Search

We synthesized, separated into enantiomers, and tested for the HIV-1 reverse transcriptase inhibitory activity a group of analogs of dimethyl-1-(1-piperidynyl)cyclobuta[b][1]benzothiophene-2,2a(7bH)-dicarboxylate (NSC-380292). Absolute configurations of the enantiomers were determined based on absolute X-ray structures and analysis of CD spectra. Within pairs of enantiomers the (R,R)-enantiomer was always much more potent HIV-1 reverse transcriptase inhibitor.

After their release from host cells, most retroviral particles undergo a maturation process, which includes viral protein cleavage, core condensation, and increased stability of the viral RNA dimer. Inactivating the viral protease prevents protein cleavage; the resulting virions lack condensed cores and contain fragile RNA dimers. Therefore, protein cleavage is linked to virion morphological change and increased stability of the RNA dimer. However, it is unclear whether protein cleavage is sufficient for mediating virus RNA maturation. We have observed a novel phenotype in a murine leukemia virus capsid mutant, which has normal virion production, viral protein cleavage, and RNA packaging. However, this mutant also has immature virion morphology and contains a fragile RNA dimer, which is reminiscent of protease-deficient mutants. To our knowledge, this mutant provides the first evidence that Gag cleavage alone is not sufficient to promote RNA dimer maturation. To extend our study further, we examined a well-defined human immunodeficiency virus type 1 (HIV-1) Gag mutant that lacks a functional PTAP motif and produces immature virions without major defects in viral protein cleavage. We found that the viral RNA dimer in the PTAP mutant is more fragile and unstable compared with those from wild-type HIV-1. Based on the results of experiments using two different Gag mutants from two distinct retroviruses, we conclude that Gag cleavage is not sufficient for promoting RNA dimer maturation, and we propose that there is a link between the maturation of virion morphology and the viral RNA dimer.

Approximately one million people in the world are dually infected with both HIV-1 and HIV-2. To identify potential interactions between these two human pathogens, we examined whether HIV-1 and HIV-2 Gag proteins can coassemble and functionally complement each other. We generated HIV-1- and HIV-2-based vectors with mutations in Gag; compared with wild-type vectors, these mutants had drastically decreased viral titers. Coexpression of the mutant HIV-1 and HIV-2 Gag could generate infectious viruses; furthermore, heterologous complementation in certain combinations showed efficiency similar to homologous complementation. Additionally, we used bimolecular fluorescence complementation analysis to directly demonstrate that HIV-1 and HIV-2 Gag can interact and coassemble. Taken together, our results indicate that HIV-1 and HIV-2 Gag polyproteins can coassemble and functionally complement each other during virus replication; to our knowledge, this is the first demonstration of its kind. These studies have important implications for AIDS treatment and the evolution of primate lentiviruses.

Initiation of reverse transcription in retroviruses occurs at a specific point in the viral genome, called the primer-binding site (PBS). The efficiency of reverse transcription initiation is not known. We previously published a paper describing reverse transcription of the retroviral vector S-2PBS containing two PBSs. Reverse transcription of this vector results in a provirus with one of four possible structures, depending, in part, on the PBSs used to initiate reverse transcription. Using Southern blotting analyses of DNA from infected cells, we measured the relative proportions of proviruses with different structures. Although the analysis allowed us to detect multiple initiation events occurring in a single virion, the measurement of frequency of such events was not possible. In this paper, we have built a probability model, which describes the reverse transcription process and predicts the outcomes of different initiation scenarios. By fitting the predicted outcomes to the observed data, we have been able to estimate the initiation efficiency in this system as approximately 0.4 initiation per PBS. In addition, we show that even though multiple models of reverse transcription can explain the observed data, all of these models predict approximately the same initiation efficiency. This initiation efficiency is discussed in relation to general replication strategies of retroviruses.

Understanding the mechanisms of HIV-1 drug resistance is critical for developing more effective antiretroviral agents and therapies. Based on our previously described dynamic copy-choice mechanism for retroviral recombination and our observations that nucleoside reverse transcriptase inhibitors (NRTIs) increase the frequency of reverse transcriptase template switching, we propose that an equilibrium exists between ( i ) NRTI incorporation, NRTI excision, and resumption of DNA synthesis and ( ii ) degradation of the RNA template by RNase H activity, leading to dissociation of the template-primer and abrogation of HIV-1 replication. As predicted by this model, mutations in the RNase H domain that reduced the rate of RNA degradation conferred high-level resistance to 3′-azido-3′-deoxythymidine and 2,3-didehydro-2,3-dideoxythymidine by as much as 180- and 10-fold, respectively, by increasing the time available for excision of incorporated NRTIs from terminated primers. These results provide insights into the mechanism by which NRTIs inhibit HIV-1 replication and imply that mutations in RNase H could significantly contribute to drug resistance either alone or in combination with NRTI-resistance mutations in reverse transcriptase.

