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. IMPORTANCE Primer 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

HIV-1 target cells in the CNS

HIV-1 replication in the central nervous system (CNS) is typically limited by the availability of target cells. HIV-1 variants that are transmitted and dominate the early stages of infection almost exclusively use the CCR5 coreceptor and are well adapted to entering, and thus infecting, cells expressing high CD4 densities similar to those found on CD4+ T cells. While the “immune privileged” CNS is largely devoid of CD4+ T cells, macrophage and microglia are abundant throughout the CNS. These cells likely express CD4 densities that are too low to facilitate efficient entry or allow sustained replication by most HIV-1 isolates. Examination of CNS viral populations reveals that late in disease the CNS of some individuals contains HIV-1 lineages that have evolved the ability to enter cells expressing low levels of CD4 and are well-adapted to entering macrophages. These macrophage-tropic (M-tropic) viruses are able to maintain sustained replication in the CNS for many generations, and their presence is associated with severe neurocognitive impairment. Whether conditions such as pleocytosis are necessary for macrophage-tropic viruses to emerge in the CNS is unknown, and extensive examinations of macrophage-tropic variants have not revealed a genetic signature of this phenotype. It is clear, however, that macrophage tropism is rare among HIV-1 isolates and is not transmitted, but is important due to its pathogenic effects on hosts. Prior to the evolution of macrophage-tropic variants, the viruses that are predominately infecting T cells (R5 T cell-tropic) may infect macrophages at a low level and inefficiently, but this could contribute to the reservoir

Compartmentalized Replication of R5 T Cell-Tropic HIV-1 in the Central Nervous System Early in the Course of Infection

Compartmentalized HIV-1 replication within the central nervous system (CNS) likely provides a foundation for neurocognitive impairment and a potentially important tissue reservoir. The timing of emergence and character of this local CNS replication has not been defined in a population of subjects. We examined the frequency of elevated cerebrospinal fluid (CSF) HIV-1 RNA concentration, the nature of CSF viral populations compared to the blood, and the presence of a cellular inflammatory response (with the potential to bring infected cells into the CNS) using paired CSF and blood samples obtained over the first two years of infection from 72 ART-naïve subjects. Using single genome amplification (SGA) and phylodynamics analysis of full-length env sequences, we compared CSF and blood viral populations in 33 of the 72 subjects. Independent HIV-1 replication in the CNS (compartmentalization) was detected in 20% of sample pairs analyzed by SGA, or 7% of all sample pairs, and was exclusively observed after four months of infection. In subjects with longitudinal sampling, 30% showed evidence of CNS viral replication or pleocytosis/inflammation in at least one time point, and in approximately 16% of subjects we observed evolving CSF/CNS compartmentalized viral replication and/or a marked CSF inflammatory response at multiple time points suggesting an ongoing or recurrent impact of the infection in the CNS. Two subjects had one of two transmitted lineages (or their recombinant) largely sequestered within the CNS shortly after transmission, indicating an additional mechanism for establishing early CNS replication. Transmitted variants were R5 T cell-tropic. Overall, examination of the relationships between CSF viral populations, blood and CSF HIV-1 RNA concentrations, and inflammatory responses suggested four distinct states of viral population dynamics, with associated mechanisms of local viral replication and the early influx of virus into the CNS. This study considerably enhances the generalizability of our results and greatly expands our knowledge of the early interactions of HIV-1 in the CNS

Multivariable analysis to determine if HIV-1 Tat dicysteine motif is associated with neurodevelopmental delay in HIV-infected children in Malawi

