HIV-1 protease catalytic efficiency effects caused by random single amino acid substitutions

Protein evolution has occurred by successive fixation of individual mutations. The probability of fixation depends on the fitness of the mutation, and the arising variant can be deleterious, neutral, or beneficial. Despite its relevance, only few studies have estimated the distribution of fitness effects caused by random single mutations on protein function. The human immunodeficiency virus type 1 (HIV-1) protease was chosen as a model protein to quantify protein's tolerability to random single mutations. After determining the enzymatic activity of 107 single random mutants, we found that 86% of single mutations were deleterious for the enzyme catalytic efficiency and 54% lethal. Only 2% of the mutations significantly increased the catalytic efficiency of the enzyme. These data demonstrate the vulnerability of HIV-1 protease to single random mutations. When a second random mutagenesis library was constructed from an HIV-1 protease carrying a highly deleterious single mutation (D30N), a higher proportion of mutations with neutral or beneficial effect were found, 26% and 9%, respectively. Importantly, antagonist epistasis was observed between deleterious mutations. In particular, the mutation N88D, lethal for the wild-type protease, restored the wild-type catalytic efficiency when combined with the highly deleterious mutation D30N. The low tolerability to single random substitutions shown here for the wild-type HIV-1 protease contrasts with its in vivo ability to generate an adaptive variation. Thus, the antagonist epistasis between deleterious or lethal mutations may be responsible for increasing the protein mutational robustness and evolvability.     

Demographic processes affect HIV-1 evolution in primary infection before the onset of selective processes

HIV-1 transmission and viral evolution in the first year of infection were studied in 11 individuals representing four transmitter-recipient pairs and three independent seroconverters. Nine of these individuals were enrolled during acute infection; all were men who have sex with men (MSM) infected with HIV-1 subtype B. A total of 475 nearly full-length HIV-1 genome sequences were generated, representing on average 10 genomes per specimen at 2 to 12 visits over the first year of infection. Single founding variants with nearly homogeneous viral populations were detected in eight of the nine individuals who were enrolled during acute HIV-1 infection. Restriction to a single founder variant was not due to a lack of diversity in the transmitter as homogeneous populations were found in recipients from transmitters with chronic infection. Mutational patterns indicative of rapid viral population growth dominated during the first 5 weeks of infection and included a slight contraction of viral genetic diversity over the first 20 to 40 days. Subsequently, selection dominated, most markedly in env and nef. Mutants were detected in the first week and became consensus as early as day 21 after the onset of symptoms of primary HIV infection. We found multiple indications of cytotoxic T lymphocyte (CTL) escape mutations while reversions appeared limited. Putative escape mutations were often rapidly replaced with mutually exclusive mutations nearby, indicating the existence of a maturational escape process, possibly in adaptation to viral fitness constraints or to immune responses against new variants. We showed that establishment of HIV-1 infection is likely due to a biological mechanism that restricts transmission rather than to early adaptive evolution during acute infection. Furthermore, the diversity of HIV strains coupled with complex and individual-specific patterns of CTL escape did not reveal shared sequence characteristics of acute infection that could be harnessed for vaccine design.     

Human immunodeficiency virus (HIV) vaccine trials: a novel assay for differential diagnosis of HIV infections in the face of vaccine-generated antibodies

All current human immunodeficiency virus (HIV) vaccine candidates contain multiple viral components and elicit antibodies that react positively in licensed HIV diagnostic tests, which contain similar viral products. Thus, vaccine trial participants could be falsely diagnosed as infected with HIV. Additionally, uninfected, seropositive vaccinees may encounter long-term social and economic harms. Moreover, this also interferes with early detection of true HIV infections during preventive HIV vaccine trials. An HIV-seropositive test result among uninfected vaccine trial participants is a major public health concern for volunteers who want to participate in future HIV vaccine trials. Based on the increased number of HIV vaccines being tested globally, it is essential to differentiate vaccine- from virus-induced antibodies. Using a whole-HIV-genome phage display library, we identified conserved sequences in Env-gp41 and Gag-p6 which are recognized soon after infection, do not contain protective epitopes, and are not part of most current HIV vaccines. We established a new HIV serodetection assay based on these peptides. To date, this assay, termed HIV-SELECTEST, demonstrates >99% specificity and sensitivity. Importantly, in testing of plasma samples from multiple HIV vaccine trials, uninfected trial participants scored negative, while all intercurrent infections were detected within 1 to 3 months of HIV infection. The new HIV-SELECTEST is a simple but robust diagnostic tool for easy implementation in HIV vaccine trials and blood banks worldwide.     