The RNase H primer grip of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) contacts the DNA primer strand and positions the template strand near the RNase H active site, influencing RNase H cleavage efficiency and specificity. Sequence alignments show that 6 of the 11 residues that constitute the RNase H primer grip have functional equivalents in murine leukemia virus (MLV) RT. We previously showed that a Y586F substitution in the MLV RNase H primer grip resulted in a 17-fold increase in substitutions within 18 nucleotides of adenine-thymine tracts, which are associated with a bent DNA conformation. To further determine the effects of the MLV RNase H primer grip on replication fidelity and viral replication, we performed additional mutational analysis. Using either β-galactosidase ( lacZ ) or green fluorescent protein ( GFP ) reporter genes, we found that S557A, A558V, and Q559L substitutions resulted in statistically significant increases in viral mutation rates, ranging from 2.1- to 3.8-fold. DNA sequencing analysis of nonfluorescent GFP clones indicated that the mutations in RNase H primer grip significantly increased the frequency of deletions between the primer-binding site (PBS) and sequences downstream of the PBS. In addition, quantitative real-time PCR analysis of reverse transcription products revealed that the mutant RTs were substantially inefficient in plus-strand DNA transfer relative to the wild-type control. These results indicate that the MLV RNase H primer grip is an important determinant of in vivo fidelity of DNA synthesis and suggest that the mutant RT was unable to copy through the DNA-RNA junction of the minus-strand DNA and the tRNA because of its bent conformation resulting in error-prone plus-strand DNA transfer.

We previously demonstrated that murine leukemia virus (MLV)-based vectors containing two primer-binding sites (PBSs) have the capacity to initiate reverse transcription more than once (Y. A. Voronin and V. K. Pathak, Virology 312: 281-294, 2003). To determine whether human immunodeficiency virus (HIV)-based vectors also have the capacity to initiate reverse transcription twice, we constructed an HIV type 1 (HIV-1)-based vector containing the HIV-1 PBS, a green fluorescent protein reporter gene ( GFP ), and a second PBS derived from HIV-2 3′ of GFP . Simultaneous initiation of reverse transcription at both the 5′ HIV-1 PBS and 3′ HIV-2 PBS was predicted to result in deletion of GFP . As in the MLV-based vectors, GFP was deleted in approximately 25% of all proviruses, indicating frequent dual initiation in HIV-based vectors containing two PBSs. Quantitative real-time PCR analysis of early reverse transcription products indicated that HIV-1 reverse transcriptase efficiently used the HIV-2 PBS. To investigate tRNA primer-RNA template interactions in vivo, we introduced several mutations in the HIV-2 U5 region. The effects of these mutations on the efficiency of reverse transcription initiation were measured by quantitative real-time PCR analysis of early reverse transcription products, with initiation at the HIV-1 PBS used as an internal control. Disruption of the lower and upper parts of the U5-inverted repeat stem reduced the efficiency of initiation 20- and 6-fold, respectively. In addition, disruption of the proposed interactions between viral RNA and tRNA Lys3 thymidine-pseudouridine-cytidine and anticodon loops decreased the efficiency of initiation seven- and sixfold, respectively. These results demonstrate the relative influence of various RNA-RNA interactions on the efficiency of initiation in vivo. Furthermore, the two-PBS vector system provides a sensitive and quantitative in vivo assay for analysis of RNA-RNA and protein-RNA interactions that can influence the efficiency of reverse transcription initiation.

HIV-1 and other retroviruses occasionally undergo hypermutation, characterized by a high rate of G-to-A substitution. Recently, the human apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (APOBEC3G), first identified as CEM15, was shown to be packaged into retroviral virions and to deaminate deoxycytidine to deoxyuridine in newly synthesized viral minus-strand DNA, thereby inducing G-to-A hypermutation. This innate mechanism of resistance to retroviral infection is counteracted by the HIV-1 viral infectivity factor (Vif), which protects the virus by preventing the incorporation of APOBEC3G into virions by rapidly inducing its ubiquitination and proteasomal degradation. To gain insights into the mechanism by which Vif protects HIV-1 from APOBEC3G, we substituted several amino acids in human APOBEC3G with equivalent residues in simian APOBEC3Gs that are resistant to HIV-1 Vif and determined the effects of the mutations on HIV-1 replication in the presence and absence of Vif. We found that a single amino acid substitution mutant of human APOBEC3G (D128K) can interact with HIV-1 Vif but is not depleted from cells; thus, it inhibits HIV-1 replication in an HIV-1 Vif-resistant manner. Interestingly, rhesus macaque simian immunodeficiency virus 239 or HIV-2 Vif coexpression depleted the intracellular steady state levels of the D128K mutant and abrogated its antiviral activity, indicating that it can be a substrate for the proteasomal pathway. The HIV-1 Vif-resistant mutant APOBEC3G could provide a gene therapy approach to combat HIV-1 infection.

