Identification of an Antiretroviral Small Molecule That Appears To Be a Host-Targeting Inhibitor of HIV-1 Assembly.

Given the projected increase in multidrug-resistant HIV-1, there is an urgent need for development of antiretrovirals that act on virus life cycle stages not targeted by drugs currently in use. Host-targeting compounds are of particular interest because they can offer a high barrier to resistance. Here, we report identification of two related small molecules that inhibit HIV-1 late events, a part of the HIV-1 life cycle for which potent and specific inhibitors are lacking. This chemotype was discovered using cell-free protein synthesis and assembly systems that recapitulate intracellular host-catalyzed viral capsid assembly pathways. These compounds inhibit replication of HIV-1 in human T cell lines and peripheral blood mononuclear cells, and are effective against a primary isolate. They reduce virus production, likely by inhibiting a posttranslational step in HIV-1 Gag assembly. Notably, the compound colocalizes with HIV-1 Gag in situ; however, unexpectedly, selection experiments failed to identify compound-specific resistance mutations in gag or pol, even though known resistance mutations developed upon parallel nelfinavir selection. Thus, we hypothesized that instead of binding to Gag directly, these compounds localize to assembly intermediates, the intracellular multiprotein complexes containing Gag and host factors that form during immature HIV-1 capsid assembly. Indeed, imaging of infected cells shows compound colocalized with two host enzymes found in assembly intermediates, ABCE1 and DDX6, but not two host proteins found in other complexes. While the exact target and mechanism of action of this chemotype remain to be determined, our findings suggest that these compounds represent first-in-class, host-targeting inhibitors of intracellular events in HIV-1 assembly.IMPORTANCE The success of antiretroviral treatment for HIV-1 is at risk of being undermined by the growing problem of drug resistance. Thus, there is a need to identify antiretrovirals that act on viral life cycle stages not targeted by drugs in use, such as the events of HIV-1 Gag assembly. To address this gap, we developed a compound screen that recapitulates the intracellular events of HIV-1 assembly, including virus-host interactions that promote assembly. This effort led to the identification of a new chemotype that inhibits HIV-1 replication at nanomolar concentrations, likely by acting on assembly. This compound colocalized with Gag and two host enzymes that facilitate capsid assembly. However, resistance selection did not result in compound-specific mutations in gag, suggesting that the chemotype does not directly target Gag. We hypothesize that this chemotype represents a first-in-class inhibitor of virus production that acts by targeting a virus-host complex important for HIV-1 Gag assembly.

Pairwise growth competition assay for determining the replication fitness of human immunodeficiency viruses.

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

A temporospatial map that defines specific steps at which critical surfaces in the Gag MA and CA domains act during immature HIV-1 capsid assembly in cells.

UNLABELLED: During HIV-1 assembly, Gag polypeptides target to the plasma membrane, where they multimerize to form immature capsids that undergo budding and maturation. Previous mutational analyses identified residues within the Gag matrix (MA) and capsid (CA) domains that are required for immature capsid assembly, and structural studies showed that these residues are clustered on four exposed surfaces in Gag. Exactly when and where the three critical surfaces in CA function during assembly are not known. Here, we analyzed how mutations in these four critical surfaces affect the formation and stability of assembly intermediates in cells expressing the HIV-1 provirus. The resulting temporospatial map reveals that critical MA residues act during membrane targeting, residues in the C-terminal CA subdomain (CA-CTD) dimer interface are needed for the stability of the first membrane-bound assembly intermediate, CA-CTD base residues are necessary for progression past the first membrane-bound intermediate, and residues in the N-terminal CA subdomain (CA-NTD) stabilize the last membrane-bound intermediate. Importantly, we found that all four critical surfaces act while Gag is associated with the cellular facilitators of assembly ABCE1 and DDX6. When correlated with existing structural data, our findings suggest the following model: Gag dimerizes via the CA-CTD dimer interface just before or during membrane targeting, individual CA-CTD hexamers form soon after membrane targeting, and the CA-NTD hexameric lattice forms just prior to capsid release. This model adds an important new dimension to current structural models by proposing the potential order in which key contacts within the immature capsid lattice are made during assembly in cells. IMPORTANCE: While much is known about the structure of the completed HIV-1 immature capsid and domains of its component Gag proteins, less is known about the sequence of events leading to formation of the HIV-1 immature capsid. Here we used biochemical and ultrastructural analyses to generate a temporospatial map showing the precise order in which four critical surfaces in Gag act during immature capsid formation in provirus-expressing cells. Because three of these surfaces make important contacts in the hexameric lattices that are found in the completed immature capsid, these data allow us to propose a model for the sequence of events leading to formation of the hexameric lattices. By providing a dynamic view of when and where critical Gag-Gag contacts form during the assembly process and how those contacts function in the nascent capsid, our study provides novel insights into how an immature capsid is built in infected cells.