Reversion of CTL escape-variant immunodeficiency viruses in vivo

Engendering cytotoxic T-lymphocyte (CTL) responses is likely to be an important goal of HIV vaccines. However, CTLs select for viral variants that escape immune detection. Maintenance of such escape variants in human populations could pose an obstacle to HIV vaccine development. We first observed that escape mutations in a heterogeneous simian immunodeficiency virus (SIV) isolate were lost upon passage to new animals. We therefore infected macaques with a cloned SIV bearing escape mutations in three immunodominant CTL epitopes, and followed viral evolution after infection. Here we show that each mutant epitope sequence continued to evolve in vivo, often re-establishing the original, CTL-susceptible sequence. We conclude that escape from CTL responses may exact a cost to viral fitness. In the absence of selective pressure upon transmission to new hosts, these original escape mutations can be lost. This suggests that some HIV CTL epitopes will be maintained in human populations.     

Human immunodeficiency virus type 1 coreceptor switching: V1/V2 gain-of-fitness mutations compensate for V3 loss-of-fitness mutations

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is mediated by the virus envelope binding to CD4 and the conformationally altered envelope subsequently binding to one of two chemokine receptors. HIV-1 envelope glycoprotein (gp120) has five variable loops, of which three (V1/V2 and V3) influence the binding of either CCR5 or CXCR4, the two primary coreceptors for virus entry. Minimal sequence changes in V3 are sufficient for changing coreceptor use from CCR5 to CXCR4 in some HIV-1 isolates, but more commonly additional mutations in V1/V2 are observed during coreceptor switching. We have modeled coreceptor switching by introducing most possible combinations of mutations in the variable loops that distinguish a previously identified group of CCR5- and CXCR4-using viruses. We found that V3 mutations entail high risk, ranging from major loss of entry fitness to lethality. Mutations in or near V1/V2 were able to compensate for the deleterious V3 mutations and may need to precede V3 mutations to permit virus survival. V1/V2 mutations in the absence of V3 mutations often increased the capacity of virus to utilize CCR5 but were unable to confer CXCR4 use. V3 mutations were thus necessary but not sufficient for coreceptor switching, and V1/V2 mutations were necessary for virus survival. HIV-1 envelope sequence evolution from CCR5 to CXCR4 use is constrained by relatively frequent lethal mutations, deep fitness valleys, and requirements to make the right amino acid substitution in the right place at the right time.     

Reversion and T Cell Escape Mutations Compensate the Fitness Loss of a CD8+ T Cell Escape Mutant in Their Cognate Transmitted/Founder Virus

Immune escape mutations that revert back to the consensus sequence frequently occur in newly HIV-1-infected individuals and have been thought to render the viruses more fit. However, their impact on viral fitness and their interaction with other immune escape mutations have not been evaluated in the background of their cognate transmitted/founder (T/F) viral genomes. To precisely determine the role of reversion mutations, we introduced reversion mutations alone or together with CD8⁺ T cell escape mutations in their unmodified cognate T/F viral genome and determined their impact on viral fitness in primary CD4⁺ T cells. Two reversion mutations, V247I and I64T, were identified in Gag and Tat, respectively, but neither had measurable effect on the fitness of their cognate T/F virus. The V247I and G248A mutations that were detected before and concurrently with the potent T cell escape mutation T242N, respectively, were selected by early T cell responses. The V247I or the G248A mutation alone partially restored the fitness loss caused by the T242N mutation. Together they could fully restore the fitness of the T242N mutant to the T/F level. These results demonstrate that the fitness loss caused by a T cell escape mutation could be compensated by preexisting or concurrent reversion and other T cell escape mutations. Our findings indicate that the overall viral fitness is modulated by the complex interplay among T cell escape, compensatory and reversion mutations to maintain the balance between immune escape and viral replication capacity.     