We previously found that azido-containing β-diketo acid derivatives (DKAs) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase (IN) (X. Zhang et al., Bioorg. Med. Chem. Lett., 13:1215-1219, 2003). To characterize the intracellular mechanisms of action of DKAs, we analyzed the antiviral activities of two potent azido-containing DKAs with either a monosubstitution or a disubstitution of azido groups, using single- and multiple-replication-cycle assays. Both azido-containing DKAs significantly inhibited HIV-1 infection in 293T, CEM-SS, and H9 cells (50% inhibitory concentration = 2 to 13 μM) and exhibited low cytotoxicity (50% cytotoxic concentration = 60 to 600 μM). Inhibition of HIV-1 IN in vivo was demonstrated by the observation that previously described L-708,906 resistance mutations in HIV-1 IN (T66I and T66I/S153Y) also conferred resistance to the azido-group-containing DKAs. In vitro assays and in vivo analysis indicated that the DKAs did not significantly inhibit the 3′ processing and selectively inhibited the strand transfer reaction. In addition, quantitative PCR indicated that two-long-terminal-repeat (2-LTR) circles were elevated in the presence of the azido-containing DKAs, confirming that HIV-1 IN was the intracellular target of viral inhibition. To gain insight into the mechanism by which the DKAs increased 2-LTR-circle formation of 3′-processed viral DNAs, we performed extensive DNA sequencing analysis of 2-LTR-circle junctions. The results indicated that the frequency of deletions at the circle junctions was elevated from 19% for the untreated controls to 32 to 41% in the presence of monosubstituted (but not disubstituted) DKAs. These results indicate that the structure of the DKAs can influence the extent of degradation of viral DNA ends by host nucleases and the frequency of deletions at the 2-LTR-circle junctions. Thus, sequencing analysis of 2-LTR-circle junctions can elucidate the intracellular mechanisms of action of HIV-1 IN inhibitors.

Cells infected with two related retroviruses can generate heterozygous virions, which are the precursors of recombinant proviruses. Although many studies have focused on the frequencies and mechanisms of retroviral recombination, little is known about the dynamics of double infection. To examine this issue, viruses generated from two HIV-1 vectors containing different markers were mixed together, and were used to infect target cells. The numbers of cells expressing none, one, or both markers were measured and were used to calculate whether double infection occurred at frequencies expected from random infection events. We found that double infection occurred significantly more frequently than predicted from random distribution; increased rates of double infection were observed in both a T cell line and primary activated CD4 + T cells. In addition to direct virus infection, we also examined the nature of cell-mediated HIV-1 double infection. Increased double infection was observed in all experiments regardless of whether a cell line or primary human dendritic cells were used for capture and transmission of HIV-1. Therefore, our results indicate that HIV-1 double infection occurs more frequently than it would at random in both direct and cell-mediated HIV-1 infections. To our knowledge, this is the first direct evidence of nonrandom double infection in HIV-1. Frequent double HIV-1 infections in infected individuals would allow the generation of recombinant viruses that could then affect their pathogenesis and evolution.

Retroviruses package two copies of viral RNA into each virion. Although each RNA contains a primer-binding site for initiation of DNA synthesis, it is unknown whether reverse transcription is initiated on both RNAs. To determine whether a single virion is capable of initiating reverse transcription more than once, we constructed a murine leukemia virus-based vector containing a second primer-binding site (PBS) derived from spleen necrosis virus and inserted the green fluorescent protein gene (GFP) between the two PBSs. Initiation of reverse transcription at either PBS results in a provirus that expresses GFP. However, initiation at both PBSs can result in the deletion of GFP, which can be detected by flow cytometry and Southern blotting analysis. Approximately 22–29% of the proviruses formed deleted the GFP in a single replication cycle, indicating the minimum proportion of virions that initiated reverse transcription on both PBSs. These results show that a significant proportion of MLV-based vectors containing two PBSs have the capacity to initiate reverse transcription more than once.

The 4-aryl-2-hydroxy-4-oxo-2-butenoic acids and their isosteric tetrazoles are among an emerging class of aryl beta-diketo (ADK)-based agents which exhibit potent inhibition of HIV-1 integrase (IN)-catalyzed strand transfer (ST) processes, while having much reduced potencies against 3'-processing (3'-P) reactions. In the current study, L-708,906 (10e) and 5CITEP (13b), which are two examples of ADK inhibitors that have been reported by Merck and Shionogi pharmaceutical companies, served as model ADK leads. Structural variations to both the "left" and "right" sides of these molecules were made in order to examine effects on HIV-1 integrase inhibitory potencies. It was found that a variety of groups could be introduced onto the left side aryl ring with maintenance of good ST inhibitory potency. However, introduction of carboxylic acid-containing substituents onto the left side aryl ring enhanced 3'-P inhibitory potency and reduced selectivity toward ST reactions. Although both L-708,906 and 5CITEP show potent inhibition of IN in biochemical assays, there is a disparity of antiviral activity in cellular assays using HIV-1-infected cells. Neither 5CITEP nor any other of the indolyl-containing inhibitors exhibit significant antiviral effects in cellular systems. Alternatively, consistent with literature reports, L-708,906 does provide antiviral protection at low micromolar concentrations. Interestingly, several analogues of L-708,906 with varied substituents on the left side aryl ring, while having good inhibitory potencies against IN in extracellular assays, are not antiviral in whole-cell systems.

