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.

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.

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.

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.

Viral interactions with the host-cell cytoskeleton: the role of retroviral proteases.

A surprisingly large number of animal viruses interact with cytoskeletal elements inside infected cells at different stages of replication. For example, a viral protease is activated during assembly of murine leukemia viruses at the cell surface, which causes both the morphological conversion of 'immature' to 'mature' particles and a drastic reduction of the actin stress fiber network.

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.

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.

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.

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.

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

HIV protease inhibitors: their anti-HIV activity and potential role in treatment.

Researchers are continuing to uncover important new information about the life cycle of the human immunodeficiency virus (HIV) and are further elucidating the products of the viral genome in their efforts to develop anti-retroviral therapies that are more effective and safer than those currently available. Although much attention has been focused on inhibiting proviral DNA synthesis with nucleoside analogues, other classes of agents are being explored that may enable clinicians to inhibit HIV transmission in vivo by targeting other stages of the viral life cycle. Among the novel approaches under investigation is inhibition of HIV protease, the proteolytic enzyme that is responsible for the cleavage of large precursor proteins in the budding virion. Preclinical studies indicate that protease inhibitors prevent the maturation of immature viral particles into infectious virions and thereby limit the spread of HIV in cell cultures. Laboratory data have also shown that these agents have good toxic-effect profiles, and recent improvements have resulted in compounds that are more bioavailable. As phase I studies of this modality are undertaken, clinical researchers will be carefully monitoring them to determine whether these favorable in vitro characteristics of protease inhibitors will be evident in the clinical arena as well.

Morphogenesis of recombinant HIV-2 gag core particles.

The gag-pol coding region of the HIV-2BEN genome was expressed in CV-1 cells infected with four recombinant vaccinia viruses (VV). These recombinant VV encoded either the whole gag-pol region or the gag gene including the protease-coding region of the pol gene or the gag gene truncated at its 3'-end or only the pol gene. The HIV-2BEN gag precursor p55, its mature cleavage products p24 and p17 as well as the pol reverse transcriptase (RT) p66 were detected in VV-infected CV-1 cells. The p55 and two intermediate cleavage products p40 and p35 were myristilated. Comparison to lysates of permanently HIV-2BEN-infected Molt 4 clone 8 cells revealed that several additional gag and pol proteins were present in the VV-infected CV-1 cells. Deletion of the gag and pol overlapping region coding for the viral protease prevented cleavage of the recombinant gag precursor. Electron microscopy of VV-infected CV-1 cells revealed budding structures and immature as well as mature retroviral particles formed by the recombinant gag proteins. Striking differences in the ability to form complete particles were observed between the different recombinant VV. Expression of the truncated gag gene led to the formation of budding structures, but completely budded circular particles were not detectable. Such particles were produced by expression of the whole gag gene and the protease. Mature virions with an internal core structure were only detected in VVgagpol-infected cells. From these findings we conclude that the 3'-end of the gag gene coding for the p16 protein is essential for the formation of complete HIV-2 particles and that the pol proteins support the assembly of the viral core.

Molecular bases for the anti-HIV-1 effect of NO. Commentary.

In infected human cells, nitric oxide (NO) has been shown to inhibit the replication of the human immunodeficiency virus-1 (HIV-1), the etiological agent of AIDS. Evidence suggests that NO may regulate HIV-1 replication by affecting the sulphydryl redox state. In this respect, it has been very recently demonstrated that NO-donors inactivate the HIV-1-encoded protease and reverse transcriptase in vitro. Further viral and host NO targets may be envisaged. Although no data are available on the anti-HIV-1 effect of NO in vivo, NO-releasing drugs, clinically used in the treatment of cardiovascular disorders, may represent a novel class of molecules for decreasing virus replication. Here, the possible molecular bases for the anti-HIV-1 effect of NO are discussed.

Potential role of the viral protease in human immunodeficiency virus type 1 associated pathogenesis.

Infection with the human immunodeficiency virus type 1 (HIV-1) results in a variety of pathological changes culminating in the acquired immune deficiency syndrome (AIDS). While most of these changes can readily be accounted for either by direct effects of HIV-1 on the immune system or by indirect effects of secondary infectious agents as a result of faulty immune surveillance, the direct cause for a number of disease states, including some neuropathies, myopathies, nephropathy, thrombocytopenia, wasting syndromes and increased incidence of cancers (primarily lymphoma) has remained an enigma. We have recently shown that the HIV-1 protease, a viral encoded enzyme necessary for virus maturation and infectivity, can cleave a variety of host cell cytoskeletal proteins in vitro. Potential substrates for the HIV-1 protease are found in all of the cell types affected in these unexplained diseases. Recent proposals suggest that elements of the cytoskeleton may play an important role in the regulation of large scale genetic regulation. We propose that some of the degenerative changes associated with infection by HIV-1 are a direct consequence of cleavage of host cell cytoskeletal proteins, which in turn may be responsible for the increased incidence of cancer in HIV-1 infected individuals as a result of the perturbation of the regulation of gene expression by cytoskeletal components.

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.

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.