addition to cytosine deamination.

IntroductionMucosal immune activation, in the context of sexual transmission of HIV-1 infection, is crucial, as the increased presence of activated T cells enhance susceptibility to infection. In this regard, it has been proposed that immunomodulatory compounds capable of modulating immune activation, such as Vitamin D (VitD) may reduce HIV-1 transmission and might be used as a safe and cost-effective strategy for prevention. Considering this, we examined the in vitro effect of the treatment of peripheral blood mononuclear cells (PBMCs) with the active form of VitD, calcitriol, on cellular activation, function and susceptibility of CD4+ T cells to HIV-1 infection.MethodsWe treated PBMCs from healthy HIV unexposed individuals (Co-HC) and frequently exposed, HIV-1 seronegative individuals (HESNs) from Colombia and from healthy non-exposed individuals from Canada (Ca-HC) with calcitriol and performed in vitro HIV-1 infection assays using X4- and R5-tropic HIV-1 strains respectively. In addition, we evaluated the activation and function of T cells and the expression of viral co-receptors, and select antiviral genes following calcitriol treatment.ResultsCalcitriol reduced the frequency of infected CD4+ T cells and the number of viral particles per cell, for both, X4- and R5-tropic viruses tested in the Co-HC and the Ca-HC, respectively, but not in HESNs. Furthermore, in the Co-HC, calcitriol reduced the frequency of polyclonally activated T cells expressing the activation markers HLA-DR and CD38, and those HLA-DR+CD38-, whereas increased the subpopulation HLA-DR-CD38+. Calcitriol treatment also decreased production of granzyme, IL-2 and MIP-1β by T cells and increased the transcriptional expression of the inhibitor of NF-kB and the antiviral genes cathelicidin (CAMP) and APOBEC3G in PBMCs from Co-HC.ConclusionOur in vitro findings suggest that VitD treatment could reduce HIV-1 transmission through a specific modulation of the activation levels and function of T cells, and the production of antiviral factors. In conclusion, VitD remains as an interesting potential strategy to prevent HIV-1 transmission that should be further explored.

Background While genome-wide association studies (GWAS) of multiple myeloma (MM) have identified variants at 23 regions influencing risk, the genes underlying these associations are largely unknown. To identify candidate causal genes at these regions and search for novel risk regions, we performed a multi-tissue transcriptome-wide association study (TWAS). Results GWAS data on 7319 MM cases and 234,385 controls was integrated with Genotype-Tissue Expression Project (GTEx) data assayed in 48 tissues (sample sizes, N = 80–491), including lymphocyte cell lines and whole blood, to predict gene expression. We identified 108 genes at 13 independent regions associated with MM risk, all of which were in 1 Mb of known MM GWAS risk variants. Of these, 94 genes, located in eight regions, had not previously been considered as a candidate gene for that locus. Conclusions Our findings highlight the value of leveraging expression data from multiple tissues to identify candidate genes responsible for GWAS associations which provide insight into MM tumorigenesis. Among the genes identified, a number have plausible roles in MM biology, notably APOBEC3C, APOBEC3H, APOBEC3D, APOBEC3F, APOBEC3G, or have been previously implicated in other malignancies. The genes identified in this TWAS can be explored for follow-up and validation to further understand their role in MM biology. Electronic supplementary material The online version of this article (10.1186/s40246-019-0231-5) contains supplementary material, which is available to authorized users.

Proteasome inhibitors (PIs) have been identified as an emerging class of HIV-1 latency-reversing agents (LRAs). These inhibitors can reactivate latent HIV-1 to produce non-infectious viruses. The mechanism underlying reduced infectivity of reactivated viruses is unknown. In this study, we analysed PI-reactivated viruses using biochemical and virological assays and demonstrated that these PIs stabilized the cellular expression of HIV-1 restriction factor, APOBEC3G, facilitating its packaging in the released viruses. Using infectivity assay and immunoblotting, we observed that the reduction in viral infectivity was due to enhanced levels of functionally active APOBEC3 proteins packaged in the virions. Sequencing of the proviral genome in the target cells revealed the presence of APOBEC3 signature hypermutations. Our study strengthens the role of PIs as bifunctional LRAs and demonstrates that the loss of infectivity of reactivated HIV-1 virions may be due to the increased packaging of APOBEC3 proteins in the virus.

APOBEC3G (A3G) belongs to the family of cytosine deaminases that play an important role in the innate immune response. Similar to other, two-domain members of the APOBEC family, A3G is prone to concentration-dependent oligomerization, which is an integral for its function in the cell. It is shown that oligomerization of A3G is related to the packing mechanism into virus particle and, is critical for the so-called roadblock model during reverse transcription of proviral ssDNA. The role of oligomerization for deaminase activity of A3G is widely discussed in the literature; however, its relevance to deaminase activity for different oligomeric forms of A3G remains unclear. Here, using Atomic Force Microscopy, we directly visualized A3G-ssDNA complexes, determined their yield and stoichiometry and in parallel, using PCR assay, measured the deaminase activity of these complexes. Our data demonstrate a direct correlation between the total yield of A3G-ssDNA complexes and their total deaminase activity. Using these data, we calculated the relative deaminase activity for each individual oligomeric state of A3G in the complex. Our results show not only similar deaminase activity for monomer, dimer and tetramer of A3G in the complex, but indicate that larger oligomers of A3G retain their deaminase activity.

Antiviral restriction factors are host cellular proteins that constitute a first line of defense blocking viral replication and propagation. In addition to interfering at critical steps of the viral replication cycle, some restriction factors also act as innate sensors triggering innate responses against infections. Accumulating evidence suggests an additional role for restriction factors in promoting antiviral cellular immunity to combat viruses. Here, we review the recent progress in our understanding on how restriction factors, particularly APOBEC3G, SAMHD1, Tetherin, and TRIM5α have the cell-autonomous potential to induce cellular resistance against HIV-1 while promoting antiviral innate and adaptive immune responses. Also, we provide an overview of how these restriction factors may connect with protein degradation pathways to modulate anti-HIV-1 cellular immune responses, and we summarize the potential of restriction factors-based therapeutics. This review brings a global perspective on the influence of restrictions factors in intrinsic, innate, and also adaptive antiviral immunity opening up novel research avenues for therapeutic strategies in the fields of drug discovery, gene therapy, and vaccines to control viral infections.

Human APOBEC3H is a single-stranded (ss)DNA deoxycytidine deaminase that inhibits replication of retroelements and HIV-1 in CD4+ T cells. When aberrantly expressed in lung or breast tissue, APOBEC3H can contribute to cancer mutagenesis. These different activities are carried out by different haplotypes of APOBEC3H. Here we studied APOBEC3H haplotype II, which is able to restrict HIV-1 replication and retroelements. We determined how the dimerization mechanism, which is mediated by a double-stranded RNA molecule, influenced interactions with and activity on ssDNA. The data demonstrate that the cellular RNA bound by APOBEC3H does not completely inhibit enzyme activity, in contrast to other APOBEC family members. Despite degradation of the cellular RNA, an approximately 12-nt RNA remains bound to the enzyme, even in the presence of ssDNA. The RNA-mediated dimer is disrupted by mutating W115 on loop 7 or R175 and R176 on helix 6, but this also disrupts protein stability. In contrast, mutation of Y112 and Y113 on loop 7 also destabilizes RNA-mediated dimerization but results in a stable enzyme. Mutants unable to bind cellular RNA are unable to bind RNA oligonucleotides, oligomerize, and deaminate ssDNA in vitro, but ssDNA binding is retained. Comparison of A3H wild type and Y112A/Y113A by fluorescence polarization, single-molecule optical tweezer, and atomic force microscopy experiments demonstrates that RNA-mediated dimerization alters the interactions of A3H with ssDNA and other RNA molecules. Altogether, the biochemical analysis demonstrates that RNA binding is integral to APOBEC3H function.