In vitro studies have indicated that retroviral nucleocapsid (NC) protein facilitates both DNA synthesis by reverse transcriptase (RT) and annealing of the nascent DNA with acceptor template. Increasing the rate of DNA synthesis is expected to reduce the frequency of RT template switching, whereas annealing the nascent DNA with acceptor template promotes template switching. We performed a mutational analysis of the murine leukemia virus (MLV) NC zinc finger domain to study its effect on RT template switching in vivo and to explore the role of NC during reverse transcription. The effects of NC mutations on RT template switching were determined by using a previously described in vivo direct-repeat deletion assay. A trans -complementation assay was also developed in which replication-defective NC mutants were rescued by coexpression of replication-defective RT mutants that provided wild-type NC in trans . We found that mutations in the MLV NC zinc finger domain increased the frequency of template switching approximately twofold. When a predicted stem-loop RNA secondary structure was introduced into the template RNA, the template-switching frequency increased 5-fold for wild-type NC and further increased up to an additional 6-fold for NC zinc finger domain mutants, resulting in an overall increase of as much as 30-fold. Thus, wild-type NC increased the efficiency with which RT was able to reverse transcribe through regions of RNA secondary structure that might serve as RT pause sites. These results provide the first in vivo evidence that NC enhances the rate of DNA synthesis by RT in regions of the template possessing stable RNA secondary structure.

Several APOBEC3 proteins, particularly APOBEC3D, APOBEC3F, and APOBEC3G, induce G-to-A hypermutations in HIV-1 genome, and abrogate viral replication in experimental systems, but their relative contributions to controlling viral replication and viral genetic variation in vivo have not been elucidated. On the other hand, an HIV-1-encoded protein, Vif, can degrade these APOBEC3 proteins via a ubiquitin/proteasome pathway. Although APOBEC3 proteins have been widely considered as potent restriction factors against HIV-1, it remains unclear which endogenous APOBEC3 protein(s) affect HIV-1 propagation in vivo. Here we use a humanized mouse model and HIV-1 with mutations in Vif motifs that are responsible for specific APOBEC3 interactions, DRMR/AAAA (4A) or YRHHY/AAAAA (5A), and demonstrate that endogenous APOBEC3D/F and APOBEC3G exert strong anti-HIV-1 activity in vivo. We also show that the growth kinetics of 4A HIV-1 negatively correlated with the expression level of APOBEC3F. Moreover, single genome sequencing analyses of viral RNA in plasma of infected mice reveal that 4A HIV-1 is specifically and significantly diversified. Furthermore, a mutated virus that is capable of using both CCR5 and CXCR4 as entry coreceptor is specifically detected in 4A HIV-1-infected mice. Taken together, our results demonstrate that APOBEC3D/F and APOBEC3G fundamentally work as restriction factors against HIV-1 in vivo, but at the same time, that APOBEC3D and APOBEC3F are capable of promoting viral diversification and evolution in vivo., Author Summary Mutation can produce three outcomes in viruses: detrimental, neutral, or beneficial. The first one leads to abrogation of virus replication because of error catastrophe, while the last one lets the virus escape from anti-viral immune system or adapt to the host. Human APOBEC3D, APOBEC3F, and APOBEC3G are cellular cytidine deaminases which cause G-to-A mutations in HIV-1 genome. Here we use a humanized mouse model and demonstrate that endogenous APOBEC3F and APOBEC3G induce G-to-A hypermutation in viral genomes and exert strong anti-HIV-1 activity in vivo. We also reveal that endogenous APOBEC3D and/or APOBEC3F induce viral diversification, which can lead to the emergence of a mutated virus that converts its coreceptor usage. Our results suggest that APOBEC3D and APOBEC3F are capable of promoting viral diversification and functional evolution in vivo.

In present scenario biometrics system are getting popular day by day and they are classified based on subject's cooperation and non-cooperation nature. Gait biometrics is one of the popularly traits used for person identification. The gait biometrics has an edge over the other biometrics traits as it works well even if the subject is not cooperative. The multiple problems such as shadow detection, removal of moving subjects in visual surveillance, less number of gallery probes in different conditions and existence of multiple views in the data are encountered due to surveillance camera and affect the performance of the gait recognition system. In this paper, we used three algorithms to overcome these problems up to an extent. In first algorithm, photometric properties based method is used to remove shadows at the time of silhouette generation from the recorded video. Then, an algorithm for synthetic gait energy image (GEI) templates generation employed to increase the corresponding gallery probes dataset and finally, singular value decomposition transformation is applied to transform the gait feature from one view to another. The performance of the proposed algorithm is experimentally validated on a benchmark dataset of indoor as well as outdoor video sequences by comparing it with the existing algorithms.