Abstract Background HIV-1 Tat protein is implicated in HIV-neuropathogenesis. Tat C31S polymorphism (TatCS) has been associated with milder neuropathology in vitro and in animal models but this has not been addressed in a cohort of HIV-infected adults or children. Methods HIV viral load (VL) in plasma and cerebrospinal fluid (CSF) were determined and plasma HIV tat gene was sequenced. Neurodevelopmental assessment was performed using Bayley Scales of Infant Development III (BSID-III), with scores standardized to Malawian norms. The association between TatCS and BSID-III scores was evaluated using multivariate linear regression. Results Neurodevelopmental assessment and HIV tat genotyping were available for 33 children. Mean age was 19.4 (SD 7.1) months, mean log VL was 5.9 copies/mL (SD 0.1) in plasma and 3.9 copies/mL (SD 0.9) in CSF. The prevalence of TatCC was 27 %. Z-scores for BSID-III subtests ranged from −1.3 to −3.9. TatCC was not associated with higher BSID-III z-scores. Conclusions The hypothesis of milder neuropathology in individuals infected with HIV TatCS was not confirmed in this small cohort of Malawian children. Future studies of tat genotype and neurocognitive disorder should be performed using larger sample sizes and investigate if this finding is due to differences in HIV neuropathogenesis between children and adults

Compartmentalized replication of R5 T cell-tropic HIV-1 in the central nervous system early in the course of infection.

Compartmentalized HIV-1 replication within the central nervous system (CNS) likely provides a foundation for neurocognitive impairment and a potentially important tissue reservoir. The timing of emergence and character of this local CNS replication has not been defined in a population of subjects. We examined the frequency of elevated cerebrospinal fluid (CSF) HIV-1 RNA concentration, the nature of CSF viral populations compared to the blood, and the presence of a cellular inflammatory response (with the potential to bring infected cells into the CNS) using paired CSF and blood samples obtained over the first two years of infection from 72 ART-naïve subjects. Using single genome amplification (SGA) and phylodynamics analysis of full-length env sequences, we compared CSF and blood viral populations in 33 of the 72 subjects. Independent HIV-1 replication in the CNS (compartmentalization) was detected in 20% of sample pairs analyzed by SGA, or 7% of all sample pairs, and was exclusively observed after four months of infection. In subjects with longitudinal sampling, 30% showed evidence of CNS viral replication or pleocytosis/inflammation in at least one time point, and in approximately 16% of subjects we observed evolving CSF/CNS compartmentalized viral replication and/or a marked CSF inflammatory response at multiple time points suggesting an ongoing or recurrent impact of the infection in the CNS. Two subjects had one of two transmitted lineages (or their recombinant) largely sequestered within the CNS shortly after transmission, indicating an additional mechanism for establishing early CNS replication. Transmitted variants were R5 T cell-tropic. Overall, examination of the relationships between CSF viral populations, blood and CSF HIV-1 RNA concentrations, and inflammatory responses suggested four distinct states of viral population dynamics, with associated mechanisms of local viral replication and the early influx of virus into the CNS. This study considerably enhances the generalizability of our results and greatly expands our knowledge of the early interactions of HIV-1 in the CNS

HIV-1 blood and CSF populations early during infection can be equilibrated or compartmentalized.

<p>Neighbor-joining phylogenetic trees depicting sequence relationships from subjects with (A) equilibrated and (B) compartmentalized viral populations. <i>env s</i>equences from the CSF (blue circles) and blood plasma (red triangles) are shown. Bootstrap values ≥35 are included (*) at the appropriate nodes. Genetic distance is indicated at the bottom of each figure (0.001, number of nucleotide substitutions per site between <i>env</i> sequences). Compartmentalized CNS populations are designated by an open circle at the appropriate node and a solid blue bracket. Clonal amplification is indicated by a solid black bar.</p

Compartmentalization can persist and evolve independently within the CSF over time.