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G, A3G), is a human anti-virus restriction protein which works deaminase-dependently and -independently. A3G is known to be ubiquitinated by HIV-1 viral infectivity factor (Vif) protein, leading to proteasomal degradation. A3G contains two zinc ions at the N-terminal domain and the C-terminal domain. Four lysine residues, K(297), K(301), K(303), and K(334), are known to be required for Vif-mediated A3G ubiquitination and degradation. Previously, we reported compound SN-1, a zinc chelator that increases steady-state expression level of A3G in the presence of Vif. In this study, we prepared Biotin-SN-1, a biotinylated derivative of SN-1, to study the SN-1-A3G interaction. A pull-down assay revealed that Biotin-SN-1 bound A3G. A zinc-abstraction experiment indicated that SN-1 binds to the zinc site of A3G. We carried out a SN-1-A3G docking study using molecular operating environment. The calculations revealed that SN-1 binds to the C-terminal domain through Zn(2+), H(216), P(247), C(288), and Y(315). Notably, SN-1-binding covers the H(257), E(259), C(288), and C(291) residues that participate in zinc-mediated deamination, and the ubiquitination regions of A3G. The binding of SN-1 presumably perturbs the secondary structure between C(288) and Y(315), leading to less efficient ubiquitination.

Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (A3G) is a host restriction factor that impedes HIV-1 replication. Viral integrity is salvaged by HIV-1 virion infectivity factor (Vif), which mediates A3G polyubiquitination and subsequent cellular depletion. Previous studies have implied that A3G polyubiquitination is essential for Vif-induced degradation. However, the contribution of polyubiquitination to the rate of A3G degradation remains unclear. Here, we show that A3G polyubiquitination is essential for degradation. Inhibition of ubiquitin-activating enzyme E1 by PYR-41 or blocking the formation of ubiquitin chains by over-expressing the lysine to arginine mutation of ubiquitin K48 (K48R) inhibited A3G degradation. Our A3G mutagenesis study showed that lysine residues 297, 301, 303, and 334 were not sufficient to render lysine-free A3G sensitive to Vif-mediated degradation. Our data also confirm that Vif could induce ubiquitin chain formation on lysine residues interspersed throughout A3G. Notably, A3G degradation relied on the lysine residues involved in polyubiquitination. Although A3G and the A3G C-terminal mutant interacted with Vif and were modified by ubiquitin chains, the latter remained more resistant to Vif-induced degradation. Furthermore, the A3G C-terminal mutant, but not the N-terminal mutant, maintained potent antiviral activity in the presence of Vif. Taken together, our results suggest that the location of A3G ubiquitin modification is a determinant for Vif-mediated degradation, implying that in addition to polyubiquitination, other factors may play a key role in the rate of A3G degradation.

Objective The human cytidine deaminase APOBEC3G (A3G) potently restricts HIV-1 but the virus, in turn, expresses a Vif protein which degrades A3G. A natural A3G-H186R variant, common in African populations, has been associated with a more rapid AIDS disease progression, but the underlying mechanism remains unknown. We hypothesized that differences in HIV-1 Vif activity towards A3G wild type and A3G-H186R contribute to the distinct clinical AIDS manifestation. Methods Vif variants were cloned from plasma samples of 26 South African HIV-1 subtype C infected patients, which either express wild type A3G or A3G-H186R. The Vif alleles were assessed for their ability to counteract A3G variants using western blot and single-cycle infectivity assays. Results We obtained a total of 392 Vif sequences which displayed an amino acid sequence difference of 6.2-19.2% between patients. The intrapatient Vif diversities from patient groups A3G, A3G and A3G were similar. Vif variants obtained from patients expressing A3G and A3G were capable of counteracting both A3G variants with similar efficiency. However, the antiviral activity of A3G-H186R was significantly reduced in both the presence and absence of Vif, indicating that the A3G-H186R variant intrinsically exerts less antiviral activity. Conclusion A3G wild type and A3G-H186R are equally susceptible to counteraction by Vif, regardless of whether the Vif variant was obtained from A3G and A3G patients. However, the A3G-H186R variant intrinsically displayed lower antiviral activity, which could explain the higher plasma viral loads and accelerated disease progression reported for patients expressing A3G.

Recently Apolipoprotein B mRNA editing enzyme, Catalytic Polypeptide-like 3G (APOBEC3G) biology has assumed importance because of its role in oncogenesis. In this context, the present study was addressed to understand the immune-modulatory role of APOBEC3G through its effect upon the T-cell plasticity phenomenon. Such an attempt revealed that APOBEC3G has the inherent capacity to regulate genes coding for STAT3, NF-κB, CCL5, IL-6, IL-4, IFN-γ, IL-10 and IL-17 coupled with downregulation of Treg cells within human peripheral blood mononuclear cells (PBMCs) without any noticeable influence upon CD4(+) and CD8(+) cell number. On the basis of these findings, we propose that APOBEC3G has the ability to induce T cell plasticity and modulate immune response.

Although all 12 subtypes of human interferon alpha (IFN-α) bind the same receptor, recent results have demonstrated that they elicit unique host responses and display distinct efficacies in the control of different viral infections. The IFN-α2 subtype is currently in HIV-1 clinical trials, but it has not consistently reduced viral loads in HIV-1 patients and is not the most effective subtype against HIV-1 in vitro . We now demonstrate in humanized mice that, when delivered at the same high clinical dose, the human IFN-α14 subtype has very potent anti-HIV-1 activity whereas IFN-α2 does not. In both postexposure prophylaxis and treatment of acute infections, IFN-α14, but not IFN-α2, significantly suppressed HIV-1 replication and proviral loads. Furthermore, HIV-1-induced immune hyperactivation, which is a prognosticator of disease progression, was reduced by IFN-α14 but not IFN-α2. Whereas ineffective IFN-α2 therapy was associated with CD8 + T cell activation, successful IFN-α14 therapy was associated with increased intrinsic and innate immunity, including significantly higher induction of tetherin and MX2, increased APOBEC3G signature mutations in HIV-1 proviral DNA, and higher frequencies of TRAIL + NK cells. These results identify IFN-α14 as a potent new therapeutic that operates via mechanisms distinct from those of antiretroviral drugs. The ability of IFN-α14 to reduce both viremia and proviral loads in vivo suggests that it has strong potential as a component of a cure strategy for HIV-1 infections. The broad implication of these results is that the antiviral efficacy of each individual IFN-α subtype should be evaluated against the specific virus being treated. IMPORTANCE The naturally occurring antiviral protein IFN-α2 is used to treat hepatitis viruses but has proven rather ineffective against HIV in comparison to triple therapy with the antiretroviral (ARV) drugs. Although ARVs suppress the replication of HIV, they fail to completely clear infections. Since IFN-α acts by different mechanism than ARVs and has been shown to reduce HIV proviral loads, clinical trials are under way to test whether IFN-α2 combined with ARVs might eradicate HIV-1 infections. IFN-α is actually a family of 12 distinct proteins, and each IFN-α subtype has different efficacies toward different viruses. Here, we use mice that contain a human immune system, so they can be infected with HIV. With this model, we demonstrate that while IFN-α2 is only weakly effective against HIV, IFN-α14 is extremely potent. This discovery identifies IFN-α14 as a more powerful IFN-α subtype for use in combination therapy trials aimed toward an HIV cure.

