APOBEC3G impairs the multimerization of the HIV-1 Vif protein in living cells.

The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Vif allows productive infection in nonpermissive cells, including most natural HIV-1 target cells, by counteracting the cellular cytosine deaminases APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G [A3G]) and A3F. Vif is also associated with the viral assembly complex and packaged into viral particles through interactions with the viral genomic RNA and the nucleocapsid domain of Pr55(Gag). Recently, we showed that oligomerization of Vif into high-molecular-mass complexes induces Vif folding and influences its binding to high-affinity RNA binding sites present in the HIV genomic RNA. To get further insight into the role of Vif multimerization in viral assembly and A3G repression, we used fluorescence lifetime imaging microscopy (FLIM)- and fluorescence resonance energy transfer (FRET)-based assays to investigate Vif-Vif interactions in living cells. By using two N-terminally tagged Vif proteins, we show that Vif-Vif interactions occur in living cells. This oligomerization is strongly reduced when the putative Vif multimerization domain ((161)PPLP(164)) is mutated, indicating that this domain is crucial, but that regions outside this motif also participate in Vif oligomerization. When coexpressed together with Pr55(Gag), Vif is largely relocated to the cell membrane, where Vif oligomerization also occurs. Interestingly, wild-type A3G strongly interferes with Vif multimerization, contrary to an A3G mutant that does not bind to Vif. These findings confirm that Vif oligomerization occurs in living cells partly through its C-terminal motif and suggest that A3G may target and perturb the Vif oligomerization state to limit its functions in the cell.

A pyrophosphatase activity associated with purified HIV-1 particles.

Treatment of HIV-1 with nucleoside reverse transcription inhibitors leads to the emergence of resistance mutations in the reverse transcriptase (RT) gene. Resistance to 3'-azido-3'-deoxythymidine (AZT) and to a lesser extent to 2'-3'-didehydro-2'-3'-dideoxythymidine is mediated by phosphorolytic excision of the chain terminator. Wild-type RT excises AZT by pyrophosphorolysis, while thymidine-associated resistance mutations in RT (TAMs) favour ATP as the donor substrate. However, in vitro, resistant RT still uses pyrophosphate more efficiently than ATP. We performed in vitro (-) strong-stop DNA synthesis experiments, with wild-type and AZT-resistant HIV-1 RTs, in the presence of physiologically relevant pyrophosphate and/or ATP concentrations and found that in the presence of pyrophosphate, ATP and AZTTP, TAMs do not enhance in vitro (-) strong-stop DNA synthesis. We hypothesized that utilisation of ATP in vivo is driven by intrinsic low pyrophosphate concentrations within the reverse transcription complex, which could be explained by the packaging of a cellular pyrophosphatase. We showed that over-expressed flagged-pyrophosphatase was associated with HIV-1 viral-like particles. In addition, we demonstrated that when HIV-1 particles were purified in order to avoid cellular microvesicle contamination, a pyrophosphatase activity was specifically associated to them. The presence of a pyrophosphatase activity in close proximity to the reverse transcription complex is most likely advantageous to the virus, even in the absence of any drug pressure.

Initiation of HIV-1 reverse transcription and functional role of nucleocapsid-mediated tRNA/viral genome interactions.

HIV-1 reverse transcription is initiated from a tRNA(Lys)(3) molecule annealed to the viral RNA at the primer binding site (PBS). The annealing of tRNA(Lys)(3) requires the opening of its three-dimensional structure and RNA rearrangements to form an efficient initiation complex recognized by the reverse transcriptase. This annealing is mediated by the nucleocapsid protein (NC). In this paper, we first review the actual knowledge about HIV-1 viral RNA and tRNA(Lys)(3) structures. Then, we summarize the studies explaining how NC chaperones the formation of the tRNA(Lys)(3)/PBS binary complex. Additional NMR data that investigated the NC interaction with tRNA(Lys)(3) D-loop are presented. Lastly, we focused on the additional interactions occurring between tRNA(Lys)(3) and the viral RNA and showed that they are dependent on HIV-1 isolates, i.e. the sequence and the structure of the viral RNA.

