pairwise-growth-competition-assay-for-determining-replication-fitness

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 ...

University of Washington

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.

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

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.

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

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.

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

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.

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

It has been hypothesized that a single mutation at a highly conserved amino acid site (HCS) can be severely deleterious to HIV in most if not all isolate-specific genetic backgrounds. Consequently, potentially universal HIV-1 vaccines exclusively targeting highly conserved regions of the viral proteome have been proposed. To test this hypothesis, we examined the impact of 10 Gag-p24 and 9 Env-gp120 HCS single mutations on viral fitness. In the original founder sequence of the subject in whom these mutations were identified, all Gag-p24 HCS mutations significantly reduced viral replication fitness, including 7 that were lethal. Similar results were obtained at 9/10 sites when the same mutations were introduced into the founder sequences of two epidemiologically unlinked subjects. In contrast, none of the 9 Env-gp120 HCS mutations were lethal in the original founder sequence, and four had no fitness cost. Hence, HCS mutations in Gag-p24 are likely to be severely deleterious in different HIV-1 subtype B backgrounds; however, some HCS mutations in both Gag-p24 and Env-gp120 fragments can be well tolerated. Therefore, when designing HIV-1 immunogens that are intended to force the virus to nonviable escape pathways, the fitness constraints on the HIV segments included should be considered beyond their conservation level.

Article	Year
Amino-acid co-variation in HIV-1 Gag subtype C: HLA-mediated selection pressure and compensatory dynamics. PloS one\| PubMed ID: 20824187	2010
HIV-1 conserved-element vaccines: relationship between sequence conservation and replicative capacity. Journal of virology\| PubMed ID: 23468488	2013
Fitness costs of mutations at the HIV-1 capsid hexamerization interface. PloS one\| PubMed ID: 23785468	2013
Factors affecting relative fitness measurements in pairwise competition assays of human immunodeficiency viruses. Journal of virological methods\| PubMed ID: 23933395	2013
Impact of mutations in highly conserved amino acids of the HIV-1 Gag-p24 and Env-gp120 proteins on viral replication in different genetic backgrounds. PloS one\| PubMed ID: 24713822	2014

Siriphan Manocheewa

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