We have previously shown that the cellular RNase MCPIP1/regnase-1 potently blocks HIV-1 infection in resting CD4+ T-cells. As simian immunodeficiency virus (SIV) encodes an accessory protein named Vpx, which enhances viral replication in resting CD4+ T-cells by degrading the cellular restriction factor SAMHD1, we investigated whether MCPIP1 restricts SIV infection and whether Vpx protein antagonizes MCPIP1-mediated restriction. In co-transfection studies, human MCPIP1 markedly reduced the production of infectious SIV, whereas MCPIP2 and MCPIP3 had little effect. MCPIP1 derived from cynomolgus monkey also inhibited human immunodeficiency virus (HIV-1) and SIV production, albeit to a lesser degree. Lastly, expression of SIV Vpx protein did not reduce MCPIP1 at the protein level, nor did it ablate the MCPIP1-mediated restriction. In conclusion, both human and cynomolgus monkey MCPIP1 restrict SIV replication. Unlike SAMHD1, MCPIP1-mediated HIV-1 restriction cannot be overcome by SIV Vpx.

The multifunctional HIV-1 accessory protein Vif counters the antiviral activities of APOBEC3G (A3G) and APOBEC3F (A3F), and some Vifs counter stable alleles of APOBEC3H (A3H). Studies in humanized mice have shown that HIV-1 lacking Vif expression is not viable. Here, we look at the relative contributions of the three APOBEC3s to viral extinction. Inoculation of bone marrow/liver/thymus (BLT) mice with CCR5-tropic HIV-1 JRCSF (JRCSF) expressing a vif gene inactive for A3G but not A3F degradation activity (JRCSFvifH42/43D) displayed either no or delayed replication. JRCSF expressing a vif gene mutated to inactivate A3F degradation but not A3G degradation (JRCSFvifW79S) always replicated to high viral loads with variable delays. JRCSF with vif mutated to lack both A3G and A3F degradation activities (JRCSFvifH42/43DW79S) failed to replicate, mimicking JRCSF without Vif expression (JRCSFΔvif). JRCSF and JRCSFvifH42/43D, but not JRCSFvifW79S or JRCSFvifH42/43DW79S, degraded APOBEC3D. With one exception, JRCSFs expressing mutant Vifs that replicated acquired enforced vif mutations. These mutations partially restored A3G or A3F degradation activity and fully replaced JRCSFvifH42/43D or JRCSFvifW79S by 10 weeks. Surprisingly, induced mutations temporally lagged behind high levels of virus in blood. In the exceptional case, JRCSFvifH42/43D replicated after a prolonged delay with no mutations in vif but instead a V27I mutation in the RNase H coding sequence. JRCSFvifH42/43D infections exhibited massive GG/AG mutations in pol viral DNA, but in viral RNA, there were no fixed mutations in the Gag or reverse transcriptase coding sequence. A3H did not contribute to viral extinction but, in combination with A3F, could delay JRCSF replication. A3H was also found to hypermutate viral DNA. IMPORTANCE Vif degradation of A3G and A3F enhances viral fitness, as virus with even a partially restored capacity for degradation outgrows JRCSFvifH42/43D and JRCSFvifW79S. Unexpectedly, fixation of mutations that replaced H42/43D or W79S in viral RNA lagged behind the appearance of high viral loads. In one exceptional JRCSFvifH42/43D infection, vif was unchanged but replication proceeded after a long delay. These results suggest that Vif binds and inhibits the non-cytosine deaminase activities of intact A3G and intact A3F, allowing JRCSFvifH42/43D and JRCSFvifW79S to replicate with reduced fitness. Subsequently, enhanced Vif function is acquired by enforced mutations. In infected cells, JRCSFΔvif and JRCSFvifH42/43DW79S are exposed to active A3F and A3G and fail to replicate. JRCSFvifH42/43D Vif degrades A3F and, in some cases, overcomes A3G mutagenic activity to replicate. Vif may have evolved to inhibit A3F and A3G by stoichiometric binding and subsequently acquired the ability to target these proteins to proteasomes.

The gradual accumulation of mutations by any of a number of mutational processes is a major driving force of divergence and evolution. Here, we investigate a potentially novel mutational process that is based on the activity of members of the AID/APOBEC family of deaminases. This gene family has been recently shown to introduce—in multiple types of cancer—enzyme-induced clusters of co-occurring somatic mutations caused by cytosine deamination. Going beyond somatic mutations, we hypothesized that APOBEC3—following its rapid expansion in primates—can introduce unique germline mutation clusters that can play a role in primate evolution. In this study, we tested this hypothesis by performing a comprehensive comparative genomic screen for APOBEC3-induced mutagenesis patterns across different hominids. We detected thousands of mutation clusters introduced along primate evolution which exhibit features that strongly fit the known patterns of APOBEC3G mutagenesis. These results suggest that APOBEC3G-induced mutations have contributed to the evolution of all genomes we studied. This is the first indication of site-directed, enzyme-induced genome evolution, which played a role in the evolution of both modern and archaic humans. This novel mutational mechanism exhibits several unique features, such as its higher tendency to mutate transcribed regions and regulatory elements and its ability to generate clusters of concurrent point mutations that all occur in a single generation. Our discovery demonstrates the exaptation of an anti-viral mechanism as a new source of genomic variation in hominids with a strong potential for functional consequences.

Interferon (IFN) is a regularly utilized therapeutic for the treatment of chronic hepatitis B and appears to induce superior HBeAg seroconversion comparing nucleos/tide analogs. However, the mechanisms underlying IFN inhibition of HBV replication, as well as poor responses to IFN are unclear. Apobec3G has been reported to be involved in regulating HBV replication. In this study, we investigated Apobec3G expression and regulatory pathways during HBV infection. We show that over-expression of A3G leads to inhibition of HBV replication. We also show that IFN induces a significant increase in A3G protein expression, which is associated with STAT3 activation. We further show that A3G expression in HBV patients is lower compared to non-infected controls, possibly by HBsAg which inhibits IFN induced A3G up-regulation in a dose dependent manner. This process is likely mediated through inhibition of STAT3-Ser727 phosphorylation. The results presented in this study indicate that STAT3 plays an important role in IFN-induced A3G production, and HBsAg may correlated with poor response to IFN treatment.

Background To suppress HIV infection, host cells have evolved numerous defenses that generally belong to the innate and adaptive immune responses. Over the last decade, extensive efforts have been focused on understanding HIV restriction factors and mechanisms of evasion. The host protein APOBEC3G (A3G) was identified as a member of cytidine deaminase family in 2002, and it was shown that, in the absence of HIV encoded Vif, A3G can block the replication of HIV-1 by introducing viral hypermutations during reverse transcription, also conferring innate immunity to the virus. Hence, therapeutic exploitation of A3G as an antiviral therapy has received an increasing amount of attention. Recent studies have developed a series of strategies to abolish the interaction between Vif and A3G or facilitate A3G expression, thus enhancing active A3G formation and delivering A3G into virion. Conclusion Here we present a review that discuss the role of A3G as a host innate immunity factor and its application in HIV therapy.

Background: Similar to other animal viruses, HIV-1 relies on the contributions of the cellular machinery to ensure efficient virus propagation. However, human cells have evolved refined mechanisms to block key steps of the virus life-cycle, thereby suppressing viral replication. These cellular proteins are generally known as restriction factors, and they provide an early antiviral defense. So far, five potent restriction factors have been shown to effectively block HIV and/or SIV replication. These are TRIM5 proteins, SAMHD-1, members of the APOBEC3 (A3) family, Mx2 and Tetherin/BST-2. Results: Here, we review the antiviral mechanisms of these and other antiviral factors, their interaction with the innate immune responses, and how their functions might be exploited to clear and prevent HIV infection. Conclusion: Since the majority of vaccine approaches against HIV have failed so far, it is imperative to start looking at alternative strategies for vaccine and therapy development. By better understanding how HIV hijacks the cellular machinery for its own benefit in completing its life-cycle, and how the virus adapts to circumvent our intrinsic immunity, we will be better equipped to design compounds that specifically interrupt virus replication and spread.