Retroviral populations exhibit a high evolutionary potential, giving rise to extensive genetic variation. Error-prone DNA synthesis catalyzed by reverse transcriptase (RT) generates variation in retroviral populations. Structural features within RTs are likely to contribute to the high rate of errors that occur during reverse transcription. We sought to determine whether amino acids within murine leukemia virus (MLV) RT that contact the deoxyribonucleoside triphosphate (dNTP) substrate are important for in vivo fidelity of reverse transcription. We utilized the previously described ANGIE P encapsidating cell line, which expresses the amphotropic MLV envelope and a retroviral vector (pGA-1). pGA-1 expresses the bacterial β-galactosidase gene ( lacZ ), which serves as a reporter of mutations. Extensive mutagenesis was performed on residues likely to interact with the dNTP substrate, and the effects of these mutations on the fidelity of reverse transcription were determined. As expected, most substitution mutations of amino acids that directly interact with the dNTP substrate significantly reduced viral titers (>10,000-fold), indicating that these residues played a critical role in catalysis and viral replication. However, the D153A and A154S substitutions, which are predicted to affect the interactions with the triphosphate, resulted in statistically significant increases in the mutation rate. In addition, the conservative substitution F155W, which may affect interactions with the base and the ribose, increased the mutation rate 2.8-fold. Substitutions of residues in the vicinity of the dNTP-binding site also resulted in statistically significant decreases in fidelity (1.3- to 2.4-fold). These results suggest that mutations of residues that contact the substrate dNTP can affect viral replication as well as alter the fidelity of reverse transcription.

Retroviral reverse transcriptases (RTs) frequently switch templates within the same RNA or between copackaged viral RNAs to generate mutations and recombination. To identify structural elements of murine leukemia virus RT important for template switching, we developed an in vivo assay in which RT template switching within direct repeats functionally reconstituted the green fluorescent protein gene. We quantified the effect of mutations in the YXDD motif, the deoxynucleoside triphosphate binding site, the thumb domain, and the RNase H domain of RT and hydroxyurea treatment on the frequencies of template switching. Hydroxyurea treatment and some mutations in RT increased the frequency of RT template switching up to fivefold, while all of the mutations tested in the RNase H domain decreased the frequency of template switching by twofold. Based on these results, we propose a dynamic copy choice model in which both the rate of DNA polymerization and the rate of RNA degradation influence the frequency of RT template switching.

Reverse transcriptase (RT) switches templates frequently during DNA synthesis; the acceptor template can be the same RNA (intramolecular) or the copackaged RNA (intermolecular). Previous results indicated that intramolecular template switching occurred far more frequently than intermolecular template switching. We hypothesized that intermolecular template-switching events (recombination) occurred at a lower efficiency because the copackaged RNA was not accessible to the RT. To test our hypothesis, the murine leukemia virus (MLV) extended packaging signal (Ψ + ) containing a dimer linkage structure (DLS) was relocated from the 5′ untranslated region (UTR) to between selectable markers, allowing the two viral RNAs to interact closely in this region. It was found that the overall maximum recombination rates of vectors with Ψ + in the 5′ UTR or Ψ + between selectable markers were not drastically different. However, vectors with Ψ + located between selectable markers reached a plateau of recombination rate at a shorter distance. This suggested a limited enhancement of recombination by Ψ + . The locations of the recombination events were also examined by using restriction enzyme markers. Recombination occurred in all four regions between the selectable markers; the region containing 5′ Ψ + including DLS did not undergo more recombination than expected from the size of the region. These experiments indicated that although the accessibility of the copackaged RNA was important in recombination, other factors existed to limit the number of viruses that were capable of undergoing intermolecular template switching. In addition, recombinants with multiple template switches were observed at a frequency much higher than expected, indicating the presence of high negative interference in the MLV-based system. This extends our observation with the spleen necrosis virus system and suggests that high negative interference may be a common phenomenon in retroviral recombination.

We have developed murine leukemia virus (MLV)-based self-inactivating and self-activating vectors to show that the previously demonstrated high-frequency direct repeat deletions are not unique to spleen necrosis virus (SNV) or the neomycin drug resistance gene. Retroviral vectors pKD-HTTK and pKD-HTpTK containing direct repeats composed of segments of the herpes simplex virus type 1 thymidine kinase (HTK) gene were constructed; in pKD-HTpTK, the direct repeat flanked the MLV packaging signal. The generation of hypoxanthine-aminopterin-thymidine-resistant colonies after one cycle of retroviral replication demonstrated functional reconstitution of the HTK gene. Quantitative Southern analysis indicated that direct repeat deletions occurred in 57 and 91% of the KD-HTTK and KD-HTpTK proviruses, respectively. These results demonstrate that (i) deletion of direct repeats occurs at similar high frequencies in SNV and MLV vectors, (ii) MLV psi can be efficiently deleted by using direct repeats, (iii) suicide genes can be functionally reconstituted during reverse transcription, and (iv) the psi region may be a hot spot for reverse transcriptase template switching events.