Lactose-ornithine bolaamphiphiles for efficient gene delivery in vitro.

The development of new nonviral vectors characterized by high transfection efficiency and low cytotoxicity remains an important challenge in the field of gene delivery. Unsymmetrical bolaamphiphiles (bolas) appear as new emerging candidates for this application. In this work, new unsymmetrical bolas, bearing neutral lactonic acid and cationic ornithine residues at the two ends of a hydrophobic spacer, were synthesized and their properties were compared to analogues bearing a gluconic acid residue. The new bolas showed DNA binding and condensation at higher N/P ratios than their gluconic analogues, probably due to their larger neutral head group. Whereas the size of the complexes of the new bolas with DNA (bolaplexes) increased with N/P, as a result of charge neutralization, their formulations with DOPE at high N/P were of small size (ca. 200 nm). These DOPE formulations showed high transfection efficiency in different cell lines (HeLa, COS-7 and HepG2), close to that of jetPEI. Their cytotoxicity was relatively low, which allowed repetitive transfection in vitro. Fluorescence imaging showed that the bolaplexes bind rapidly to cell surface and internalize mainly through endocytosis. This work suggests a new type of efficient nonviral vectors based on bolaamphiphiles.

New unsymmetrical bolaamphiphiles: synthesis, assembly with DNA, and application for gene delivery.

The success in gene therapy relies strongly on new efficient gene delivery vectors. Nonviral vectors based on lipids and polymers constitute an important alternative to the viral vectors. However, the key problem with these vectors is the poor structural control of their DNA complexes. In the present work, following new design we synthesized unsymmetrical bolaamphiphiles, molecules bearing neutral sugar (gluconic acid) and dicationic ornithine head groups connected by different long hydrophobic spacers. Within this design, a positively charged headgroup is expected to bind DNA, the hydrophobic spacer is to drive the formation of a monolayer membrane shell around DNA, while the neutral group is to be exposed outside of the complex. Our fluorescence and gel electrophoresis data showed that self-assembly of bolas and their interaction with DNA depend strongly on the bola structure. The size of bola/DNA complexes (bolaplexes) estimated from dynamic light scattering data was ∼100 nm at low N/P (cationic nitrogen/DNA phosphate molar ratio), while at higher N/Ps it was significantly larger due to neutralization of their surface charge. Atomic force microscopy studies revealed nanostructural rod-shaped or spherical morphology of the bolaplexes. Transfection efficiency of the bolaplexes in vitro was significant when either DOPE or chloroquine were used as helping agents, suggesting that the key barrier for their internalization is the endosomal escape. Finally, all bolas showed low cytotoxicity (cell viability >80%). The present results show that bolas are prospective candidates for construction of nonviral gene delivery vectors. We believe that further optimization of polar head groups and a hydrophobic spacer in the bolas will lead to vectors with controlled small size and high transfection efficiency.

Antiretroviral activity of ancestral TRIM5alpha.

The antiretroviral protein TRIM5alpha is known to have evolved different restriction capacities against various retroviruses, driven by positive Darwinian selection. However, how these different specificities have evolved in the primate lineages is not fully understood. Here we used ancestral protein resurrection to estimate the evolution of antiviral restriction specificities of TRIM5alpha on the primate lineage leading to humans. We used TRIM5alpha coding sequences from 24 primates for the reconstruction of ancestral TRIM5alpha sequences using maximum-likelihood and Bayesian approaches. Ancestral sequences were transduced into HeLa and CRFK cells. Stable cell lines were generated and used to test restriction of a panel of extant retroviruses (human immunodeficiency virus type 1 [HIV-1] and HIV-2, simian immunodeficiency virus [SIV] variants SIV(mac) and SIV(agm), and murine leukemia virus [MLV] variants N-MLV and B-MLV). The resurrected TRIM5alpha variant from the common ancestor of Old World primates (Old World monkeys and apes, approximately 25 million years before present) was effective against present day HIV-1. In contrast to the HIV-1 restriction pattern, we show that the restriction efficacy against other retroviruses, such as a murine oncoretrovirus (N-MLV), is higher for more recent resurrected hominoid variants. Ancestral TRIM5alpha variants have generally limited efficacy against HIV-2, SIV(agm), and SIV(mac). Our study sheds new light on the evolution of the intrinsic antiviral defense machinery and illustrates the utility of functional evolutionary reconstruction for characterizing recently emerged protein differences.