The allelic variations in the AIDS restriction genes have been associated with the acquisition of HIV-1 and its progression. The distribution of antiviral gene variants significantly differs between populations. Therefore, we aimed to evaluate the distribution of variant allele of 186H/R in exon4 of APOBEC3G between HIV infected individuals and healthy controls among western Indian.In the present cross-sectional study, we enrolled a total of 153 HIV-infected patients confirmed and 156 unrelated healthy individuals. Polymorphism for 186H/R in exon4 of APOBEC3G gene was genotyped by PCR-RFLP. With the frequency of 186HR heterozygous genotype of APOBEC3G was found to be 13% in healthy controls and none in HIV infected cases. The frequency of 186HH common genotype of APOBEC3G was observed higher in HIV infected individuals compared with healthy controls (100% vs 91.7%). The variant genotype 186RR in APOBEC3G was not found in both the groups. The frequency of 186R allele of APOBEC3G was found 4.16% in healthy controls and nil in HIV-infected cases. The frequency of 186H allele of APOBEC3G was found to be higher in HIV-infected cases compared with healthy controls (100% vs 95.83%). The frequency of 186R allele in exon4 of APOBEC3G was found to be 4.16% in healthy controls. This observation differs from the previous report published from North India stating the absence of 186R allele of APOBEC3G in the North Indian individuals. The variant 186H/R in exon4 of APOBEC3G was neither associated with risk of acquisition of HIV-1 nor its progression.

Plasma drug-resistant minority human immunodeficiency virus type 1 variants (DRMVs) increase the risk of virological failure to first-line non-nucleoside reverse transcriptase inhibitor antiretroviral therapy (ART). The origin of DRMVs in ART-naive patients, however, remains unclear. In a large pan-European case-control study investigating the clinical relevance of pre-existing DRMVs using 454 pyrosequencing, the six most prevalent plasma DRMVs detected corresponded to G-to-A nucleotide mutations (V90I, V106I, V108I, E138K, M184I and M230I). Here, we evaluated if such DRMVs could have emerged from apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3G/F (APOBEC3G/F) activity. Out of 236 ART-naive subjects evaluated, APOBEC3G/F hypermutation signatures were detected in plasma viruses of 14 (5.9%) individuals. Samples with minority E138K, M184I, and M230I mutations, but not those with V90I, V106I or V108I, were significantly associated with APOBEC3G/F activity (Fisher's P0.005), defined as the presence of0.5% of sample sequences with an APOBEC3G/F signature. Mutations E138K, M184I and M230I co-occurred in the same sequence as APOBEC3G/F signatures in 3/9 (33%), 5/11 (45%) and 4/8 (50%) of samples, respectively; such linkage was not found for V90I, V106I or V108I. In-frame STOP codons were observed in 1.5% of all clonal sequences; 14.8% of them co-occurred with APOBEC3G/F signatures. APOBEC3G/F-associated E138K, M184I and M230I appeared within clonal sequences containing in-frame STOP codons in 2/3 (66%), 5/5 (100%) and 4/4 (100%) of the samples. In a re-analysis of the parent case control study, the presence of APOBEC3G/F signatures was not associated with virological failure. In conclusion, the contribution of APOBEC3G/F editing to the development of DRMVs is very limited and does not affect the efficacy of non-nucleoside reverse transcriptase inhibitor ART.

Purpose: APOBEC3 DNA cytosine deaminase family members normally defend against viruses and transposons. However, deregulated APOBEC3 activity causes mutations in cancer. Because of broad expression profiles and varying mixtures of normal and cancer cells in tumors, including immune cell infiltration, it is difficult to determine where different APOBEC3s are expressed. Here, we ask whether correlations exist between APOBEC3 expression and T-cell infiltration in high-grade serous ovarian cancer (HGSOC), and assess whether these correlations have prognostic value. Experimental Design: Transcripts for APOBEC3G, APOBEC3B, and the T-cell markers, CD3D, CD4, CD8A, GZMB, PRF1, and RNF128 were quantified by RT-qPCR for a cohort of 354 HGSOC patients. Expression values were correlated with each other and clinical parameters. Two additional cohorts were used to extend HGSOC clinical results. Immunoimaging was used to colocalize APOBEC3G and the T-cell marker CD3. TCGA data extended expression analyses to additional cancer types. Results: A surprising positive correlation was found for expression of APOBEC3G and several T cell genes in HGSOC. Immunohistochemistry and immunofluorescent imaging showed protein colocalization in tumor-infiltrating T lymphocytes. High APOBEC3G expression correlated with improved outcomes in multiple HGSOC cohorts. TCGA data analyses revealed that expression of APOBEC3D and APOBEC3H also correlates with CD3D across multiple cancer types. Conclusions: Our results identify APOBEC3G as a new candidate biomarker for tumor-infiltrating T lymphocytes and favorable prognoses for HGSOC. Our data also highlight the complexity of the tumor environment with respect to differential APOBEC family gene expression in both tumor and surrounding normal cell types. Clin Cancer Res; 22(18); 4746–55. ©2016 AACR.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G, A3G) exert antiviral defense as an important factor of innate immunity. A variety of cytokines such as IFN-γ、IL2、IL15、IL7 could induce the transcription of A3G. However, the regulation of other nuclear factor on the transcription of A3G have not been reported at the present. To gain new insights into the transcriptional regulation of this restriction factor, we cloned and characterized the promoter region of A3G and investigate the modulation of USF1 gene on the transcription of A3G. We identified a 232 bp region that was sufficient to regulate the activity of full promoter. Transcriptional start sites (TSS) were identified by the luciferase reporter assays of plasmids containing full or shorter fragments of the A3G promoter. The results demonstrated that the core promoter of A3G is located within the region -159/-84 relative to the TSS. Transcriptional activity of A3G core promoter regulated by USF1 was dependent on an E-box (located at position -91/-86 relative to the major TSS) and was abolished after mutation of this DNA element. USF1 gene can take part in basal transcription regulation of the human A3G gene in hepatocyte, and the identified E-box represented a binding site for the USF1.

SummaryHuman cells express natural antiviral proteins, such as APOBEC3G (A3G), that potently restrict HIV replication. As a counter-defense, HIV encodes the accessory protein Vif, which binds A3G and mediates its proteasomal degradation. Our structural knowledge on how Vif and A3G interact is limited, because a co-structure is not available. We identified specific points of contact between Vif and A3G by using functional assays with full-length A3G, patient-derived Vif variants, and HIV forced evolution. These anchor points were used to model and validate the Vif-A3G interface. The resultant co-structure model shows that the negatively charged β4-α4 A3G loop, which contains primate-specific variation, is the core Vif binding site and forms extensive interactions with a positively charged pocket in HIV Vif. Our data present a functional map of this viral-host interface and open avenues for targeted approaches to block HIV replication by obstructing the Vif-A3G interaction.

APOBEC3B is a newly identified source of mutation in many cancers, including breast, head/neck, lung, bladder, cervical, and ovarian. APOBEC3B is a member of the APOBEC3 family of enzymes that deaminate DNA cytosine to produce the pro-mutagenic lesion, uracil. Several APOBEC3 family members function to restrict virus replication. For instance, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H combine to restrict HIV-1 in human lymphocytes. HIV-1 counteracts these APOBEC3s with the viral protein Vif, which targets the relevant APOBEC3s for proteasomal degradation. While APOBEC3B does not restrict HIV-1 and is not targeted by HIV-1 Vif in CD4-positive T cells, we asked whether related lentiviral Vif proteins could degrade APOBEC3B. Interestingly, several SIV Vif proteins are capable of promoting APOBEC3B degradation, with SIVmac239 Vif proving the most potent. This likely occurs through the canonical polyubiquitination mechanism as APOBEC3B protein levels are restored by MG132 treatment and by altering a conserved E3 ligase-binding motif. We further show that SIVmac239 Vif can prevent APOBEC3B mediated geno/cytotoxicity and degrade endogenous APOBEC3B in several cancer cell lines. Our data indicate that the APOBEC3B degradation potential of SIV Vif is an effective tool for neutralizing the cancer genomic DNA deaminase APOBEC3B. Further optimization of this natural APOBEC3 antagonist may benefit cancer therapy.

