Kieran Cashin

Longitudinal Analysis of CCR5 and CXCR4 Usage in a Cohort of Antiretroviral Therapy-Naïve Subjects with Progressive HIV-1 Subtype C Infection

HIV-1 subtype C (C-HIV) is responsible for most HIV-1 cases worldwide. Although the pathogenesis of C-HIV is thought to predominantly involve CCR5-restricted (R5) strains, we do not have a firm understanding of how frequently CXCR4-using (X4 and R5X4) variants emerge in subjects with progressive C-HIV infection. Nor do we completely understand the molecular determinants of coreceptor switching by C-HIV variants. Here, we characterized a panel of HIV-1 envelope glycoproteins (Envs) (n = 300) cloned sequentially from plasma of 21 antiretroviral therapy (ART)-naïve subjects who experienced progression from chronic to advanced stages of C-HIV infection, and show that CXCR4-using C-HIV variants emerged in only one individual. Mutagenesis studies and structural models suggest that the evolution of R5 to X4 variants in this subject principally involved acquisition of an “Ile-Gly” insertion in the gp120 V3 loop and replacement of the V3 “Gly-Pro-Gly” crown with a “Gly-Arg-Gly” motif, but that the accumulation of additional gp120 “scaffold” mutations was required for these V3 loop changes to confer functional effects. In this context, either of the V3 loop changes could confer possible transitional R5X4 phenotypes, but when present together they completely abolished CCR5 usage and conferred the X4 phenotype. Our results show that the emergence of CXCR4-using strains is rare in this cohort of untreated individuals with advanced C-HIV infection. In the subject where X4 variants did emerge, alterations in the gp120 V3 loop were necessary but not sufficient to confer CXCR4 usage.

Alternative Coreceptor Requirements for Efficient CCR5- and CXCR4-Mediated HIV-1 Entry into Macrophages

ABSTRACT Macrophage tropism of human immunodeficiency virus type 1 (HIV-1) is distinct from coreceptor specificity of the viral envelope glycoproteins (Env), but the virus-cell interactions that contribute to efficient HIV-1 entry into macrophages, particularly via CXCR4, are not well understood. Here, we characterized a panel of HIV-1 Envs that use CCR5 (n = 14) or CXCR4 (n = 6) to enter monocyte-derived macrophages (MDM) with various degrees of efficiency. Our results show that efficient CCR5-mediated MDM entry by Env-pseudotyped reporter viruses is associated with increased tolerance of several mutations within the CCR5 N terminus. In contrast, efficient CXCR4-mediated MDM entry was associated with reduced tolerance of a large deletion within the CXCR4 N terminus. Env sequence analysis and structural modeling identified amino acid variants at positions 261 and 263 within the gp41-interactive region of gp120 and a variant at position 326 within the gp120 V3 loop that were associated with efficient CXCR4-mediated MDM entry. Mutagenesis studies showed that the gp41 interaction domain variants exert a significant but strain-specific influence on CXCR4-mediated MDM entry, suggesting that the structural integrity of the gp120-gp41 interface is important for efficient CXCR4-mediated MDM entry of certain HIV-1 strains. However, the presence of Ile326 in the gp120 V3 loop stem, which we show by molecular modeling is located at the gp120-coreceptor interface and predicted to interact with the CXCR4 N terminus, was found to be critical for efficient CXCR4-mediated MDM entry of divergent CXCR4-using Envs. Together, the results of our study provide novel insights into alternative mechanisms of Env-coreceptor engagement that are associated with efficient CCR5- and CXCR4-mediated HIV-1 entry into macrophages.

CoRSeqV3-C: a novel HIV-1 subtype C specific V3 sequence based coreceptor usage prediction algorithm

BackgroundThe majority of HIV-1 subjects worldwide are infected with HIV-1 subtype C (C-HIV). Although C-HIV predominates in developing regions of the world such as Southern Africa and Central Asia, C-HIV is also spreading rapidly in countries with more developed economies and health care systems, whose populations are more likely to have access to wider treatment options, including the CCR5 antagonist maraviroc (MVC). The ability to reliably determine C-HIV coreceptor usage is therefore becoming increasingly more important. In silico V3 sequence based coreceptor usage prediction algorithms are a relatively rapid and cost effective method for determining HIV-1 coreceptor specificity. In this study, we elucidated the V3 sequence determinants of C-HIV coreceptor usage, and used this knowledge to develop and validate a novel, user friendly, and highly sensitive C-HIV specific coreceptor usage prediction algorithm.ResultsWe characterized every phenotypically-verified C-HIV gp120 V3 sequence available in the Los Alamos HIV Database. Sequence analyses revealed that compared to R5 C-HIV V3 sequences, CXCR4-using C-HIV V3 sequences have significantly greater amino acid variability, increased net charge, increased amino acid length, increased frequency of insertions and substitutions within the GPGQ crown motif, and reduced frequency of glycosylation sites. Based on these findings, we developed a novel C-HIV specific coreceptor usage prediction algorithm (CoRSeqV3-C), which we show has superior sensitivity for determining CXCR4 usage by C-HIV strains compared to all other available algorithms and prediction rules, including Geno2pheno[coreceptor] and WebPSSMSINSI-C, which has been designed specifically for C-HIV.ConclusionsCoRSeqV3-C is now openly available for public use at http://www.burnet.edu.au/coreceptor. Our results show that CoRSeqV3-C is the most sensitive V3 sequence based algorithm presently available for predicting CXCR4 usage of C-HIV strains, without compromising specificity. CoRSeqV3-C may be potentially useful for assisting clinicians to decide the best treatment options for patients with C-HIV infection, and will be helpful for basic studies of C-HIV pathogenesis.