CTL responses of high functional avidity and broad variant cross-reactivity are associated with HIV control

Cytotoxic T lymphocyte (CTL) responses targeting specific HIV proteins, in particular Gag, have been associated with relative control of viral replication in vivo. However, Gag-specific CTL can also be detected in individuals who do not control the virus and it remains thus unclear how Gag-specific CTL may mediate the beneficial effects in some individuals but not in others. Here, we used a 10mer peptide set spanning HIV Gag-p24 to determine immunogen-specific T-cell responses and to assess functional properties including functional avidity and cross-reactivity in 25 HIV-1 controllers and 25 non-controllers without protective HLA class I alleles. Our data challenge the common belief that Gag-specific T cell responses dominate the virus-specific immunity exclusively in HIV-1 controllers as both groups mounted responses of comparable breadths and magnitudes against the p24 sequence. However, responses in controllers reacted to lower antigen concentrations and recognized more epitope variants than responses in non-controllers. These cross-sectional data, largely independent of particular HLA genetics and generated using direct ex-vivo samples thus identify T cell responses of high functional avidity and with broad variant reactivity as potential functional immune correlates of relative HIV control.     

Initial Fitness Recovery of HIV-1 Is Associated with Quasispecies Heterogeneity and Can Occur without Modifications in the Consensus Sequence

Background

Fitness recovery of HIV-1 “in vitro” was studied using viral clones that had their fitness decreased as a result of plaque-to-plaque passages.

Principal Findings

After ten large population passages, the viral populations showed an average increase of fitness, although with wide variations among clones. While 5 clones showed significant fitness increases, 3 clones showed increases that were only marginally significant (p<0.1), and 4 clones did not show any change. Fitness recovery was not accompanied by an increase in p24 production, but was associated with an increase in viral titer. Few mutations (an average of 2 mutations per genome) were detected in the consensus nucleotide sequence of the entire genome in all viral populations. Five of the populations did not fix any mutation, and three of them displayed marginally significant fitness increases, illustrating that fitness recovery can occur without detectable alterations of the consensus genomic sequence. The investigation of other possible viral factors associated with the initial steps of fitness recovery, showed that viral quasispecies heterogeneity increased between the initial clones and the passaged populations. A direct statistical correlation between viral heterogeneity and viral fitness was obtained.

Conclusions

Thus, the initial fitness recovery of debilitated HIV-1 clones was mediated by an increase in quasispecies heterogeneity. This observation, together with the invariance of the consensus sequence despite fitness increases demonstrates the relevance of quasispecies heterogeneity in the evolution of HIV-1 in cell culture.     

Can modulation of viral fitness represent a target for anti-HIV-1 strategies?

Prolonged use of anti-retroviral compounds in human immunodeficiency virus type 1 (HIV-1) infection selects for drug-resistant and often mutidrug-resistant viral variants. Drug-resistance mutations may also affect viral fitness. Interestingly, recent research has indicated that some of the unfit drug-resistant variants may be less pathogenic, suggesting that decreased viral fitness is beneficial for the host and may be driven by specific treatments during anti-HIV-1 infection. A second potential antiviral strategy starting with profound inteference with viral fitness aims at forcing viruses towards lethal mutagenesis (the so-called "error catastrophe"). This review summarizes the methods for addressing HIV-1 fitness in vitro and ex vivo, the current understanding of clinical implications of reduced HIV-1 fitness, and the potential use of anti-HIV-1 strategies aiming at modulating viral fitness. Finally, it is emphasized how the peculiar features of HIV-1 quasispecies (displaying two different forms of memory, a replicative and a non-replicative form) may sharply influence the design of future diagnostic methodologies for fitness analysis.     