Factors affecting relative fitness measurements in pairwise competition assays of human immunodeficiency viruses.

Cell culture growth competition assays of human immunodeficiency virus type 1 (HIV-1) are used to estimate viral fitness and quantify the impact of mutations conferring drug resistance and immunological escape. A comprehensive study of growth competition assays was conducted and identified experimental parameters that can impact measurements of relative fitness including multiplicity of infection, viral input ratio, number, timing and interval of time points used to evaluate selective outgrowth, and the algorithm for calculating fitness values. An optimized protocol is developed here that is a multi-point growth competition assay that resolves reproducibly small differences in viral fitness. The optimized protocol uses an MOI of 0.005, a consistent ratio of mutant: parental viruses (70:30), and a multipoint [1+s 4,7] algorithm that uses data points within the logarithmic phase of viral growth for assessing fitness differences.

Fitness costs of mutations at the HIV-1 capsid hexamerization interface.

The recently available x-ray crystal structure of HIV-1 capsid hexamers has provided insight into the molecular interactions crucial for the virus's mature capsid formation. Amino acid changes at these interaction points are likely to have a strong impact on capsid functionality and, hence, viral infectivity and replication fitness. To test this hypothesis, we introduced the most frequently observed single amino acid substitution at 30 sites: 12 at the capsid hexamerization interface and 18 at non-interface sites. Mutations at the interface sites were more likely to be lethal (Fisher's exact test p = 0.027) and had greater negative impact on viral replication fitness (Wilcoxon rank sum test p = 0.040). Among the interface mutations studied, those located in the cluster of hydrophobic contacts at NTD-NTD interface and those that disrupted NTD-CTD inter-domain helix capping hydrogen bonds were the most detrimental, indicating that these interactions are particularly important for maintaining capsid structure and/or function. These functionally constrained sites provide potential targets for novel HIV drug development and vaccine immunogen design.

HIV-1 conserved-element vaccines: relationship between sequence conservation and replicative capacity.

To overcome the problem of HIV-1 variability, candidate vaccine antigens have been designed to be composed of conserved elements of the HIV-1 proteome. Such candidate vaccines could be improved with a better understanding of both HIV-1 evolutionary constraints and the fitness cost of specific mutations. We evaluated the in vitro fitness cost of 23 mutations engineered in the HIV-1 subtype B Gag-p24 Center-of-Tree (COT) protein through fitness competition assays. While some mutations at conserved sites exacted a high fitness cost, as expected under the assumption that the most conserved residue confers the highest fitness, there was no overall strong relationship between sequence conservation and replicative capacity. By comparing sites that have evolved since the beginning of the epidemic to those that have remain unchanged, we found that sites that have evolved over time were more likely to correspond to HLA-associated sites and that their mutation had limited fitness costs. Our data showed no transcendent link between high conservation and high fitness cost, indicating that merely focusing on conserved segments of HIV-1 would not be sufficient for a successful vaccine strategy. Nonetheless, a subset of sites exacted a high fitness cost upon mutation--these sites have been under selective pressure to change since the beginning of the epidemic but have proved virtually nonmutable and could constitute preferred targets for vaccine design.

