Deep Sequencing of the HIV-1 env Gene Reveals Discrete X4 Lineages and Linkage Disequilibrium between X4 and R5 Viruses in the V1/V2 and V3 Variable Regions

Abstract

HIV-1 requires the CD4 receptor and a coreceptor (CCR5 [R5 phenotype] or CXCR4 [X4 phenotype]) to enter cells. Coreceptor tropism can be assessed by either phenotypic or genotypic analysis, the latter using bioinformatics algorithms to predict tropism based on the env V3 sequence. We used the Primer ID sequencing strategy with the MiSeq sequencing platform to reveal the structure of viral populations in the V1/V2 and C2/V3 regions of the HIV-1 env gene in 30 late-stage and 6 early-stage subjects. We also used endpoint dilution PCR followed by cloning of env genes to create pseudotyped virus to explore the link between genotypic predictions and phenotypic assessment of coreceptor usage. We found out that the most stringently sequence-based calls of X4 variants (Geno2Pheno false-positive rate [FPR] of <2%) formed distinct lineages within the viral population, and these were detected in 24 of 30 late-stage samples (80%), which was significantly higher than what has been seen previously by using other approaches. Non-X4 lineages were not skewed toward lower FPR scores in X4-containing populations. Phenotypic assays showed that variants with an intermediate FPR (2 to 20%) could be either X4/dual-tropic or R5 variants, although the X4 variants made up only about 25% of the lineages with an FPR of <10%, and these variants carried a distinctive sequence change. Phylogenetic analysis of both the V1/V2 and C2/V3 regions showed evidence of recombination within but very little recombination between the X4 and R5 lineages, suggesting that these populations are genetically isolated. IMPORTANCEPrimer ID sequencing provides a novel approach to study genetic structures of viral populations. X4 variants may be more prevalent than previously reported when assessed by using next-generation sequencing (NGS) and with a greater depth of sampling than single-genome amplification (SGA). Phylogenetic analysis to identify lineages of sequences with intermediate FPR values may provide additional information for accurately predicting X4 variants by using V3 sequences. Limited recombination occurs between X4 and R5 lineages, suggesting that X4 and R5 variants are genetically isolated and may be replicating in different cell types or that X4/R5 recombinants have reduced fitness. H uman immunodeficiency virus type 1 (HIV-1) requires the CD4 receptor and a coreceptor to infect host cells. Entry is mediated by the viral envelope protein (Env), which is processed to give two associated subunits, gp120 and gp41, that assemble into a trimeric structure embedded in the viral envelope/membrane. The binding of gp120 to CD4 triggers a structural change that exposes its variable region 3 (V3) loop, which is part of the coreceptor domain (1, 2). The majority of transmitted/founder (T/F) viruses and viruses isolated from the clinically latent stage in HIV-infected subjects use CCR5 as the coreceptor, making the virus CCR5 tropic (or an R5 virus). The virus can evolve to use CXCR4 (CXCR4 tropic [or an X4 virus]), and viruses that have switched core...

New Frontiers in Measuring and Characterizing the HIV Reservoir

“…This diversity is primarily attributed to the absence of a proofreading mechanism in the HIV reverse transcriptase enzyme, as well as high levels of viremia and recombination (van Zyl et al, 2018). Other factors that contribute to this diversity include: host immune response (APOBEC enzyme hypermutations) (Bruner et al, 2016), length of infection (accumulation of mutations and depletion of CD4 T cell targets) (Arrildt et al, 2015;Zhou et al, 2016), viral compartmentalization (Yukl and Wong, 2015;van Zyl et al, 2018;Miller et al, 2019), and selection for viral quasispecies that are able to evade killing by host immune mechanisms (Phillips et al, 1991;Wei et al, 2003;Kearney et al, 2009;van Zyl et al, 2018).…”

The evolution of HIV-1 entry phenotypes as a guide to changing target cells

“…, we include an example of such an analysis taken from Zhou et al. showing the persistence of R5 viruses at high levels in the blood even when X4 viruses are present (although we do not know what the ratio of these variants might be in tissues or how much different tissues contribute to plasma viremia). The more accurate characterization of the three entry phenotypes of HIV‐1 puts R5 T cell‐tropic viruses at the center of the HIV‐1 story, gives a better definition beyond “neurotropic” to the types of viruses that are found in the CNS, explains why they may have evolved there, and places both X4 T cell‐tropic viruses and M‐tropic viruses as usually minor evolutionary variants that are rarely transmitted and appear when CD4+/CCR5+ T cells are depleted.…”

“…For comparison, a group of three “early‐stage subjects” are included showing limited diversity associated with the transmission of a single variant and R5‐predicted phenotypes. Taken from Zhou et al . with permission.…”

HIV evolution and diversity in ART-treated patients

“…HIV diversity is also influenced by a large population size and high recombination rate [ 1 – 4 ]. Other factors that contribute to the high genetic diversity of HIV are host APOBEC-mediated substitutions [ 5 , 6 ] and changes in the population of susceptible cells over the duration of infection [ 7 , 8 ] and across different anatomical compartments, such as the brain [ 9 – 11 ]. HIV evolution is driven, in large part, by the selection of expressed variants that carry mutations allowing escape from cell killing or virus neutralization by host immune responses [ 12 – 15 ].…”