How retroviruses regulate the amount of RNA genome packaged into each virion has remained a long-standing question. Our previous study showed that most HIV-1 particles contain two copies of viral RNA, indicating that the number of genomes packaged is tightly regulated. In this report, we examine the mechanism that controls the number of RNA genomes encapsidated into HIV-1 particles. We hypothesize that HIV-1 regulates genome packaging by either the mass or copy number of the viral RNA. These two distinct mechanisms predict different outcomes when the genome size deviates significantly from that of wild type. Regulation by RNA mass would result in multiple copies of a small genome or one copy of a large genome being packaged, whereas regulation by copy number would result in two copies of a genome being packaged independent of size. To distinguish between these two hypotheses, we examined the packaging of viral RNA that was larger (≈17 kb) or smaller (≈3 kb) than that of wild-type HIV-1 (≈9 kb) and found that most particles packaged two copies of the viral genome regardless of whether they were 17 kb or 3 kb. Therefore, HIV-1 regulates RNA genome encapsidation not by the mass of RNA but by packaging two copies of RNA. To further explore the mechanism that governs this regulation, we examined the packaging of viral RNAs containing two packaging signals that can form intermolecular dimers or intramolecular dimers (self-dimers) and found that one self-dimer is packaged. Therefore, HIV-1 recognizes one dimeric RNA instead of two copies of RNA. Our findings reveal that dimeric RNA recognition is the key mechanism that regulates HIV-1 genome encapsidation and provide insights into a critical step in the generation of infectious viruses., Author Summary Viruses must package their genomes in particles to pass their genetic information to the next generation. Although many aspects of RNA packaging are well-studied, how retroviruses regulate the number of genomes in the particle is currently unknown. Based on the dimeric nature of retroviral genomes in particles, it was often assumed that two copies of RNA were packaged into one particle. This assumption was validated recently when we demonstrated that most HIV-1 particles contain two copies of viral RNA, which revealed that the number of genomes packaged is tightly controlled. In this report, we examined the mechanism that regulates the amount of RNAs encapsidated into HIV-1 particles. Our results showed that RNA packaging is not regulated by the mass of the viral RNA as two copies of small or large genomes are packaged. However, packaging of two copies of RNA can be perturbed; HIV-1 can encapsidate one copy of its genome when the RNA contains two packaging/dimerization signals that allow for intramolecular dimer (self-dimer) formation. These studies revealed that HIV-1 regulates genome packaging by recognizing the dimeric RNA structure, and suggest that the interaction of viral protein Gag and dimeric RNA serves as the nucleation point of virus assembly.

Retroviruses mutate at a high rate in vivo during viral replication. Mutations may occur during proviral transcription by RNA polymerase II, during minus-strand DNA synthesis (RNA template) by viral reverse transcriptase, or during plus-strand DNA synthesis (DNA template) by reverse transcriptase. To determine the contributions of different stages of replication to the retroviral mutation rates, we developed a spleen necrosis virus-based in vivo system to selectively identify mutations occurring during the early stage (RNA transcription plus minus-strand synthesis) and the late stage (plus-strand synthesis plus DNA repair). A lacZalpha reporter gene was inserted into the long terminal repeat (LTR) of a spleen necrosis virus shuttle vector, and proviruses were recovered from infected cells as plasmids containing either one or both LTRs. Plasmids containing both LTRs generated a mutant phenotype only if the lacZalpha genes in both LTRs were mutated, which is most likely to occur during the early stage. Mutant phenotypes were identified from plasmids containing one LTR regardless of the stage at which the mutations occurred. Thus, mutant frequencies obtained after recovery of plasmids containing both LTRs or one LTR provided early-stage and total mutation rates, respectively. Analysis of 56,409 proviruses suggested that the retroviral mutation rates during the early and late stages of replication were equal or within twofold of each other. In addition, two mutants with A-to-G hypermutations were discovered, suggesting a role for mammalian double-stranded RNA adenosine deaminase enzyme in retroviral mutations. These experiments provide a system to selectively identify mutations in the early stage of retroviral replication and to provide upper and lower limits to the in vivo mutation rates during minus-strand and plus-strand synthesis, respectively.

A critical step in retroviral reverse transcription is the initiation of plus-strand DNA synthesis at the polypurine tract (PPT) and strand transfer of the PPT-primed strong-stop DNA to the 5' end of the viral DNA. An attachment site (att) immediately 3' to the PPT is essential for proper integration of proviral DNA into the host chromosome. Plus-strand DNA synthesis is discontinuous in many retroviruses, indicating that sequences upstream of the PPT are also used to initiate plus-strand DNA synthesis (internally initiated DNA). Strand transfer of internally initiated DNA would result in "dead" viral DNA that lacks the att site needed for integration. Strand transfer of the internally initiated DNA could occur if DNA synthesis failed to initiate at the PPT or if the PPT-primed DNA was displaced before strand transfer. We sought to determine the efficiency of DNA synthesis initiating at the PPT and the proportions of PPT-primed DNA and internally initiated DNAs that are utilized for strand transfer. We constructed spleen necrosis virus-based retroviral vectors containing an internal PPT and an att site 5' of the normal PPT and att site. After one replication cycle of the retroviral vectors, the structures of the resulting proviruses were determined by Southern blotting. The analysis suggested that the PPT is an efficient and rapid initiator of plus-strand DNA synthesis and that internally initiated DNAs are rarely utilized for strand transfer. We hypothesize that efficient synthesis and strand transfer of PPT-primed DNA evolved to prevent lethal strand transfers of internally initiated DNAs.