Pokeweed antiviral protein (PAP) is an RNA glycosidase that inhibits production of human immunodeficiency virus type 1 (HIV-1) when expressed in human culture cells. Previously, we showed that the expression of PAP reduced the levels of several viral proteins, including virion infectivity factor (Vif). However, the mechanism causing Vif reduction and the consequences of the inhibition were not determined. Here we show that the Vif mRNA is directly depurinated by PAP. Because of depurination at two specific sites within the Vif ORF, Vif levels decrease during infections and the progeny viruses that are generated are ∼ 10-fold less infectious and compromised for proviral integration. These results are consistent with PAP activity inhibiting translation of Vif, which in turn reduces the effect of Vif to inactivate the host restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like editing complex 3G). Our findings identify Vif mRNA as a new substrate for PAP and demonstrate that derepression of innate immunity against HIV-1 contributes to its antiviral activity.

In 2014, two novel and promising benzimidazole-based APOBEC3G stabilizers MM-1 and MM-2 (MMs) were uncovered with an elusive mechanism of action. Vif-APOBEC3G axis has been recognized as a novel therapeutic target for anti HIV-1 drug development. The unexplored mechanism of MMs hindered their further development into lead compounds. To recognize their underlying mechanism we adopted an exhaustive in silico workflow by which we tested their ability to interrupt Vif complex network formation. The preliminary outcome guided us to a high likelihood of MMs interaction within Elongin C binding site, which in turn, perturbs Vif/Elongin C binding and ultimately undermines Vif action. To validate our estimation, we synthesized only MM-1 as a model to complement our study by in vitro assay for a real-time understanding. An immunoprecipitation experiment confirmed the capacity of MM-1 to interrupt Vif/Elongin C interaction. This is an integral study that lies at the interface between theoretical and experimental approaches showing the potential of molecular modelling to address issues related to drug development.

Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) family members are cytidine deaminases that play crucial roles in innate responses to retrovirus infection. The mechanisms by which some of these enzymes restrict human immunodeficiency virus type 1 (HIV-1) replication have been extensively investigated in vitro. However, little is known regarding how APOBEC3 proteins affect the pathogenesis of HIV-1 infection in vivo and how antiretroviral therapy influences their expression. In this work, a longitudinal analysis was performed to evaluate APOBEC3G/3A expression in peripheral blood mononuclear cells of antiretroviral-naive HIV-1-infected individuals treated with cenicriviroc (CVC) or efavirenz (EFV) at baseline and 4, 12, 24, and 48 weeks post-treatment follow-up. While APOBEC3G expression was unaffected by therapy, APOBEC3A levels increased in CVC but not EFV arm at week 48 of treatment. APOBEC3G expression correlated directly with CD4+ cell count and CD4+/CD8+ cell ratio, whereas APOBEC3A levels inversely correlated with plasma soluble CD14. These findings suggest that higher APOBEC3G/3A levels may be associated with protective effects against HIV-1 disease progression and chronic inflammation and warrant further studies.

The northern pig-tailed macaques (NPMs) lack TRIM5α, an antiviral restriction factor, and instead have TRIM5-CypA. In our previous study, we demonstrated that HIV-1NL4−3 successfully infected NPMs and formed a long-term viral reservoir in vivo. However, the HIV-1-infected NPMs showed relatively high viremia in the first 6 weeks of infection, which declined thereafter suggesting that HIV-1 NL4−3 infection in these animals was only partly permissive. To optimize HIV-1 infection in NPMs therefore, we generated HIV-1NL4−R3A and stHIV-1sv, and infected NPMs with these viruses. HIV-1NL4−R3A and stHIV-1sv can replicate persistently in NPMs during 41 weeks of acute infection stage. Compared to the HIV-1NL4−R3A, stHIV-1sv showed a notably higher level of replication, and the NPMs infected with the latter induced a more robust neutralizing antibody but a weaker cellular immune response. In addition, IFN-I signaling was significantly up-regulated with the viral replication, and was higher in the stHIV-1sv infected macaques. Consequently, the sequences of pro-viral env showed fewer G-A hyper-mutations in stHIV-1sv, suggesting that vif gene of SIV could antagonize the antiviral effects of APOBEC3 proteins in NPMs. Taken together, NPMs infected with HIV-1NL4−R3A and stHIV-1sv show distinct virological and immunological features. Furthermore, interferon-related gene expression might play a role in controlling primary HIV-1NL4−R3A and stHIV-1sv replication in NPMs. This result suggests NPM is a potential HIV/AIDS animal model.

The apolipoprotein B editing enzyme catalytic subunit 3 (APOBEC3) is a family of DNA cytosine deaminases that mutate and inactivate viral genomes by single-strand DNA editing, thus providing an innate immune response against a wide range of DNA and RNA viruses. In particular, APOBEC3A (A3A), a member of the APOBEC3 family, is induced by human cytomegalovirus (HCMV) in decidual tissues where it efficiently restricts HCMV replication, thereby acting as an intrinsic innate immune effector at the maternal-fetal interface. However, the widespread incidence of congenital HCMV infection implies that HCMV has evolved to counteract APOBEC3-induced mutagenesis through mechanisms that still remain to be fully established. Here, we have assessed gene expression and deaminase activity of various APOBEC3 gene family members in HCMV-infected primary human foreskin fibroblasts (HFFs). Specifically, we show that APOBEC3G (A3G) gene products and, to a lesser degree, those of A3F but not of A3A, are upregulated in HCMV-infected HFFs. We also show that HCMV-mediated induction of A3G expression is mediated by interferon beta (IFN-β), which is produced early during HCMV infection. However, knockout or overexpression of A3G does not affect HCMV replication, indicating that A3G is not a restriction factor for HCMV. Finally, through a bioinformatics approach, we show that HCMV has evolved mutational robustness against IFN-β by limiting the presence of A3G hot spots in essential open reading frames (ORFs) of its genome. Overall, our findings uncover a novel immune evasion strategy by HCMV with profound implications for HCMV infections. IMPORTANCE APOBEC3 family of proteins plays a pivotal role in intrinsic immunity defense mechanisms against multiple viral infections, including retroviruses, through the deamination activity. However, the currently available data on APOBEC3 editing mechanisms upon HCMV infection remain unclear. In the present study, we show that particularly the APOBEC3G (A3G) member of the deaminase family is strongly induced upon infection with HCMV in fibroblasts and that its upregulation is mediated by IFN-β. Furthermore, we were able to demonstrate that neither A3G knockout nor A3G overexpression appears to modulate HCMV replication, indicating that A3G does not inhibit HCMV replication. This may be explained by HCMV escape strategy from A3G activity through depletion of the preferred nucleotide motifs (hot spots) from its genome. The results may shed light on antiviral potential of APOBEC3 activity during HCMV infection, as well as the viral counteracting mechanisms under A3G-mediated selective pressure.

Background Activation induced deaminase (AID) and apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3) are deaminases that mutate C to U on single-stranded DNA (ssDNA). AID is expressed primarily in germinal center B-cells, where it facilitates affinity maturation and class-switch recombination. APOBEC3 are a family of anti-viral proteins that act as part of the intrinsic immune response. In both cases, there are particular sequence motifs, also known as “mutation motifs”, to which these deaminases prefer to bind and mutate. Results We present a program, the cytidine deaminase under-representation reporter (CDUR) designed to statistically determine whether a given sequence has an under/over-representation of these mutation motifs. CDUR shows consitency with other studies of mutation motifs, as we show by analyzing sequences from the adeno-associated virus 2 (AAV2) and human papillomavirus (HPV). Conclusion Using various shuffling mechanisms to generate different null model distributions, we can tailor CDUR to correct for metrics such as GC-content, dinucleotide frequency, and codon bias.