Role of common human TRIM5alpha variants in HIV-1 disease progression.

BACKGROUND: The retroviral restriction factor tripartite motif protein (TRIM)5alpha, is characterized by marked amino acid diversity among primates, including specific clusters of residues under positive selection. The identification of multiple non-synonymous changes in humans suggests that TRIM5alpha variants might be relevant to retroviral pathogenesis. Previous studies have shown that such variants are unlikely to modify susceptibility to HIV-1 infection, or the course of early infection. However, the longterm effect of carrying Trim5alpha variants on disease progression in individuals infected with HIV-1 has not previously been investigated. METHODS: In a cohort of 979 untreated individuals infected with HIV-1 with median follow up 3.2 years and 9,828 CD4 T cell measurements, we analysed common amino acid variations: H43Y, V112F, R136Q, G249D, and H419Y. The rate of CD4 T cell decline before treatment was used as the phenotype. In addition, we extended previous work on the in vitro susceptibility of purified donor CD4 T cells (n = 125) to HIV-1 infection, and on the susceptibility of HeLa cells that were stably transduced with the different TRIM5 variants. Haplotypes were analysed according to the most parsimonious evolutionary structure, where two main human TRIM5alpha groups can be defined according to the residue at amino acid 136. Humans present both Q136 and R136 at similar frequency, and additional TRIM5alpha amino acid variants are almost exclusively derived from R136-carrying haplotypes. RESULTS: We observed modest differences in disease progression for evolutionary branches carrying R136-derived haplotypes, and with the non-synonymous polymorphisms G249D and H419Y. In vitro analysis of susceptibility of donor CD4 T cells, and of the various transduced HeLa cell lines supported the absence of significant differential restriction of HIV-1 infection by the various huTRIM5alpha alleles. CONCLUSION: Common human variants of TRIM5alpha have no effect or modest effect on HIV-1 disease progression. These variants occur at sites conserved throughout evolution, and are remote from clusters of positive selection in the primate lineage. The evolutionary value of the substitutions remains unclear.

Mg2+ dependency of HIV-1 reverse transcription, inhibition by nucleoside analogues and resistance.

Metal ions are essential for DNA polymerase and RNase H activities of HIV-1 reverse transcriptase (RT). RT studies are routinely performed at 6-8 mM Mg2+, despite the fact that the in vivo concentration might be as low as 0.2 mM. We studied the influence of MgCl2 and ATP, which likely binds a significant fraction of the magnesium pool in vivo, on the DNA polymerase and RNase H activities of HIV-1 RT, its inhibition by nucleoside RT inhibitors (NRTIs) and primer unblocking by AZT-resistant RT. At low Mg2+ concentration, reverse transcription of a natural template strongly increased despite a dramatically reduced intrinsic polymerase activity under such conditions. Low Mg2+ concentrations affected the RNA stability and indirectly decreased its degradation by the RNase H activity. The reduced RNA degradation prevented premature dissociation of the template and primer strands that otherwise generated dead-end DNA products. In addition, low Mg2+ dramatically decreased the incorporation of NRTIs into DNA and increased nucleotide excision by AZT-resistant RT. The latter effect is also most likely owing to the diminished cleavage of the RNA template. Thus, differences in the free Mg2+ concentration between different cell types or during the cell cycle might strongly affect HIV-1 replication and its inhibition.