A broad spectrum of mutations occurs at a high rate during a single round of retrovirus replication (V.K. Pathak and H. M. Temin, Proc. Natl. Acad. Sci. USA 87:6019-6023, 1990). We have now determined that this high rate of spontaneous mutation can be further increased by 5-azacytidine (AZC) treatment or by regions of potential RNA secondary structure. We found a 13-fold increase in the mutation rate after AZC treatment of retrovirus-producing cells and target cells. The AZC-induced substitutions were located at the same target sites as previously identified spontaneous substitutions. The concordance of the AZC-induced and spontaneous substitutions indicates the presence of reverse transcription "pause sites," where the growing point is error prone. An analysis of nucleotides that neighbored substitutions revealed that transversions occur primarily by transient template misalignment, whereas transitions occur primarily by misincorporation. We also introduced a 34-bp potential stem-loop structure as an in-frame insertion within a lacZ alpha gene that was inserted in the long terminal repeat (LTR) U3 region and determined whether this potential secondary structure increased the rate of retrovirus mutations. We found a threefold increase in the retrovirus mutation rate. Fifty-seven of 96 mutations were deletions associated with the potential stem-loop. We also determined that these deletion mutations occurred primarily during minus-strand DNA synthesis by comparing the frequencies of mutations in recovered provirus plasmids containing both LTRs and in provirus plasmids containing only one LTR.

Background Although extensive HIV drug resistance information is available for the first 400 amino acids of its reverse transcriptase, the impact of antiretroviral treatment in C-terminal domains of Pol (thumb, connection and RNase H) is poorly understood. Methods and Findings We wanted to characterize conserved regions in RT C-terminal domains among HIV-1 group M subtypes and CRF. Additionally, we wished to identify NRTI-related mutations in HIV-1 RT C-terminal domains. We sequenced 118 RNase H domains from clinical viral isolates in Brazil, and analyzed 510 thumb and connection domain and 450 RNase H domain sequences collected from public HIV sequence databases, together with their treatment status and histories. Drug-naive and NRTI-treated datasets were compared for intra- and inter-group conservation, and differences were determined using Fisher's exact tests. One third of RT C-terminal residues were found to be conserved among group M variants. Three mutations were found exclusively in NRTI-treated isolates. Nine mutations in the connection and 6 mutations in the RNase H were associated with NRTI treatment in subtype B. Some of them lay in or close to amino acid residues which contact nucleic acid or near the RNase H active site. Several of the residues pointed out herein have been recently associated to NRTI exposure or increase drug resistance to NRTI. Conclusions This is the first comprehensive genotypic analysis of a large sequence dataset that describes NRTI-related mutations in HIV-1 RT C-terminal domains in vivo. The findings into the conservation of RT C-terminal domains may pave the way to more rational drug design initiatives targeting those regions.

We determined the in vivo forward mutation rate in a single replication cycle for spleen necrosis virus (SNV). A method was developed to clone integrated proviruses of retroviral shuttle vectors by exploiting the tight binding of the lac operator to the lac repressor protein. The vectors contained the lacZ alpha gene as a reporter of mutations. Thirty-seven of the 16,867 proviruses recovered contained five classes of mutations, including substitutions and frameshifts. Runs of 9 and 10 identical base pairs and a direct repeat of 110 base pairs were mutational hotspots. In addition, two copies of a provirus contained 15 G-to-A substitutions. Such proviruses, which we name hypermutants, may arise through the action of an error-prone polymerase and could significantly contribute to the genetic variation in retroviral populations.

A human liver cDNA encoding the beta subunit of protein synthesis initiation factor 2 (eIF-2) was isolated and sequenced. The 1416 bp cDNA encodes a protein of 333 amino acids (38,404 daltons) with characteristics that resemble authentic purified eIF-2 beta. De novo synthesized eIF-2 beta from cDNA transcripts incorporates into endogenous rabbit eIF-2 complexes. The protein possesses putative GTP-binding sites, a zinc finger motif, and a highly charged N-terminal region composed of three basic polylysine blocks separated by acidic domains. The polylysine blocks and the zinc finger motif suggest that eIF-2 beta interacts with RNA. A yeast protein, Sui3, isolated as an extragenic suppressor of his4 initiation codon mutations, exhibits extensive sequence identity with human eIF-2 beta, especially in the polylysine and zinc finger domains, thereby reinforcing the view that these elements are important for function.

A host cytidine deaminase, APOBEC3G (A3G), inhibits replication of human immunodeficiency virus type 1 (HIV-1) by incorporating into virions in the absence of the virally encoded Vif protein (Deltavif virions), at least in part by causing G-to-A hypermutation. To gain insight into the antiretroviral function of A3G, we determined the quantities of A3G molecules that are incorporated in Deltavif virions. We combined three experimental approaches-reversed-phase high-pressure liquid chromatography (HPLC), scintillation proximity assay (SPA), and quantitative immunoblotting-to determine the molar ratio of A3G to HIV-1 capsid protein in Deltavif virions. Our studies revealed that the amount of the A3G incorporated into Deltavif virions was proportional to the level of its expression in the viral producing cells, and the ratio of the A3G to Gag in the Deltavif virions produced from activated human peripheral blood mononuclear cells (PBMC) was approximately 1:439. Based on previous estimates of the stoichiometry of HIV-1 Gag in virions (1400-5000), we conclude that approximately 7 (+/-4) molecules of A3G are incorporated into Deltavif virions produced from human PBMCs. These results indicate that virion incorporation of only a few molecules of A3G is sufficient to inhibit HIV-1 replication.