Reliable Genotypic Tropism Tests for the Major HIV-1 Subtypes

Over the past decade antiretroviral drugs have dramatically improved the prognosis for HIV-1 infected individuals, yet achieving better access to vulnerable populations remains a challenge. The principal obstacle to the CCR5-antagonist, maraviroc, from being more widely used in anti-HIV-1 therapy regimens is that the pre-treatment genotypic “tropism tests” to determine virus susceptibility to maraviroc have been developed primarily for HIV-1 subtype B strains, which account for only 10% of infections worldwide. We therefore developed PhenoSeq, a suite of HIV-1 genotypic tropism assays that are highly sensitive and specific for establishing the tropism of HIV-1 subtypes A, B, C, D and circulating recombinant forms of subtypes AE and AG, which together account for 95% of HIV-1 infections worldwide. The PhenoSeq platform will inform the appropriate use of maraviroc and future CCR5 blocking drugs in regions of the world where non-B HIV-1 predominates, which are burdened the most by the HIV-1 pandemic.

Differences in coreceptor specificity contribute to alternative tropism of HIV-1 subtype C for CD4(+) T-cell subsets, including stem cell memory T-cells.

BackgroundCD4+ memory T-cells are a major target for infection by HIV-1, whereby latent provirus can establish and endure suppressive antiretroviral therapies. Although HIV-1 subtype C strains (C-HIV) account for the majority of HIV-1 infections worldwide, the susceptibility of CD4+ memory T-cells to infection by CCR5- (R5) and CXCR4-using (X4) C-HIV is unknown. Here, we quantified the susceptibility of naïve and memory CD4+ T-cell subsets, including stem cell memory T-cells (TSCM), to infection by HIV-1 subtype C (C-HIV) strains from treatment-naïve subjects who progressed from chronic to advanced stages of disease whilst either maintaining CCR5-using (R5) viruses (subjects 1503 and 1854), or who experienced emergence of dominant CXCR4-using (X4) strains (subject 1109).FindingsWe show that R5 and X4 C-HIV viruses preferentially target memory and naïve CD4+ T-cell subsets, respectively. While TSCM were susceptible to infection by both R5 and X4 C-HIV viruses, the proportion of infected CD4+ T-cells that were TSCM was higher for R5 strains. Mutagenesis studies of subject 1109 viruses established the V3 region of env as the determinant underlying the preferential targeting of naïve CD4+ T-cells by emergent X4 C-HIV variants in this subject. In contrast, the tropism of R5 C-HIV viruses for CD4+ T-cell subsets was maintained from chronic to advanced stages of disease in subjects 1503 and 1854.ConclusionsThis study provides new insights into the natural history of tropism alterations for CD4+ T-cell subsets by C-HIV strains during progression from chronic to advanced stages of infection. Although not preferentially targeted, our data suggest that TSCM and other memory CD4+ T-cells are likely to be viral reservoirs in subjects with X4 C-HIV infection.

Quantifying Susceptibility of CD4+ Stem Memory T-Cells to Infection by Laboratory Adapted and Clinical HIV-1 Strains

CD4+ T cells are principal targets for human immunodeficiency virus type 1 (HIV-1) infection. CD4+ T cell subsets are heterogeneous cell populations, divided by functional and phenotypic differences into naïve and memory T cells. The memory CD4+ T cells are further segregated into central, effector and transitional memory cell subsets by functional, phenotypic and homeostatic characteristics. Defining the distribution of HIV-1 infection in different T cell subsets is important, as this can play a role in determining the size and composition of the viral reservoir. Both central memory and transitional memory CD4+ T cells have been described as long-lived viral reservoirs for HIV. Recently, the newly described stem memory T cell subset has also been implicated as a long-lived HIV reservoir. Using green fluorescent protein (GFP) reporter strains of HIV-1 and multi parameter flow cytometry, we developed an assay to simultaneously quantify the susceptibility of stem memory (TSCM), central memory, effector memory, transitional memory and naïve CD4+ T cell subsets, to HIV-1 infection in vitro. We show that TSCM are susceptible to infection with laboratory adapted and clinical HIV-1 strains. Our system facilitates the quantitation of HIV-1 infection in alternative T cell subsets by CCR5- and CXCR4-using viruses across different HIV-1 subtypes, and will be useful for studies of HIV-1 pathogenesis and viral reservoirs.

