HIV protease cleaves the antiviral m6A reader protein YTHDF3 in the viral particle.

N6-methyladenosine (m6A) is the most abundant HIV RNA modification but the interplay between the m6A reader protein YTHDF3 and HIV replication is not well understood. We found that knockout of YTHDF3 in human CD4+ T-cells increases infection supporting the role of YTHDF3 as a restriction factor. Overexpression of the YTHDF3 protein in the producer cells reduces the infectivity of the newly produced viruses. YTHDF3 proteins are incorporated into HIV particles in a nucleocapsid-dependent manner permitting the m6A reader protein to limit infection in the new target cell at the step of reverse transcription. Importantly, HIV protease cleaves the virion-incorporated full-length YTHDF3 protein, a process which is blocked by HIV protease inhibitors used to treat HIV infected patients. Mass-spectrometry confirmed the proteolytic processing of YTHDF3 in the virion. Thus, HIV protease cleaves the virion-encapsidated host m6A effector protein in addition to the viral polyproteins to ensure optimal infectivity of the mature virion.

Gag-Pol Transframe Domain p6* Is Essential for HIV-1 Protease-Mediated Virus Maturation.

HIV-1 protease (PR) is encoded by pol, which is initially translated as a Pr160gag-pol polyprotein by a ribosomal frameshift event. Within Gag-Pol, truncated p6gag is replaced by a transframe domain (referred to as p6* or p6pol) located directly upstream of PR. p6* has been proposed as playing a role in modulating PR activation. Overlapping reading frames between p6* and p6gag present a challenge to researchers using genetic approaches to studying p6* biological functions. To determine the role of p6* in PR activation without affecting the gag reading frame, we constructed a series of Gag/Gag-Pol expression vectors by duplicating PR with or without p6* between PR pairs, and observed that PR duplication eliminated virus production due to significant Gag cleavage enhancement. This effect was mitigated when p6* was placed between the two PRs. Further, Gag cleavage enhancement was markedly reduced when either one of the two PRs was mutationally inactivated. Additional reduction in Gag cleavage efficiency was noted following the removal of p6* from between the two PRs. The insertion of a NC domain (wild-type or mutant) directly upstream of PR or p6*PR did not significantly improve Gag processing efficiency. With the exception of those containing p6* directly upstream of an active PR, all constructs were either noninfectious or weakly infectious. Our results suggest that (a) p6* is essential for triggering PR activation, (b) p6* has a role in preventing premature virus processing, and

The triple threat of HIV-1 protease inhibitors.

Newly released human immunodeficiency virus type 1 (HIV-1) particles obligatorily undergo a maturation process to become infectious. The HIV-1 protease (PR) initiates this step, catalyzing the cleavage of the Gag and Gag-Pro-Pol structural polyproteins. Proper organization of the mature virus core requires that cleavage of these polyprotein substrates proceeds in a highly regulated, specific series of events. The vital role the HIV-1 PR plays in the viral life cycle has made it an extremely attractive target for inhibition and has accordingly fostered the development of a number of highly potent substrate-analog inhibitors. Though the PR inhibitors (PIs) inhibit only the HIV-1 PR, their effects manifest at multiple different stages in the life cycle due to the critical importance of the PR in preparing the virus for these subsequent events. Effectively, PIs masquerade as entry inhibitors, reverse transcription inhibitors, and potentially even inhibitors of post-reverse transcription steps. In this chapter, we review the triple threat of PIs: the intermolecular cooperativity in the form of a cooperative dose-response for inhibition in which the apparent potency increases with increasing inhibition; the pleiotropic effects of HIV-1 PR inhibition on entry, reverse transcription, and post-reverse transcription steps; and their potency as transition state analogs that have the potential for further improvement that could lead to an inability of the virus to evolve resistance in the context of single drug therapy.

Casp8p41 generated by HIV protease kills CD4 T cells through direct Bak activation.

Previous studies have shown that human immunodeficiency virus (HIV) protease cleaves procaspase 8 to a fragment, termed Casp8p41, that lacks caspase activity but nonetheless contributes to T cell apoptosis. Herein, we show that Casp8p41 contains a domain that interacts with the BH3-binding groove of pro-apoptotic Bak to cause Bak oligomerization, Bak-mediated membrane permeabilization, and cell death. Levels of active Bak are higher in HIV-infected T cells that express Casp8p41. Conversely, targeted mutations in the Bak-interacting domain diminish Bak binding and Casp8p41-mediated cell death. Similar mutations in procaspase 8 impair the ability of HIV to kill infected T cells. These observations support a novel paradigm in which HIV converts a normal cellular constituent into a direct activator that functions like a BH3-only protein.