Recombination can assort polymorphic alleles to increase diversity in the HIV-1 population. To better understand the recombination potential of subtype A HIV-1, we generated viruses containing sequences from two variants circulating in Russia and analyzed the polymerase gene (pol) of the recombinants after one round of HIV-1 replication using single-genome sequencing. We observed that recombination occurred throughout pol and could easily assort alleles containing mutations that conferred resistance to currently approved antivirals. We measured the recombination rate in various regions of pol including a G-rich region that has been previously proposed to be a recombination hot spot. Our study does not support a recombination hot spot in this G-rich region. Importantly, of the 58 proviral sequences containing crossover event(s) in pol, we found that each sequence was a unique genotype indicating that recombination is a powerful genetic mechanism in assorting the genomes of subtype A HIV-1 variants.

The phosphorylation of the alpha-subunit of initiation factor eIF-2 leads to an inhibition of protein synthesis in mammalian cells. We have performed site-directed mutagenesis on a cDNA encoding the alpha-subunit of human eIF-2 and have replaced the candidate sites of phosphorylation, Ser-48 and Ser-51, with alanines. The cDNAs were expressed in vitro by SP6 polymerase transcription and rabbit reticulocyte lysate translation, and the radiolabeled protein products were analyzed by high-resolution two-dimensional gel electrophoresis. The wild-type and Ser-48 mutant proteins became extensively phosphorylated by eIF-2 kinases present in the reticulocyte lysate, and when additional heme-controlled repressor or double-stranded RNA-activated kinase was present, phosphorylation of the proteins was enhanced. The Ser-51 mutant showed little covalent modification by the endogenous enzymes and showed no increase in the acidic variant with additional eIF-2 kinases, thereby suggesting that Ser-51 is the site of phosphorylation leading to repression of protein synthesis.

Translational control is defined as a change in the efficiency of translation of mRNAs. This may involve a quantitative change in the overall amount of protein synthesized, called global control. Alternatively, the change may affect the synthesis of specific proteins, called mRNA-specific control. In either case, efficiency of translation is usually measured by radiolabeled amino acid incorporation into protein, or by the number and activity of ribosomes. Since the rate limiting step in protein synthesis appears to be at the initiation phase, most control mechanisms are thought to regulate this step. We therefore focus on the pathway of initiation and address studies of how global rates of protein synthesis may be controlled.

The process of protein synthesis on ribosomes is promoted by soluble protein factors that act during the initiation, elongation and termination phases of translation (for reviews, see Moldave, 1985; Pain, 1986). A striking characteristic of these factors is that most of them bind GTP or GDP. Since the structure and mechanism of action of these proteins have been studied extensively for the past 20 years, considerable knowledge of their mechanisms of action has accummulated which may be relevant to understanding how G-proteins function in general. We are concerned here with one of the mammalian factors, initiation factor eIF2.

Rearrangement of the int-1 and int-2 regions of mouse chromosomes was compared in the C3H and BALB/cfC3H hyperplastic alveolar nodule and its hyperplastic outgrowth (HPO) model systems by examining the DNA of the different stages of the neoplastic progression, with use of the Southern blot technique. Rearrangement of int region DNAs associated with proviral amplification occurred more frequently in spontaneous tumors (19 of 27) than in tumors from HPOs (7 of 37) and rarely occurred in HPOs (1 of 29). However, the int-1 rearrangement maintained in 1 BALB/cfC3H HPO line through 11 transplant generations suggests that the int-1 rearrangement is neither sufficient nor necessary for progression to mouse mammary carcinoma.

Phosphorylation of the alpha subunit of the eucaryotic translation initiation factor (eIF-2 alpha) by the double-stranded RNA-activated inhibitor (DAI) kinase correlates with inhibition of translation initiation. The importance of eIF-2 alpha phosphorylation in regulating translation was studied by expression of specific mutants of eIF-2 alpha in COS-1 cells. DNA transfection of certain plasmids could activate DAI kinase and result in poor translation of plasmid-derived mRNAs. In these cases, translation of the plasmid-derived mRNAs was improved by the presence of DAI kinase inhibitors or by the presence of a nonphosphorylatable mutant (serine to alanine) of eIF-2 alpha. The improved translation mediated by expression of the nonphosphorylatable eIF-2 alpha mutant was specific to plasmid-derived mRNA and did not affect global mRNA translation. Expression of a serine-to-aspartic acid mutant eIF-2 alpha, created to mimic the phosphorylated serine, inhibited translation of the mRNAs derived from the transfected plasmid. These results substantiate the hypothesis that DAI kinase activation reduces translation initiation through phosphorylation of eIF-2 alpha and reinforce the importance of phosphorylation of eIF-2 alpha as a way to control initiation of translation in intact cells.