Structural variability of the initiation complex of HIV-1 reverse transcription.

HIV-1 reverse transcription is initiated from a tRNA(3)(Lys) molecule annealed to the viral RNA at the primer binding site (PBS), but the structure of the initiation complex of reverse transcription remains controversial. Here, we performed in situ structural probing, as well as in vitro structural and functional studies, of the initiation complexes formed by highly divergent isolates (MAL and NL4.3/HXB2). Our results show that the structure of the initiation complex is not conserved. In MAL, and according to sequence analysis in 14% of HIV-1 isolates, formation of the initiation complex is accompanied by complex rearrangements of the viral RNA, and extensive interactions with tRNA(3)(Lys) are required for efficient initiation of reverse transcription. In NL4.3, HXB2, and most isolates, tRNA(3)(Lys) annealing minimally affects the viral RNA structure and no interaction outside the PBS is required for optimal initiation of reverse transcription. We suggest that in MAL, extensive interactions with tRNA(3)(Lys) are required to drive the structural rearrangements generating the structural elements ultimately recognized by reverse transcriptase. In NL4.3 and HXB2, these elements are already present in the viral RNA prior to tRNA(3)(Lys) annealing, thus explaining that extensive interactions with the primer are not required. Interestingly, such interactions are required in HXB2 mutants designed to use a non-cognate tRNA as primer (tRNA(His)). In the latter case, the extended interactions are required to counteract a negative contribution associate with the alternate primer.

Primer unblocking by HIV-1 reverse transcriptase and resistance to nucleoside RT inhibitors (NRTIs).

During zidovudine and stavudine treatment, HIV-1 selects several mutations (thymidine-associated mutations, TAMs) in the reverse transcriptase gene that confer high- and moderate-levels of resistance, respectively, to these nucleoside reverse transcriptase inhibitors (NRTIs). The mechanism of the resistance provided by these mutations has long remained elusive. However, recent data showed that ATP-phosphorolysis, a reaction analogous to pyrophosphorolysis (the reverse of the nucleotide incorporation reaction) in which ATP is the pyrophosphate donor, is central to this mechanism by allowing repair of the chain-terminated primer. A detailed structural and mechanistic model accounting for the specificity of the ATP-phosphorolysis and its inhibition by the next complementary nucleotide is now available. In the context of multiresistant viruses, the TAMs are also associated with resistance to abacavir, and to a lesser extent to didanisone, zalcitabine and tenofovir. When associated with the TAMs, a dipeptide insertion in the fingers of reverse transcriptase increases the ATP-phosphorolysis of most chain terminators, stressing the increasing importance of this mechanism. However, some non-nucleoside reverse transcriptase inhibitors (NNRTIs) inhibit this process. In addition, point mutations conferring resistance to NNRTIs (Y181C and L100I) or NRTIs (K65R, L74V, and M184V) partially resensitize the resistant viruses to AZT by inhibiting ATP-phosphorolysis. These findings allow rationalizing the beneficial effects of some drug combinations and should contribute to improve drug cocktails. The development of NRTIs that would not allow the ATP-mediated excision to take place should prove beneficial for future treatments, even though high-level resistance to multiple NRTIs can ultimately develop in the absence of any significant primer unblocking.

Structure-function relationships of the initiation complex of HIV-1 reverse transcription: the case of mutant viruses using tRNA(His) as primer.