Evolution of the human immunodeficiency virus type 1 protease: effects on viral replication capacity and protease robustness.

The rapid spread of human immunodeficiency virus type 1 (HIV-1) in humans has been accompanied by continuous extensive genetic diversification of the virus. The aim of this study was to investigate the impact of HIV-1 diversification on HIV-1 replication capacity (RC) and mutational robustness. Thirty-three HIV-1 protease sequences were amplified from three groups of viruses: two naïve sample groups isolated 15 years apart plus a third group of protease inhibitor-(PI) resistant samples. The amplified proteases were recombined with an HXB2 infectious clone and RC was determined in MT-4 cells. RC was also measured in these three groups after random mutagenesis in vitro using error-prone PCR. No significant RC differences were observed between recombinant viruses from either early or recent naïve isolates (P = 0.5729), even though the proteases from the recent isolates had significantly lower sequence conservation scores compared with a subtype B ancestral sequence (P<0.0001). Randomly mutated recombinant viruses from the three groups exhibited significantly lower RC values than the corresponding wild-type viruses (P<0.0001). There was no significant difference regarding viral infectivity reduction between viruses carrying randomly mutated naïve proteases from early or recent sample isolates (P = 0.8035). Interestingly, a significantly greater loss of RC was observed in the PI-resistant protease group (P = 0.0400). These results demonstrate that protease sequence diversification has not affected HIV-1 RC or protease robustness and indicate that proteases carrying PI resistance substitutions are less robust than naïve proteases.

[What's going on post-budding?].

In general, the retrovirus particles become infectious on post-budding with cleavages of structural protein Gag by viral protease. Protease defective mutants bud particles normally, but the particles are non-infectious and called donuts-like particle because of their morphology. The viral genomes inside the donuts-like particles form very fragile dimer, which are far different from those in wild-type particles. The ordered particle maturation process is essential for infectivity of virus, but its mechanism largely remains unclear. We have constructed HIV-1 Gag cleavage site mutants to enable the steady state observation of virion maturation steps, and precisely study Gag processing, RNA dimerization, virion morphology and infectivity. As results, we found that these process progressed synchronously, but each transition point did not coincide completely. The mutual relationship between viral protein and RNA maturation is discussed for a further understanding of the retroviral life cycle.

[Establishment and application of a high-throughput screening assay for premature activation of HIV-1 precursors].

Strict regulation of HIV-1 PR function is critical for efficient production of mature viral particles. During viral protein expression and viral assembly, HIV-1 PR located within Gag-Pol precursor must be inactive to prevent premature cytoplasmic processing of the viral Gag and Gag-Pol precursors. Premature activation of HIV-1 precursors leads to major defects in viral assembly and production of viral particles. A cell-level premature activation of HIV-1 precursors assay using bioluminescence resonance energy transfer (BRET) was established. Three thousand compounds were screened to evaluate this assay. The results showed that the assay is sensitive, specific and stable (Z' factor is 0.905).

The HIV-1 protease substitution K55R: a protease-inhibitor-associated substitution involved in restoring viral replication.

OBJECTIVES: The identification and in vitro characterization of novel protease mutations strongly associated with known protease resistance mutations. METHODS: The association between pairs of protease amino acid substitutions was identified using a database of protease sequences derived from protease inhibitor-experienced patients (n = 803). In vitro characterization included drug susceptibility and viral replication studies performed on recombinant viruses harbouring site-directed mutations. RESULTS: The K55R mutation, which is not a natural polymorphism, was identified to be strongly associated with protease mutations M46I/L and to a lesser extent L24I, I54V and V82A/T/S/F. In vitro characterization of the K55R substitution indicated a primary role for this substitution in increasing replicative capacity in the presence of specific protease mutations. CONCLUSIONS: The K55R mutation is a secondary drug resistance mutation that can improve viral replication capacity in the presence of other primary protease mutations.

Insights into amprenavir resistance in E35D HIV-1 protease mutation from molecular dynamics and binding free-energy calculations.