The present article describes techniques for classical simulations of proteins and protein-nucleic acid complexes, revealing their dynamics and protein-substrate binding energies. The approach is based on classical atomistic molecular dynamics (MD) simulations of the experimentally determined structures of the complexes. MD simulations can provide dynamics of complexes in realistic solvents on microsecond timescales, and the free energy methods are able to provide Gibbs free energies of binding of substrates, such as nucleic acids, to proteins. The chapter describes methodologies for the preparation of computer models of biomolecular complexes and free energy perturbation methodology for evaluating Gibbs free energies of binding. The applications are illustrated with examples of snapshots of proteins and their complexes with nucleic acids, as well as the precise Gibbs free energies of binding.

Several members of the APOBEC3 DNA cytosine deaminase family can potently inhibit Vif-deficient human immunodeficiency virus type 1 (HIV-1) by catalyzing cytosine deamination in viral cDNA and impeding reverse transcription. HIV-1 counteracts restriction with the virally encoded Vif protein, which targets relevant APOBEC3 proteins for proteasomal degradation. HIV-1 Vif is optimized for degrading the restrictive human APOBEC3 repertoire, and, in general, lentiviral Vif proteins specifically target the restricting APOBEC3 enzymes of each host species. However, simian immunodeficiency virus SIV mac239 Vif elicits a curiously wide range of APOBEC3 degradation capabilities that include degradation of several human APOBEC3s and even human APOBEC3B, a non-HIV-1-restricting APOBEC3 enzyme. To better understand the molecular determinants of the interaction between SIV mac239 Vif and human APOBEC3B, we analyzed an extensive series of mutants. We found that SIV mac239 Vif interacts with the N-terminal domain of human APOBEC3B and, interestingly, that this occurs within a structural region homologous to the HIV-1 Vif interaction surface of human APOBEC3G. An alanine scan of SIV mac239 Vif revealed several residues required for human APOBEC3B degradation activity. These residues overlap HIV-1 Vif surface residues that interact with human APOBEC3G and are distinct from those that engage APOBEC3F or APOBEC3H. Overall, these studies indicate that the molecular determinants of the functional interaction between human APOBEC3B and SIV mac239 Vif resemble those between human APOBEC3G and HIV-1 Vif. These studies contribute to the growing knowledge of the APOBEC-Vif interaction and may help guide future efforts to disrupt this interaction as an antiviral therapy or exploit the interaction as a novel strategy to inhibit APOBEC3B-dependent tumor evolution. IMPORTANCE Primate APOBEC3 proteins provide innate immunity against retroviruses such as HIV and SIV. HIV-1, the primary cause of AIDS, utilizes its Vif protein to specifically counteract restrictive human APOBEC3 enzymes. SIV mac239 Vif exhibits a much wider range of anti-APOBEC3 activities that includes several rhesus macaque enzymes and extends to multiple proteins in the human APOBEC3 repertoire, including APOBEC3B. Understanding the molecular determinants of the interaction between SIV mac239 Vif and human APOBEC3B adds to existing knowledge on the APOBEC3-Vif interaction and has potential to shed light on what processes may have shaped Vif functionality over evolutionary time. An intimate understanding of this interaction may also lead to a novel cancer therapy because, for instance, creating a derivative of SIV mac239 Vif that specifically targets human APOBEC3B could be used to suppress tumor genomic DNA mutagenesis by this enzyme, slow ongoing tumor evolution, and help prevent poor clinical outcomes.

Cytidine deaminases inhibit replication of a broad range of DNA viruses by deaminating cytidines on single-stranded DNA (ssDNA) to generate uracil. While several lines of evidence have revealed hepatitis B virus (HBV) genome editing by deamination, it is still unclear which nucleic acid intermediate of HBV is modified. Hepatitis B virus has a relaxed circular double-stranded DNA (rcDNA) genome that is reverse transcribed within virus cores from a RNA template. The HBV genome also persists as covalently closed circular DNA (cccDNA) in the nucleus of an infected cell. In the present study, we found that in HBV-producing HepAD38 and HepG2.2.15 cell lines, endogenous cytidine deaminases edited 10 to 25% of HBV rcDNA genomes, asymmetrically with almost all mutations on the 5′ half of the minus strand. This region corresponds to the last half of the minus strand to be protected by plus-strand synthesis. Within this half of the genome, the number of mutations peaks in the middle. Overexpressed APOBEC3A and APOBEC3G could be packaged in HBV capsids but did not change the amount or distribution of mutations. We found no deamination on pregenomic RNA (pgRNA), indicating that an intact genome is encapsidated and deaminated during or after reverse transcription. The deamination pattern suggests a model of rcDNA synthesis in which pgRNA and then newly synthesized minus-sense single-stranded DNA are protected from deaminase by interaction with the virus capsid; during plus-strand synthesis, when enough dsDNA has been synthesized to displace the remaining minus strand from the capsid surface, the single-stranded DNA becomes deaminase sensitive. IMPORTANCE Host-induced mutation of the HBV genome by APOBEC proteins may be a path to clearing the virus. We examined cytidine-to-thymidine mutations in the genomes of HBV particles grown in the presence or absence of overexpressed APOBEC proteins. We found that genomes were subjected to deamination activity during reverse transcription, which takes place within the virus capsid. These observations provide a direct insight into the mechanics of reverse transcription, suggesting that newly synthesized dsDNA displaces ssDNA from the capsid walls, making the ssDNA accessible to deaminase activity.

We found earlier that ectopic expression of the cytidine deaminase APOBEC3G (A3G) in Vero cells inhibits measles virus (MV), respiratory syncytial virus, and mumps virus, while the mechanism of inhibition remained unclear. A microarray analysis revealed that in A3G-transduced Vero cells, several cellular transcripts were differentially expressed, suggesting that A3G regulates the expression of host factors. One of the most upregulated host cell factors, REDD1 (regulated in development and DNA damage response-1, also called DDIT4), reduced MV replication ∼10-fold upon overexpression in Vero cells. REDD1 is an endogenous inhibitor of mTORC1 (mammalian target of rapamycin complex-1), the central regulator of cellular metabolism. Interestingly, rapamycin reduced the MV replication similarly to REDD1 overexpression, while the combination of both did not lead to further inhibition, suggesting that the same pathway is affected. REDD1 silencing in A3G-expressing Vero cells abolished the inhibitory effect of A3G. In addition, silencing of A3G led to reduced REDD1 expression, confirming that its expression is regulated by A3G. In primary human peripheral blood lymphocytes (PBL), expression of A3G and REDD1 was found to be stimulated by phytohemagglutinin (PHA) and interleukin-2. Small interfering RNA (siRNA)-mediated depletion of A3G in PHA-stimulated PBL reduced REDD1 expression and increased viral titers, which corroborates our findings in Vero cells. Silencing of REDD1 also increased viral titers, confirming the antiviral role of REDD1. Finally, pharmacological inhibition of mTORC1 by rapamycin in PHA-stimulated PBL reduced viral replication to the level found in unstimulated lymphocytes, indicating that mTORC1 activity supports MV replication as a proviral host factor.IMPORTANCE Knowledge about host factors supporting or restricting virus replication is required for a deeper understanding of virus-cell interactions and may eventually provide the basis for therapeutic intervention. This work was undertaken predominantly to explain the mechanism of A3G-mediated inhibition of MV, a negative-strand RNA virus that is not affected by the deaminase activity of A3G acting on single-stranded DNA. We found that A3G regulates the expression of several cellular proteins, which influences the capacity of the host cell to replicate MV. One of these, REDD1, which modulates the cellular metabolism in a central position by regulating the kinase complex mTORC1, was identified as the major cellular factor impairing MV replication. These findings show interesting aspects of the function of A3G and the dependence of the MV replication on the metabolic state of the cell. Interestingly, pharmacological inhibition of mTORC1 can be utilized to inhibit MV replication in Vero cells and primary human peripheral blood lymphocytes.