A sensitive real-time PCR based assay to estimate the impact of amino acid substitutions on the competitive replication fitness of human immunodeficiency virus type 1 in cell culture.

Fixation of mutations in human immunodeficiency virus type 1 (HIV-1), such as those conferring drug resistance and immune escape, can result in a change in replication fitness. To assess these changes, a real-time TaqMan PCR detection assay and statistical methods for data analysis were developed to estimate sensitively relative viral fitness in competitive viral replication experiments in cell culture. Chimeric viruses with the gene of interest in an HIV-1NL4-3 backbone were constructed in two forms, vifA (native vif gene in NL4-3) and vifB (vif gene with six synonymous nucleotide differences from vifA). Subsequently, mutations of interest were introduced into the chimeric viruses in NL4-3VifA backbones, and the mutants were competed against the chimera with the isogenic viral sequence in the NL4-3VifB backbone in cell culture. In order to assess subtle fitness differences, culture supernatants were sampled longitudinally, and the viruses differentially quantified using vifA- and vifB-specific primers in real-time PCR assays. Based on an exponential net growth model, the growth rate of each virus was determined and the fitness cost of the mutation(s) distinguishing the two viruses represented as the net growth rate difference between the mutant and the native variants. Using this assay, the fitness impact of eight amino acid substitutions was quantitated at highly conserved sites in HIV-1 Gag and Env.

Amino-acid co-variation in HIV-1 Gag subtype C: HLA-mediated selection pressure and compensatory dynamics.

BACKGROUND: Despite high potential for HIV-1 genetic variation, the emergence of some mutations is constrained by fitness costs, and may be associated with compensatory amino acid (AA) co-variation. To characterize the interplay between Cytotoxic T Lymphocyte (CTL)-mediated pressure and HIV-1 evolutionary pathways, we investigated AA co-variation in Gag sequences obtained from 449 South African individuals chronically infected with HIV-1 subtype C. METHODOLOGY/PRINCIPAL FINDINGS: Individuals with CTL responses biased toward Gag presented lower viral loads than individuals with under-represented Gag-specific CTL responses. Using methods that account for founder effects and HLA linkage disequilibrium, we identified 35 AA sites under Human Leukocyte Antigen (HLA)-restricted CTL selection pressure and 534 AA-to-AA interactions. Analysis of two-dimensional distances between co-varying residues revealed local stabilization mechanisms since 40% of associations involved neighboring residues. Key features of our co-variation analysis included sites with a high number of co-varying partners, such as HLA-associated sites, which had on average 55% more connections than other co-varying sites. CONCLUSIONS/SIGNIFICANCE: Clusters of co-varying AA around HLA-associated sites (especially at typically conserved sites) suggested that cooperative interactions act to preserve the local structural stability and protein function when CTL escape mutations occur. These results expose HLA-imprinted HIV-1 polymorphisms and their interlinked mutational paths in Gag that are likely due to opposite selective pressures from host CTL-mediated responses and viral fitness constraints.

Nef induces multiple genes involved in cholesterol synthesis and uptake in human immunodeficiency virus type 1-infected T cells.

Several recent reports indicate that cholesterol might play an important role in human immunodeficiency virus type 1 (HIV-1) replication. We investigated the effects of HIV-1 infection on cholesterol biosynthesis and uptake using microarrays. HIV-1 increased gene expression of cholesterol genes in both transformed T-cell lines and primary CD4(+) T cells. Consistent with our microarray data, (14)C-labeled mevalonate and acetate incorporation was increased in HIV-1-infected cells. Our data also demonstrate that changes in cholesterol biosynthesis and uptake are only observed in the presence of functional Nef, suggesting that increased cholesterol synthesis may contribute to Nef-mediated enhancement of virion infectivity and viral replication.