<p>Neighbor-joining phylogenetic trees showing how compartmentalization can: (A) be persistent with multiple clonal expansions allowing recombination; (B) consist of sequential transient clonal expansions; and (C) be established with a transmitted variant. <i>env</i> sequences from the CSF are labeled with circles (C, colors designated in figure) and <i>env</i> sequences from the blood plasma are labeled with triangles (P, colors designated in figure). Days p.i. are noted. Bootstrap values ≥ 50 are indicated (*) at the appropriate nodes to highlight the more significant branch points. Genetic distance is indicated at the top of each phylogenetic tree (0.001, number of nucleotide substitutions per site between <i>env</i> sequences.) Compartmentalized populations are indicated at the appropriate node by an open circle and emphasized using a blue bar. BEAST-generated TMRCAs of the entire viral population are noted adjacent to the subject ID, and the TMRCAs of the different compartmentalized linages (subject 9040 and 9021) and transmitted parental lineages (subject 7146) are also noted.</p

Subject population virologic, clinical and phylogenetic characteristics.

<p>^aTime point(s) beyond 2 years p.i. analyzed for subject 9018 and 9040 were not included in any overall population analyses.</p><p>^bEstimated.</p><p>^c,dVL, viral load; HIV-1 RNA (log₁₀ copies/ml).</p><p>^eCells/μl.</p><p>^fCSF white blood cell (WBC) count, cell/μl.</p><p>^gCSF/plasma albumin ratio.</p><p>^hThree statistical analyses of genetic compartmentalization between viral populations in the blood plasma and CSF: Slatkin-Maddison test (SM) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004720#ppat.1004720.ref018" target="_blank">18</a>], Wright’s measure of population subdivision (F_st) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004720#ppat.1004720.ref019" target="_blank">19</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004720#ppat.1004720.ref020" target="_blank">20</a>] and the Nearest-neighbor statistic (S_nn) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004720#ppat.1004720.ref021" target="_blank">21</a>]. <i>P</i> values <0.05 indicated statistically significant genetic compartmentalization.</p><p>ⁱHIV-1 population characteristics in the CSF compartment (compart). Eq, equilibrated blood plasma and CSF populations; Cp (compartmentalized), significant compartmentalization by three compartmentalization analyses; Ap, clonal amplification of ≥3 variants detected in the CSF.[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004720#ppat.1004720.ref018" target="_blank">18</a>]</p><p>^jTMRCA (Time to Most Recent Common Ancestor) of the entire viral population, analyzed by BEAST [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004720#ppat.1004720.ref015" target="_blank">15</a>]. An asterisk (*) indicates transmission of > 1 variant.</p><p>^kTMRCA for the compartmentalized (Comp) CSF population for compartmentalized subjects.</p><p>^lPatient 9018 diagnosed with neurosyphilis at indicated date.</p><p>^mSignificant compartmentalization scores were due to a compartmentalized lineage in the plasma. After removing this plasma lineage, the remaining plasma and CSF sequences were equilibrated.</p><p>Subject population virologic, clinical and phylogenetic characteristics.</p

All viruses required high levels of CD4 for efficient entry, indicative of primarily being selected for replication in CD4+ T cells.

<p>Single-cycle infection of HIV-1 Env-pseudotyped reporter viruses on CD4^lowCCR5^high 293-Affinofile cells [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004720#ppat.1004720.ref028" target="_blank">28</a>]. Receptor expression was measured as follows: CD4^low = 972 receptors/cell, CD4^high = 72,041 receptors/cell, CCR5^high = 15,636 receptors/cell. The data are averaged from triplicate wells for each of 2 to 3 <i>env</i> clones that were generated per indicated amplicon. Amplicons were selected for cloning to represent different portions of the phylogenetic tree. Subjects with no evidence of CNS compartmentalization (i.e. equilibrated) are shown in panel (A); and subjects with CNS compartmentalization at one or more time points are in panel (B). Longitudinal time points are indicated (T1, T2, T3, etc.) and samples with marked pleocytosis are noted (*).</p

Background demographic and clinical characteristics of study participants at baseline.

<p>Values shown are medians (interquartile ranges) except where noted</p><p>^aSubjects with sufficient viral RNA loads in the CSF (>1,000 copies of viral RNA/ml; to ensure adequate sampling) for further SGA analysis at one or more time points within the first two years.</p><p>Background demographic and clinical characteristics of study participants at baseline.</p