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

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.

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

The deoxycytidine analog KP1212, and its prodrug KP1461, are prototypes of a new class of antiretroviral drugs designed to increase viral mutation rates, with the goal of eventually causing the collapse of the viral population. Here we present an extensive analysis of viral sequences from HIV-1 infected volunteers from the first "mechanism validation" phase II clinical trial of a mutagenic base analog in which individuals previously treated with antiviral drugs received 1600 mg of KP1461 twice per day for 124 days. Plasma viral loads were not reduced, and overall levels of viral mutation were not increased during this short-term study, however, the mutation spectrum of HIV was altered. A large number (N = 105 per sample) of sequences were analyzed, each derived from individual HIV-1 RNA templates, after 0, 56 and 124 days of therapy from 10 treated and 10 untreated control individuals (>7.1 million base pairs of unique viral templates were sequenced). We found that private mutations, those not found in more than one viral sequence and likely to have occurred in the most recent rounds of replication, increased in treated individuals relative to controls after 56 (p = 0.038) and 124 (p = 0.002) days of drug treatment. The spectrum of mutations observed in the treated group showed an excess of A to G and G to A mutations (p = 0.01), and to a lesser extent T to C and C to T mutations (p = 0.09), as predicted by the mechanism of action of the drug. These results validate the proposed mechanism of action in humans and should spur development of this novel antiretroviral approach.

Mutation of HIV-1 genomes in a clinical population treated with the mutagenic nucleoside KP1461.

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.

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.

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
Nef induces multiple genes involved in cholesterol synthesis and uptake in human immunodeficiency virus type 1-infected T cells. Journal of virology\| PubMed ID: 16014965	2005
Mutation of HIV-1 genomes in a clinical population treated with the mutagenic nucleoside KP1461. PloS one\| PubMed ID: 21264288	2011
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. Journal of virological methods\| PubMed ID: 23201292	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

Ushnal Rao

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