Identification of Interferon-Stimulated Genes with Antiretroviral Activity

SummaryInterferons (IFNs) exert their anti-viral effects by inducing the expression of hundreds of IFN-stimulated genes (ISGs). The activity of known ISGs is insufficient to account for the antiretroviral effects of IFN, suggesting that ISGs with antiretroviral activity are yet to be described. We constructed an arrayed library of ISGs from rhesus macaques and tested the ability of hundreds of individual macaque and human ISGs to inhibit early and late replication steps for 11 members of the retroviridae from various host species. These screens uncovered numerous ISGs with antiretroviral activity at both the early and late stages of virus replication. Detailed analyses of two antiretroviral ISGs indicate that indoleamine 2,3-dioxygenase 1 (IDO1) can inhibit retroviral replication by metabolite depletion while tripartite motif-56 (TRIM56) accentuates ISG induction by IFNα and inhibits the expression of late HIV-1 genes. Overall, these studies reveal numerous host proteins that mediate the antiretroviral activity of IFNs

Extreme genetic fragility of the HIV-1 capsid

Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency >3%, and were also present in the mutant library, had fitness levels that were >40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies

Non-viable HIV-1 CA mutants.

<p>Mutants were considered non-viable if they exhibited <2% of WT fitness in the spreading replication assay as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003461#ppat-1003461-t001" target="_blank">Table 1</a>.</p

Summary of the effects of random mutations in HIV-1 CA.

<p>(A) Estimation of the frequency of various outcomes, based on <i>in vitro</i> and <i>in vivo</i> data, for random single nucleotide substitutions (A) and random single amino acid changes (B). The charts indicate changes that are synonymous (A only), nonsynonymous and nonsense (determined computationally using the CA sequence from pNHGcapNM). Mutants encoding stop codons in CA were assumed to be non-viable and synonymous changes were assumed to be viable. Because only mutants with >40% fitness <i>in vitro</i> are frequently observed in natural populations, this threshold was considered as sufficiently fit to thrive <i>in vivo</i> (the proportion of random mutants that exhibit less than this 40% fitness threshold was estimated using data from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003461#ppat-1003461-t001" target="_blank">Tables 1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003461#ppat-1003461-t002" target="_blank">2</a>, and is indicated by the red circumferential line). The proportion of mutants actually expected to thrive (nonsynonymous viable <i>in vitro</i> and <i>in vivo</i>) and expected not to thrive (nonsynonymous viable <i>in vitro</i> only) was estimated using the data from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003461#ppat-1003461-g009" target="_blank">Figure 9</a>. The fate of the remaining mutants was estimated using the data from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003461#ppat-1003461-g006" target="_blank">Figure 6</a> (nonsynonymous, non-infectious particles or nonsynonymous, attenuated (>5-fold deficit) particle formation).</p

Occurrence of CA mutations in natural HIV-1 subtype B populations.

<p>(A) Plot of the frequency with which the library mutations occur in 1000 natural HIV-1 subtype B CA sequences (Y-axis) versus the fitness of the same mutations in a spreading replication assay (X-axis). The vertical dashed line indicates 40% of WT replicative fitness, below which mutants rarely occurred in nature. Mutants that exhibited less than 0.01% of WT infectivity, and were never observed in nature would appear at the origin of this graph and are not plotted. (B) List of the library mutants that occurred at a frequency greater than 3% in 1000 subtype B isolates, and their corresponding fitness values. (C) List of the library mutants with greater than 40% of WT fitness that occurred infrequently (<0.3%) or never in 1000 subtype B sequences, and their corresponding fitness values. (D). Location of residues in the CA hexamer where mutations resulted in minimal fitness costs (>40% of WT replication) but that occurred in less than 0.3% of subtype B isolates. The leftmost image shows the hexamer viewed from the exterior of the intact conical capsid, the center image show the hexamer viewed from within the plane of the capsid lattice and the rightmost image shows the hexamer viewed from the interior of the intact conical capsid.</p

Distribution of single amino acid substitutions in CA and their effects on fitness.