Drug resistance is a very important factor contributing to the failure of current HIV therapies. The ability to understand the resistance mechanism of HIV-protease mutants may be useful in developing more effective and longer lasting treatment regimens. In this paper, we report the first computational study of the clinically relevant E35D mutation of HIV-1 protease in its unbound conformation and complexed with the clinical inhibitor amprenavir and a sample substrate (Thr-Ile-Met-Met-Gln-Arg). Our data, collected from 10 ns molecular-dynamics simulations, show that the E35D mutation results in an increased flexibility of the flaps, thereby affecting the conformational equilibrium between the closed and semi-open conformations of the free protease. The E35D mutation also causes a significant reduction of the calculated binding free energies both for substrate and amprenavir, thus giving a plausible explanation for its ability to increase the level of resistance. One possible explanation for the emergence of this mutation, despite its unfavorable effect on substrate affinity, might be the role of E35D as an escape mutation, which favors escape from the immune system in addition to conferring drug resistance.

Amino acid insertions at position 35 of HIV-1 protease interfere with virus replication without modifying antiviral drug susceptibility.

Among 1330 patients undergoing highly active antiretroviral therapy (HAART), 3 showed 1 or 2 amino acid (aa) insertions at position 35 of the HIV-1 protease gene. Protease genes containing aa insertions, either in the presence (ins35G+res.muts, ins35TN+res.muts) or absence (ins35G, ins35TN) of other resistance mutations, were introduced into the wild-type HIV-1 strain NL4-3. The introduction of ins35G and ins35TN in the wild-type protease confirmed that these mutations were per se not responsible of decreased drug susceptibility. The replication rate of mutant recombinant viruses was determined by HIV RNA quantification in supernatants of cell cultures in comparison with a recombinant HIV-1 strain with wild-type protease. Recombinant ins35G and ins35TN HIV-1 strains did not display increased resistance to currently used protease inhibitors (PIs). Comparison of ins35TN+res.muts and ins35G+res.muts with respect to the corresponding recombinant rescue mutants showed that ins35TN decreased the replication rate of the PI-resistant strain, while ins35G had a protective effect.

Diversification and specialization of HIV protease function during in vitro evolution.

Our goal is to understand how enzymes adapt to utilize novel substrates. We and others have shown that directed evolution tends to generate enzyme variants with broadened substrate specificity. Broad-specificity enzymes are generally deleterious to living cells, so this observed trend might be an artifact of the most commonly employed high throughput screens. Here, we demonstrate a more natural and effective screening strategy for directed evolution. The gene encoding model enzyme HIV protease was randomly mutated, and the resulting library was expressed in Escherichia coli cells to eliminate cytotoxic broad-specificity variants. The surviving variants were screened for clones with activity against a reporter enzyme. The wild-type human immunodeficiency virus type I protease (HIV PR) is cytotoxic and exhibits no detectable activity in reactions with beta-galactosidase (BGAL). In contrast, the selected variants were nontoxic and exhibited greater activity and specificity against BGAL than did the wild-type HIV PR in reactions with any substrate. A single round of whole gene random mutagenesis and conventional high-throughput screening does not usually effect complete inversions of substrate specificity. This suggests that a combination of positive and purifying selection engenders more rapid adaptation than positive selection alone.

Processing sites in the human immunodeficiency virus type 1 (HIV-1) Gag-Pro-Pol precursor are cleaved by the viral protease at different rates.

We have examined the kinetics of processing of the HIV-1 Gag-Pro-Pol precursor in an in vitro assay with mature protease added in trans. The processing sites were cleaved at different rates to produce distinct intermediates. The initial cleavage occurred at the p2/NC site. Intermediate cleavages occurred at similar rates at the MA/CA and RT/IN sites, and to a lesser extent at sites upstream of RT. Late cleavages occurred at the sites flanking the protease (PR) domain, suggesting sequestering of these sites. We observed paired intermediates indicative of half- cleavage of RT/RH site, suggesting that the RT domain in Gag-Pro-Pol was in a dimeric form under these assay conditions. These results clarify our understanding of the processing kinetics of the Gag-Pro-Pol precursor and suggest regulated cleavage. Our results further suggest that early dimerization of the PR and RT domains may serve as a regulatory element to influence the kinetics of processing within the Pol domain.

Ordered processing of the human immunodeficiency virus type 1 GagPol precursor is influenced by the context of the embedded viral protease.