Reverse transcription of HIV-1 RNA is initiated from the 3' end of a tRNA3Lys molecule annealed to the primer binding site (PBS). An additional interaction between the anticodon loop of tRNA3Lys and a viral A-rich loop is required for efficient initiation of reverse transcription of the HIV-1 MAL isolate. In the HIV-1 HXB2 isolate, simultaneous mutations of the PBS and the A-rich loop (mutant His-AC), but not of the PBS alone (mutant His) allows the virus to stably utilize tRNA(His) as primer. However, mutant His-AC selects additional mutations during cell culture, generating successively His-AC-GAC and His-AC-AT-GAC. Here, we wanted to establish direct relationships between the evolution of these mutants in cell culture, their efficiency in initiating reverse transcription and the structure of the primer/template complexes in vitro. The initiation of reverse transcription of His and His-AC RNAs was dramatically reduced. However, His-AC-GAC RNA, which incorporated three adaptative point mutations, was reverse transcribed more efficiently than the wild type RNA. Incorporation of two additional mutations decreased the efficiency of the initiation of reverse transcription, which remained at the wild type level. Structural probing showed that even though both His-AC and His-AC-GAC RNAs can potentially interact with the anticodon loop of tRNA(His), only the latter template formed a stable interaction. Thus, our results showed that the selection of adaptative mutations by HIV-1 mutants utilizing tRNA(His) as primer was initially dictated by the efficiency of the initiation of reverse transcription, which relied on the existence of a stable interaction between the mutated A-rich loop and the anticodon loop of tRNA(His).

Does the HIV-1 primer activation signal interact with tRNA3(Lys) during the initiation of reverse transcription?

Reverse transcription of HIV-1 RNA is primed by a tRNA3(Lys) molecule bound at the primer binding site (PBS). Complex intermolecular interactions were proposed between tRNA3(Lys) and the RNA of the HIV-1 Mal isolate. Recently, an alternative interaction was proposed between the TPsiC stem of tRNA3(Lys) and a primer activation signal (PAS) of the Lai and Hxb2 RNAs, suggesting major structural variations in the reverse transcription complex of different HIV-1 strains. Here, we analyzed mutants of the Hxb2 RNA that prevent the interaction between the PAS and tRNA3(Lys) or/and a complementary sequence in the viral RNA. We compared the kinetics of reverse transcription of the wild type and mutant Hxb2 RNAs, using either tRNA3(Lys) or an 18mer oligoribonucleotide complementary to the PBS, which cannot interact with the PAS, as primers. We also used chemical probing to test the structure of the mutant and wild type RNAs, as well as the complex formed between the later RNA and tRNA3(Lys). These experiments, together with the analysis of long term replication data of mutant viruses obtained by C. Morrow and coworkers (Birmingham, USA) that use alternate tRNAs as primers, strongly suggest that the interaction between the Hxb2 PAS and tRNA3(Lys) does not exist. Instead, the effects of the vRNA mutations on reverse transcription seem to be linked to incorrect folding of the mutant RNAs.

Direct and indirect contributions of RNA secondary structure elements to the initiation of HIV-1 reverse transcription.

Initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription requires specific recognition between the viral RNA (vRNA), tRNA(3)(Lys), which acts as primer, and reverse transcriptase (RT). The specificity of this ternary complex is mediated by intricate interactions between the HIV-1 RNA and tRNA(3)(Lys). Here, we compared the relative importance of the secondary structure elements of this complex in the initiation process. To this aim, we used the previously published three-dimensional model of the initiation complex to rationally introduce a series of deletions and substitutions in the vRNA. When necessary, we used chemical probing to check the structure of the tRNA(3)(Lys)-mutant vRNA complexes. For each of them, we measured the binding affinity of RT and the kinetics of initial extension of tRNA(3)(Lys) and of synthesis of the (-) strand strong stop DNA. Our results were overall in keeping with the three-dimensional model of the initiation complex. Surprisingly, we found that disruption of the intermolecular template-primer interactions, which are not directly recognized by RT, more severely affected reverse transcription than deletions or disruption of one of the intramolecular helices to which RT directly binds. Perturbations of the highly constrained junction between the intermolecular helix formed by the primer binding site and the 3' end of tRNA(3)(Lys) and the helix immediately upstream also had dramatic effects on the initiation of reverse transcription. Taken together, our results demonstrate the overwhelming importance of the overall three-dimensional structure of the initiation complex and identify structural elements that constitute promising targets for anti-initiation-specific drugs.