Systems biological analysis has recently revealed how innate immune variants as well as gut microbiota impact the individual response to immunization. HIV-infected (HIVC) patients have a worse response rate after standard vaccinations, possibly due to the immune exhaustion, increased gut permeability and microbial translocation. In the last decade, dendritic cells (DC)-based immunotherapy has been proposed as an alternative approach to control HIV plasma viral load, however clinical trials showed a heterogeneity of immunization response. Hypothesizing that host genetics may importantly affects the outcome of immunotherapy in HIVC patients, genetic polymorphisms’ distribution and gene expression modulation were analyzed in a phase I/II clinical trial of DC-based immunotherapy according to immunization response, and quality of vaccine product (DC). Polymorphisms in genes previously associated with progression of HIV infection to AIDS (i.e.: PARD3B, CCL5) contribute to a better response to immunotherapy in HIVC individuals, possibly through a systemic effect on host immune system, but also directly on vaccine product. Genes expression profile after immunization correlates with different degrees of immune chronic activation/exhaustion of HIVC patients (i.e. PD1, IL7RA, EOMES), but also with anti-viral response and DC quality (i.e.: APOBEC3G, IL8, PPIA), suggested that an immunocompetent individual would have a better vaccine response. These findings showed once more that host genetics can affect the response to DC-based immunotherapy in HIVC individuals, contributing to the heterogeneity of response observed in concluded trials; and it can be used as predictor of immunization success.

HIV-1 replication normally requires Vif-mediated neutralization of APOBEC3 antiviral enzymes. Viruses lacking Vif succumb to deamination-dependent and -independent restriction processes. Here, HIV-1 adaptation studies were leveraged to ask whether viruses with an irreparable vif deletion could develop resistance to restrictive levels of APOBEC3G. Several resistant viruses were recovered with multiple amino acid substitutions in Env, and these changes alone are sufficient to protect Vif-null viruses from APOBEC3G-dependent restriction in T cell lines. Env adaptations cause decreased fusogenicity, which results in higher levels of Gag-Pol packaging. Increased concentrations of packaged Pol in turn enable faster virus DNA replication and protection from APOBEC3G-mediated hypermutation of viral replication intermediates. Taken together, these studies reveal that a moderate decrease in one essential viral activity, namely Env-mediated fusogenicity, enables the virus to change other activities, here, Gag-Pol packaging during particle production, and thereby escape restriction by the antiviral factor APOBEC3G. We propose a new paradigm in which alterations in viral homeostasis, through compensatory small changes, constitute a general mechanism used by HIV-1 and other viral pathogens to escape innate antiviral responses and other inhibitions including antiviral drugs., Author summary APOBEC3G is a virus restriction factor that blocks the replication of Vif-deficient HIV-1 by deamination-dependent and -independent mechanisms. The HIV-1 accessary protein Vif counteracts APOBEC3G through a proteasome-mediated degradation pathway. However, viruses often possess multiple distinct mechanisms to evade innate immune responses, and it was unknown whether HIV-1 possesses alternative mechanisms for escaping restriction by APOBEC3G. To investigate this possibility, HIV-1 with a non-revertable vif deletion was adapted in stepwise cultures to increasing amounts of APOBEC3G. Three independent APOBEC3G resistant viral isolates acquired amino acid substitutions in Env. Mechanistic studies showed that these Env adaptations cause decreased fusogenicity, which re-optimizes viral homeostasis by allowing increased Gag-Pol packaging and higher rates of reverse transcription, which in turn protect viral DNA from lethal hypermutation by APOBEC3G. Thus, these results demonstrate a novel Env-dependent mechanism mediated by RT that HIV-1 can utilize to escape APOBEC3G-mediated restriction. Sequence comparisons suggest that transmitting isolates may also utilize this mechanism. More broadly, our results suggest a new paradigm in which relatively small changes in essential viral processes and overall viral homeostasis can have rather large phenotypic consequences such as enabling resistance to potent antiviral measures.

The aberrant expression of RNA-binding proteins (RBPs) plays a crucial role in the occurrence and progression of human cancer. However, the key functions of RBPs in the metastasis of colorectal cancer have not yet been fully elucidated. Here, we integrated multi-omics data and identified four differentially expressed RBPs (APOBEC3G, EEF1A2, EIF5AL1 and CELF3) in patients with colorectal cancer metastasis. To clarify the underlying molecular mechanisms, we systematically analyzed the genomic features and downstream regulatory relationships of the four RBPs. In a genomic level, the copy number variations of APOBEC3G, EEF1A2, and CELF3 demonstrated significantly differential distributions between metastatic and nonmetastatic patients. Besides that, combining sequence and expression information, we identified 436 putative RNA targets regulated by the four RBPs through strict multistep bioinformatics screening. For the downstream analysis, the evidence from functional enrichment analysis and public literature indicated the roles of these target genes in the carcinogenesis and progression of colorectal cancer. Furthermore, through the machine learning algorithm and statistical analysis, we obtained two gene candidates that had obvious effects on the metastasis and overall survival status of patients with colorectal cancer. In summary, our study comprehensively explored the influence of APOBEC3G, EEF1A2, EIF5AL1, and CELF3 in colorectal cancer metastasis, which may offer favorable perspectives for clinical diagnosis and therapy.

Background The aim of this study was to investigate the presence of minority variants (MVs) in high-risk human papillomavirus (HPV) types (HPV-16, -52, and -58) from cervical and anal smears. Methods Whole HPV genome ultra-deep sequencing (UDS) was performed on cervical and anal smears collected during patient follow-up. Bioinformatics analyses were performed using Bowtie2 (Geneious). Results We assessed 55 HPV-16-positive, 20 HPV-52-positive, and 17 HPV-58-positive samples, with significant differences in patient characteristics for the 2 anatomic sites. HPV-16 MVs were detected in 20 samples (36%), with no difference between cervical and anal samples. We did not find an association between the presence of MVs and cytovirological parameters. Seven HPV-16 genomes (13%) were apolipoprotein B messenger RNA editing, catalytic polypeptide-like 3 (APOBEC) edited. Among the cervical HPV-16-positive samples, most MVs (55%) resulted from APOBEC-related mutations. MVs were detected in 10 HPV-52-positive (50%) and 12 HPV-58-positive (71%) samples, with no difference between cervical and anal samples. No APOBEC-related mutations were found on HPV-58 or HPV-52 genomes. Conclusions Overall, high-risk HPV MVs were found in about half of all cases in both anal and cervical samples. Interestingly, we reported for the first time a differential impact of APOBEC3 mutagenic activity depending on high-risk HPV type.

