Abstract
BackgroundThe HIV-1 pandemic is not the result of a static pathogen but a large genetically diverse and dynamic viral population. The virus is characterized by a highly mutable genome rendering efforts to design a universal vaccine a significant challenge and drives the emergence of drug resistant variants upon antiviral pressure. Gaining a comprehensive understanding of the mutational tolerance of each HIV-1 genomic position is therefore of critical importance.ResultsHere we combine high-density mutagenesis with the power of next-generation sequencing to gauge the replication capacity and therefore mutational tolerability of single point mutations across the entire HIV-1 genome. We were able to achieve the evaluation of point mutational effects on viral replicative capacity for 5,553 individual HIV-1 nucleotide positions – representing 57% of the viral genome. Replicative capacity was assessed at 3,943 nucleotide positions for a single alternate base change, 1,459 nucleotide positions for two alternate base changes, and 151 nucleotide positions for all three possible alternate base changes. This resulted in the study of how a total of 7,314 individual point mutations impact HIV-1 replication on a single experimental platform. We further utilize the dataset for a focused structural analysis on a capsid inhibitor binding pocket.ConclusionThe approach presented here can be applied to any pathogen that can be genetically manipulated in a laboratory setting. Furthermore, the methodology can be utilized under externally applied selection conditions, such as drug or immune pressure, to identify genetic elements that contribute to drug or host interactions, and therefore mutational routes of pathogen resistance and escape.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0124-6) contains supplementary material, which is available to authorized users.
Highlights
The human immunodeficiency virus-1 (HIV-1) pandemic is not the result of a static pathogen but a large genetically diverse and dynamic viral population
Our replication capacity (RC) data may overlap with sequence conservation information that is readily available from patient derived HIV-1 sequence databases, but conservation of a particular nucleotide position does not strictly equate to a high RC for that viral position [23,24], but a direct RC advantage of a select ancestral strain in that particular patient environment. quantitative high resolution genetics (qHRG) provides a complementary, direct, and functionally-based approach to impartially identify amino acid residues that are critical for viral replication in a defined cellular environment
To complement data collected from naturally occurring variations in clinical samples, our approach can be applied to study the dynamics of viral mutant populations in different growth conditions with precise control of experimental conditions to directly ascertain the mechanistic interplay between virus and host
Summary
The HIV-1 pandemic is not the result of a static pathogen but a large genetically diverse and dynamic viral population. Gaining a comprehensive understanding of the mutational tolerance of each HIV-1 genomic position is of critical importance. ~35 million people are living with human immunodeficiency virus-1 (HIV-1) infection, the pathogen responsible for acquired immunodeficiency syndrome (AIDS), with tens of millions having died of AIDSrelated causes worldwide since the pandemic began The virus rapidly evolves due to the high error rate of the viral reverse transcriptase (RT) enzyme at 3.4 × 10−5 mutations per site per generation coupled with a rapid generation output rate of ~1 × 1010 virions per patient per day [1,2,3,4], and the propensity of RT to mediate RNA recombination via.
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