Derivation of non-infectious envelope proteins from virions isolated from plasma negative for HIV antibodies

Natural membrane-bound HIV-1 envelope proteins (mHIVenv) could be used to produce an effective subunit vaccine against HIV infection, akin to effective vaccination against HBV infection using the hepatitis B surface antigen. The quaternary structure of mHIVenv is postulated to elicit broadly neutralizing antibodies protective against HIV-1 transmission. The founder virus transmitted to infected individuals during acute HIV-1 infection is genetically homogeneous and restricted to CCR5-tropic phenotype. Therefore, isolates of plasma-derived HIV-1 (PHIV) from infected blood donors while negative for antibodies to HIV proteins were selected for expansion in primary lymphocytes as an optimized cell substrate (OCS). Virions in the culture supernatants were purified by removing contaminating microvesicles using immunomagnetic beads coated with anti-CD45. Membrane cholesterol was extracted from purified virions with beta-cyclodextrin to permeabilize them and expel p24, RT and viral RNA, and permit protease-free Benzonase to hydrolyze the residual viral/host DNA/RNA without loss of gp120. The resultant mHIVenv, containing gp120 bound to native gp41 in immunoreactive form, was free from infectivity in vitro in co-cultures with OCS and in vivo after inoculating SCID-hu Thy/Liv mice. These data should help development of mHIVenv as a virally safe immunogen and enable preparation of polyclonal hyper-immune globulins for immunoprophylaxis against HIV-1 infection.     

Changes in both gp120 and gp41 can account for increased growth potential and expanded host range of human immunodeficiency virus type 1.

Virus derived from an infectious molecular clone of the ELI strain of human immunodeficiency virus type 1 (HIV-1) replicates well in peripheral blood mononuclear cells and in some CD4-positive cell lines but exhibits a delayed time course of infection in CEM and H9 cells and fails to infect SupT1 and U937 cells. If the virus that emerges from infected H9 cells is used to infect CEM and H9 cells, the time course of infection is accelerated and the virus is able to infect U937 and SupT1 cells. In this study, we used the technique of polymerase chain reaction-single-strand conformation polymorphism to localize changes in both the extracellular gp120 and the transmembrane gp41 components of the envelope gene associated with adaptation to growth in tissue culture cell lines. Specifically, mutations were identified both in a region of gp120 implicated in CD4 binding and in the amino-terminal portion of gp41 adjacent to the region involved in fusion. No changes were found in the V3 loop of gp120, a region previously shown to be involved in viral tropism. When these mutations were introduced into the original molecular clone, they conferred an enhanced replicative capacity on ELI. These results demonstrate that two additional determinants in the HIV-1 envelope protein influence viral tropism and growth in vitro. They also may have important implications for the generation of viruses with increased growth potential and expanded host range seen in the late stages of HIV disease.     

Recognition of a highly conserved region of human immunodeficiency virus type 1 gp120 by an HLA-Cw4-restricted cytotoxic T-lymphocyte clone.

Human immunodeficiency virus type 1 (HIV-1) isolates exhibit extensive sequence variation, particularly in the gp120 subunit of the envelope glycoprotein, and the degree of this variation has raised questions as to whether conserved regions of the HIV-1 envelope can be recognized by the host immune response. A CD8+ cytotoxic T-lymphocyte (CTL) clone specific for the HIV-1 envelope was derived by culturing peripheral blood mononuclear cells from an HIV-1 seropositive subject in the presence of a CD3-specific monoclonal antibody, interleukin-2, and irradiated allogeneic peripheral blood mononuclear cells. Lysis of target cells was restricted by an HLA-C molecule, Cw4, which has not been previously shown to present viral antigen to CTL. Mapping of the specificity of this CTL clone by using synthetic HIV-1 peptides localized the epitope to an 8-amino-acid region of gp120 (amino acids 376 to 383) which is conserved among approximately 90% of sequenced viral isolates. Examination of the recognition of variant peptides by this CTL clone demonstrated that a single, nonconservative amino acid substitution within the 8-amino-acid minimal epitope could abrogate lysis of targets incubated with the variant peptide. The identification of a CTL epitope in a highly conserved region of gp120 documents the ability of cellular immune responses of infected persons to respond to relatively invariant portions of this highly variable envelope glycoprotein. However, the ability of even a single-amino-acid change in gp120 to abolish lysis by CTL supports the hypothesis that sequence variation in HIV-1 may serve as a mechanism of immune escape. In addition, the identification of an HLA-C molecule presenting viral antigen to CTL supports a functional role for these molecules.