Human immunodeficiency virus-1 (HIV-1) is known to interact with multiple host cellular proteins during its replication in the target cell. While many of these host cellular proteins facilitate viral replication, a number of them are reported to inhibit HIV-1 replication at various stages of its life cycle. These host cellular proteins, which are known as restriction factors, constitute an integral part of the host’s first line of defence against the viral pathogen. Since the discovery of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G) as an HIV-1 restriction factor, several human proteins have been identified that exhibit anti-HIV-1 restriction. While each restriction factor employs a distinct mechanism of inhibition, the HIV-1 virus has equally evolved complex counter strategies to neutralize their inhibitory effect. APOBEC3G, tetherin, sterile alpha motif and histidine-aspartate domain 1 (SAMHD1), and trim-5α are some of the best known HIV-1 restriction factors that have been studied in great detail. Recently, six novel restriction factors were discovered that exhibit significant antiviral activity: endoplasmic reticulum α1,2-mannosidase I (ERManI), translocator protein (TSPO), guanylate-binding protein 5 (GBP5), serine incorporator (SERINC3/5) and zinc-finger antiviral protein (ZAP). The focus of this review is to discuss the antiviral mechanism of action of these six restriction factors and provide insights into the probable counter-evasion strategies employed by the HIV-1 virus. The recent discovery of new restriction factors substantiates the complex host–pathogen interactions occurring during HIV-1 pathogenesis and makes it imperative that further investigations are conducted to elucidate the molecular basis of HIV-1 replication.

Background MOV10 protein has ATP-dependent 5′–3′ RNA helicase activity and belongs to the UPF1p superfamily. It can inhibit human immunodeficiency virus type 1 (HIV-1) replication at multiple stages and interact with apolipoprotein-B-mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G), a member of the cytidine deaminase family that exerts potent inhibitory effects against HIV-1 infection. However, HIV-1-encoded virion infectivity factor (Vif) protein specifically mediates the degradation of A3G via the ubiquitin–proteasome system (UPS). Results We demonstrate that MOV10 counteracts Vif-mediated degradation of A3G by inhibiting the assembly of the Vif-CBF-β-Cullin 5-ElonginB-ElonginC complex. Through interference with UPS, MOV10 enhances the level of A3G in HIV-1-infected cells and virions, and synergistically inhibits the replication and infectivity of HIV-1. In addition, the DEAG-box of MOV10 is required for inhibition of Vif-mediated A3G degradation as the DEAG-box mutant significantly loses this ability. Conclusions Our results demonstrate a novel mechanism involved in the anti-HIV-1 function of MOV10. Given that both MOV10 and A3G belong to the interferon antiviral system, their synergistic inhibition of HIV-1 suggests that these proteins may play complicated roles in antiviral functions.

Long interspersed nuclear element-1 (L1) retrotransposons are normally suppressed in somatic tissues mainly by DNA methylation and antiviral defense. However, L1s can be desuppressed in cancers to act as insertional mutagens and cause genomic instability by creating DNA double strand breaks and chromosomal rearrangements. Whereas the frequency of somatic L1 insertions varies greatly among individual tumors, much remains to be learned about underlying genetic, cellular, or environmental factors. Here, our pan-gastrointestinal cancer genome analyses for stomach, colorectal, and esophageal tumors identified multiple correlates of L1 activity. Clinical indicators of tumor progression, such as tumor grade and patient age, showed positive association. Potential L1 expression suppressors such as TP53 and DNMT1, a DNA methyltransferase, were inactivated in tumors with frequent L1 insertions. Importantly, tumors with high immune activity, for example, due to viral infection or high tumor-antigen load, tended to carry a low number of L1 insertions in their genomes with high expression levels of L1 suppressors such as APOBEC3s and SAMHD1. Our analysis of the transcriptional effects of intragenic retrotransposon insertions demonstrated an increased risk of gene disruption in retrotransposition-prone cancers. In particular, we found a splicing-disrupting L1 insertion in an exon of MOV10, a key L1 suppressor, which caused exon skipping with evidence of nonsense-mediated decay in a tumor with a high L1 insertion load. Our results indicate that cancer immunity may contribute to genome stability by suppressing L1 retrotransposition particularly in gastrointestinal cancers.

APOBEC3G (A3G) DNA deaminase activity requires a holoenzyme complex whose assembly on nascent viral reverse transcripts initiates with A3G dimers binding to ssDNA followed by formation of higher-order A3G homo oligomers. Catalytic activity is inhibited when A3G binds to RNA. Our prior studies suggested that RNA inhibited A3G binding to ssDNA. In this report, near equilibrium binding and gel shift analyses showed that A3G assembly and disassembly on ssDNA was an ordered process involving A3G dimers and multimers thereof. Although, fluorescence anisotropy showed that A3G had similar nanomolar affinity for RNA and ssDNA, RNA stochastically dissociated A3G dimers and higher-order oligomers from ssDNA, suggesting a different modality for RNA binding. Mass spectrometry mapping of A3G peptides cross-linked to nucleic acid suggested ssDNA only bound to three peptides, amino acids (aa) 181–194 in the N-terminus and aa 314–320 and 345–374 in the C-terminus that were part of a continuous exposed surface. RNA bound to these peptides and uniquely associated with three additional peptides in the N- terminus, aa 15–29, 41–52 and 83–99, that formed a continuous surface area adjacent to the ssDNA binding surface. The data predict a mechanistic model of RNA inhibition of ssDNA binding to A3G in which competitive and allosteric interactions determine RNA-bound versus ssDNA-bound conformational states.

APOBEC3 (A3) proteins are host-encoded restriction factors that inhibit retrovirus infection by mutagenic deamination of cytosines in minus-strand DNA replication intermediates. APOBEC3F (A3F) and APOBEC3G (A3G) are two of the most potent A3 enzymes in humans with each having a different target DNA specificity. A3G prefers to deaminate cytosines preceded by a cytosine (5'-CC), whereas A3F preferentially targets cytosines preceded by a thymine (5'-TC). Here we performed a detailed comparative analysis of retrovirus-encoded gene sequences edited by A3F and A3G, with the aim of correlating the context and intensity of the mutations with their effects on gene function. Our results revealed that, when there are few (TGG) tryptophan codons in the sequence, both enzymes alter gene function with a similar efficiency when given equal opportunities to deaminate in their preferred target DNA context. In contrast, tryptophan-rich genes are efficiently inactivated in the presence of a low mutational burden, through termination codon generation by A3G but not A3F. Overall, our results clearly demonstrated that the target DNA specificity of an A3 enzyme along with the intensity of the mutational burden and the tryptophan content of the gene being targeted are the factors that have the most forceful influence on whether A3-induced mutations will favour either terminal inactivation or genetic diversification of a retrovirus.

Sites of HIV/TB coinfection are characterized by increased HIV-1 replication and a TH1 profile. However, expression of HIV-1 restriction factors, such as APOBEC3G (A3G) in situ, is unknown. Using an RT-profiler focused on genes related to HIV-1 expansion, we examined pleural fluid mononuclear cells (PFMCs) from patients with HIV/TB coinfection in comparison to HIV-uninfected patients with TB disease. Significant expression of interferon (IFN)-γ and restriction factors A3G and A3F and TRIM5α in PFMCs was found. Genes correlating significantly with the expression of IFN-γ included A3G and A3F. However, pleural fluid HIV-1 viral load and HIV-1 gag/pol mRNA in PFMCs did not correlate with A3G activity.

The human APOBEC3G (A3G) DNA cytosine deaminase restricts and hypermutates DNA-based parasites including HIV-1. The viral infectivity factor (Vif) prevents restriction by triggering A3G degradation. Although the structure of the A3G catalytic domain is known, the structure of the N-terminal Vif-binding domain has proven more elusive. Here, we used evolution- and structure-guided mutagenesis to solubilize the Vif-binding domain of A3G, thus permitting structural determination by NMR spectroscopy. A smaller zinc-coordinating pocket and altered helical packing distinguish the structure from previous catalytic-domain structures and help to explain the reported inactivity of this domain. This soluble A3G N-terminal domain is bound by Vif; this enabled mutagenesis and biochemical experiments, which identified a unique Vif-interacting surface formed by the α1-β1, β2-α2 and β4-α4 loops. This structure sheds new light on the Vif-A3G interaction and provides critical information for future drug development.