Ordered and accurate processing of the human immunodeficiency virus type 1 (HIV-1) GagPol polyprotein precursor by a virally encoded protease is an indispensable step in the appropriate assembly of infectious viral particles. The HIV-1 protease (PR) is a 99-amino-acid enzyme that is translated as part of the GagPol precursor. Previously, we have demonstrated that the initial events in precursor processing are accomplished by the PR domain within GagPol in cis, before it is released from the polyprotein. Despite the critical role that ordered processing of the precursor plays in viral replication, the forces that define the order of cleavage remain poorly understood. Using an in vitro assay in which the full-length HIV-1 GagPol is processed by the embedded PR, we examined the effect of PR context (embedded within GagPol versus the mature 99-amino-acid enzyme) on precursor processing. Our data demonstrate that the PR domain within GagPol is constrained in its ability to cleave some of the processing sites in the precursor. Further, we find that this constraint is dependent upon the presence of a proline as the initial amino acid in the embedded PR; substitution of an alanine at this position produces enhanced cleavage at additional sites when the precursor is processed by the embedded, but not the mature, PR. Overall, our data support a model in which the selection of processing sites and the order of precursor processing are defined, at least in part, by the structure of GagPol itself.

The paradox of HIV nef function: resolution of the contradictions.

The function of nef, an accessory protein of HIV, has been highly controversial. Careful studies by respected investigators have ascribed diametrically opposed functions to nef with some groups claiming nef increases replication and viral infectivity while others argue nef inhibits replication and reduces infectivity. Replicative homeostasis resolves this superficially irreconcilable paradox, and indicates both positions could be true, if the exact function of nef is dependent on whether the nef tested is wild-type or variant with respect to the polymerase. The basis of resolution of the enigma posed by nef function is fundamental to and understanding of viral pathobiology.

Protein evolution in viral quasispecies under selective pressure: a thermodynamic and phylogenetic analysis.

The evolution of RNA viruses under antiviral pressure is characterized by high mutation rates and strong selective forces that induce extremely rapid changes of protein sequences. This makes the course of molecular evolution directly observable on time scales of months. Here we study the interplay between selection for drug resistance and selection for thermodynamic stability in the protease (PR) and the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) clones extracted from two patients with complex treatment histories. This analysis shows that folding thermodynamic properties may fluctuate very strongly in the course of quasispecies evolution under selective pressure. For the first case, our data suggest that folding efficiency of the RT is sacrificed at the advantage of drug resistance, while the corresponding PR seems to undergo selection for thermodynamic stability in the absence of substitutions associated to resistance. The PR of the second case is not submitted to antiviral pressure during the period analyzed and seems to initiate random fluctuations that lead to the accidental increase of its folding efficiency. In summary, joint consideration of sequence evolution and thermodynamic parameters can represent a more comprehensive approach for the study of the evolution of RNA viruses.

Structural requirements for recognition of the human immunodeficiency virus type 1 core during host restriction in owl monkey cells.

Human immunodeficiency virus type 1 (HIV-1) infection of simian cells is restricted at an early postentry step by host factors whose mechanism of action is unclear. These factors target the viral capsid protein (CA) and attenuate reverse transcription, suggesting that they bind to the HIV-1 core and interfere with its uncoating. To identify the relevant binding determinants in the capsid, we tested the capacity of viruses containing Gag cleavage site mutations and amino acid substitutions in CA to inhibit restriction of a wild type HIV-1 reporter virus in owl monkey cells. The results demonstrated that a stable, polymeric capsid and a correctly folded amino-terminal CA subunit interface are essential for saturation of host restriction in target cells by HIV-1 cores. We conclude that the owl monkey cellular restriction machinery recognizes a polymeric array of CA molecules, most likely via direct engagement of the HIV-1 capsid in target cells prior to uncoating.

Combating susceptibility to drug resistance: lessons from HIV-1 protease.

Drug resistance is a major obstacle in modern medicine. However, resistance is rarely considered in drug development and may inadvertently be facilitated, as many designed inhibitors contact residues that can mutate to confer resistance, without significantly impairing function. Contemporary drug design often ignores the detailed atomic basis for function and primarily focuses on disrupting the target's activity, which is necessary but not sufficient for developing a robust drug. In this study, we examine the impact of drug-resistant mutations in HIV-1 protease on substrate recognition and demonstrate that most primary active site mutations do not extensively contact substrates, but are critical to inhibitor binding. We propose a general, structure-based strategy to reduce the probability of drug resistance by designing inhibitors that interact only with those residues that are essential for function.

Variability at human immunodeficiency virus type 1 subtype C protease cleavage sites: an indication of viral fitness?