<p>(A) Plot of HIV-1 fitness values for CA mutants with the location of the mutation arranged on the X-axis from left (N-terminal residue) to right (C-terminal residue), and their corresponding fitness (as a % of WT in a spreading replication assay) as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003461#ppat-1003461-t001" target="_blank">Tables 1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003461#ppat-1003461-t002" target="_blank">2</a>. (B, C) Location of single amino acid mutations in an X-ray crystal structure of the CA hexamer (PDB: 3GV2). Leftmost images show the hexamer viewed from the exterior of the intact capsid, center images show the hexamer viewed from within the plane of the capsid lattice and rightmost images show the hexamer viewed from the interior of the intact conical capsid. Residues that gave greater than 2% of WT fitness in a spreading replication assay when mutated (viable mutants) are shown in green (B). Residues that gave less than 2% of WT fitness in a spreading replication assay when mutated (non-viable mutants) are shown in red (C). (D) Comparison of fitness and residue exposure, using solvent accessible surface area (determined using UCSF Chimera with PDB 3GV2). Mutated residues were placed in two groups that had either (i) a solvent accessible surface area <50 Ų or (ii) a solvent accessible surface area >50 Ų. The corresponding fitness measurement for each mutant is plotted.</p

All ts CA mutants exhibit protease-dependent reduction in extracellular particle yield at the non-permissive temperature.

<p>(A). Western blot analysis (using an anti-CA antibody) of cell lysates and virions generated by transfected 293T cells. Two lanes are shown for each mutant: on the left is the sample from cells that were incubated at 35°C following transfection (Lo) and on the right is the sample from cells that were incubated at 39.5°C following transfection (Hi). Numbers below each lane indicate fluorescence intensities (LiCOR) associated with the CA protein pelleted from virion-containing supernatant. (B) Western blot analysis using an anti-HIV-1 MA antibody (p17) of cell lysates and virions, for the same panel as shown in A. Numbers below each lane indicate fluorescence intensities (LiCOR) associated with the MA protein pelleted from virion-containing supernatant. (C) Western blot analysis for the same panel of ts CA mutants as in A, but expressed in the context of a protease-defective proviral plasmid. Numbers below each lane indicate fluorescence intensities (LiCOR) associated with the CA protein pelleted from virion-containing supernatant.</p

Most CA mutants do not exhibit cell cycle dependent infectivity defects.

<p>The infectivity of 58 CA mutants exhibiting readily measurable infectivity in a single cycle assay was measured using untreated TZM-bl target cells (x-axis), and following arrest of the cell cycle with aphidicolin (arrests cell cycle in the S-phase) at 2 µg/ml (y-axis).</p

‘Rare but fit’ mutants behave indistinguishably from frequently occurring mutants in primary cells.

<p>(A) Percentage of infected macrophages following infection with VSV-G pseudotyped WT virus (NHGcapNM, filled squares) or derivatives containing mutations that occurred either frequently (filled triangles) or rarely (filled circles) in natural sequences, as indicated. (B) Percentage of infected stimulated CD4+ T cells, as indicated. (C) Spreading replication, following infection of stimulated peripheral blood mononuclear cells with WT virus or derivatives containing mutations that occurred either frequently or rarely in natural sequences, as indicated by the number of infected (GFP+) cells at the indicated times, after infection at an MOI of 0.1.</p

Most non-infectious constitutively non-viable CA mutants exhibit attenuated particle formation.

<p>(A) Western blots, probed with an anti-CA antibody, of cell lysates and virions for all constitutively non-viable CA mutants (those exhibiting <2% of WT fitness in a spreading replication assay). It should be noted that multiple substitutions between residues 159 and 168 resulted in diminished signal using the monoclonal anti-CA antibody (derived from hybridoma 183-H12-5C). Indeed CA carrying an R167Q mutation is infectious, but is not recognised by this antibody (unpublished observations) indicating that the epitope of the antibody lies in this region. This suggests that the E159V substitution (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003461#ppat-1003461-g006" target="_blank">Figure 6A</a>) might also disrupt antigen recognition, accounting for the absence of signal in this lane. (B) Location of mutations in the CA hexamer that are constitutively non-viable but exhibit near WT levels of particle formation. The leftmost image shows the hexamer viewed from the exterior of the intact conical capsid, the center image shows the hexamer viewed from within the plane of the conical capsid lattice and the rightmost image shows the hexamer viewed from the interior of the intact conical capsid.</p