Naturally occurring polymorphisms in the protease of human immunodeficiency virus type 1 (HIV-1) subtype C would be expected to lead to adaptive (compensatory) changes in protease cleavage sites. To test this hypothesis, we examined the prevalences and patterns of cleavage site polymorphisms in the Gag, Gag-Pol, and Nef cleavage sites of C compared to those in non-C subtypes. Codon-based maximum-likelihood methods were used to assess the natural selection and evolutionary history of individual cleavage sites. Seven cleavage sites (p17/p24, p24/p2, NC/p1, NC/TFP, PR/RT, RT/p66, and p66/IN) were well conserved over time and in all HIV-1 subtypes. One site (p1/p6(gag)) exhibited moderate variation, and four sites (p2/NC, TFP/p6(pol), p6(pol)/PR, and Nef) were highly variable, both within and between subtypes. Three of the variable sites are known to be major determinants of polyprotein processing and virion production. P2/NC controls the rate and order of cleavage, p6(gag) is an important phosphoprotein required for virion release, and TFP/p6(pol), a novel cleavage site in the transframe domain, influences the specificity of Gag-Pol processing and the activation of protease. Overall, 58.3% of the 12 HIV-1 cleavage sites were significantly more diverse in C than in B viruses. When analyzed as a single concatenated fragment of 360 bp, 96.0% of group M cleavage site sequences fell into subtype-specific phylogenetic clusters, suggesting that they coevolved with the virus. Natural variation at C cleavage sites may play an important role, not only in regulation of the viral cycle but also in disease progression and response to therapy.

Human immunodeficiency virus type 1 protease regulation of tat activity is essential for efficient reverse transcription and replication.

The human immunodeficiency virus type 1 (HIV-1) Tat protein enhances reverse transcription, but it is not known whether Tat acts directly on the reverse transcription complex or through indirect mechanisms. Since processing of Tat by HIV protease (PR) might mask its presence and, at least in part, explain this lack of data, we asked whether Tat can be cleaved by PR. We used a rabbit reticulocyte lysate (RRL) system to make Tat and PR. HIV-1 PR is expressed as a Gag-Pol fusion protein, and a PR-inactivated Gag-Pol is also expressed as a control. We showed that Tat is specifically cleaved in the presence of PR, producing a protein of approximately 5 kDa. This result suggested that the cleavage site was located in or near the Tat basic domain (amino acids 49 to 57), which we have previously shown to be important in reverse transcription. We created a panel of alanine-scanning mutations from amino acids 45 to 54 in Tat and evaluated functional parameters, including transactivation, reverse transcription, and cleavage by HIV-1 PR. We showed that amino acids 49 to 52 (RKKR) are absolutely required for Tat function in reverse transcription, that mutation of this domain blocks cleavage by HIV-1 PR, and that other pairwise mutations in this region modulate reverse transcription and proteolysis in strikingly similar degrees. Mutation of Tat Y47G48 to AA also down-regulated Tat-stimulated reverse transcription but had little effect on transactivation or proteolysis by HIV PR, suggesting that Y47 is critical for reverse transcription. We altered the tat gene of the laboratory strain NL4-3 to Y47D and Y47N so that overlapping reading frames were not affected and showed that Y47D greatly diminished virus replication and conveyed a reverse transcription defect. We hypothesize that a novel, cleaved form of Tat is present in the virion and that it requires Y47 for its role in support of efficient reverse transcription.

Selection of high-level resistance to human immunodeficiency virus type 1 protease inhibitors.

Protease inhibitors represent some of the most potent agents available for therapeutic strategies designed to inhibit human immunodeficiency virus type 1 (HIV-1) replication. Under certain circumstances the virus develops resistance to the inhibitor, thereby negating the benefits of this therapy. We have carried out selections for high-level resistance to each of three protease inhibitors (indinavir, ritonavir, and saquinavir) in cell culture. Mutations accumulated over most of the course of the increasing selective pressure. There was significant overlap in the identity of the mutations selected with the different inhibitors, and this gave rise to high levels of cross-resistance. Virus particles from the resistant variants all showed defects in processing at the NC/p1 protease cleavage site in Gag. Selections with pairs of inhibitors yielded similar patterns of resistance mutations. A virus that could replicate at near-toxic levels of the three protease inhibitors combined was selected. The pro sequence of this virus was similar to that of the viruses that had been selected for high-level resistance to each of the drugs singly. Finally, a molecular clone carrying the eight most common resistance mutations seen in these selections was characterized. The sequence of this virus was relatively stable during selection for revertants in spite of displaying poor processing at the NC/p1 site and having significantly reduced fitness. These results reveal patterns of drug resistance that extend to near the limits of attainable selective pressure with these inhibitors and confirm the patterns of cross-resistance for these three inhibitors and the attenuation of virion protein processing and fitness that accompanies high-level resistance.