Linkages between HIV-1 specificity for CCR5 or CXCR4 and in vitro usage of alternative coreceptors during progressive HIV-1 subtype C infection

BackgroundHuman immunodeficiency virus type 1 (HIV-1) subtype C (C-HIV) is spreading rapidly and is now responsible for >50% of HIV-1 infections worldwide, and >95% of infections in southern Africa and central Asia. These regions are burdened with the overwhelming majority of HIV-1 infections, yet we know very little about the pathogenesis of C-HIV. In addition to CCR5 and CXCR4, the HIV-1 envelope glycoproteins (Env) may engage a variety of alternative coreceptors for entry into transfected cells. Whilst alternative coreceptors do not appear to have a broad role in mediating the entry of HIV-1 into primary cells, characterizing patterns of alternative coreceptor usage in vitro can provide valuable insights into mechanisms of Env-coreceptor engagement that may be important for HIV-1 pathogenesis.ResultsHere, we characterized the ability of luciferase reporter viruses pseudotyped with HIV-1 Envs (n = 300) cloned sequentially from plasma of 21 antiretroviral therapy (ART)-naïve subjects experiencing progression from chronic to advanced C-HIV infection over an approximately 3-year period, who either exclusively maintained CCR5-using (R5) variants (n = 20 subjects) or who experienced a coreceptor switch to CXCR4-using (X4) variants (n = 1 subject), to utilize alternative coreceptors for entry. At a population level, CCR5 usage by R5 C-HIV Envs was strongly linked to usage of FPRL1, CCR3 and CCR8 as alternative coreceptors, with the linkages to FPRL1 and CCR3 usage becoming statistically more robust as infection progressed from chronic to advanced stages of disease. In contrast, acquisition of an X4 Env phenotype at advanced infection was accompanied by a dramatic loss of FPRL1 usage. Env mutagenesis studies confirmed a direct link between CCR5 and FPRL1 usage, and showed that the V3 loop crown, but not other V3 determinants of CCR5-specificity, was the principal Env determinant governing the ability of R5 C-HIV Envs from one particular subject to engage FPRL1.ConclusionsOur results suggest that, in the absence of coreceptor switching, the ability of R5 C-HIV viruses to engage certain alternative coreceptors in vitro, in particular FPRL1, may reflect an altered use of CCR5 that is selected for during progressive C-HIV infection, and which may contribute to C-HIV pathogenicity.

Covariance of Charged Amino Acids at Positions 322 and 440 of HIV-1 Env Contributes to Coreceptor Specificity of Subtype B Viruses, and Can Be Used to Improve the Performance of V3 Sequence-Based Coreceptor Usage Prediction Algorithms

The ability to determine coreceptor usage of patient-derived human immunodeficiency virus type 1 (HIV-1) strains is clinically important, particularly for the administration of the CCR5 antagonist maraviroc. The envelope glycoprotein (Env) determinants of coreceptor specificity lie primarily within the gp120 V3 loop region, although other Env determinants have been shown to influence gp120-coreceptor interactions. Here, we determined whether conserved amino acid alterations outside the V3 loop that contribute to coreceptor usage exist, and whether these alterations improve the performance of V3 sequence-based coreceptor usage prediction algorithms. We demonstrate a significant covariant association between charged amino acids at position 322 in V3 and position 440 in the C4 Env region that contributes to the specificity of HIV-1 subtype B strains for CCR5 or CXCR4. Specifically, positively charged Lys/Arg at position 322 and negatively charged Asp/Glu at position 440 occurred more frequently in CXCR4-using viruses, whereas negatively charged Asp/Glu at position 322 and positively charged Arg at position 440 occurred more frequently in R5 strains. In the context of CD4-bound gp120, structural models suggest that covariation of amino acids at Env positions 322 and 440 has the potential to alter electrostatic interactions that are formed between gp120 and charged amino acids in the CCR5 N-terminus. We further demonstrate that inclusion of a “440 rule” can improve the sensitivity of several V3 sequence-based genotypic algorithms for predicting coreceptor usage of subtype B HIV-1 strains, without compromising specificity, and significantly improves the AUROC of the geno2pheno algorithm when set to its recommended false positive rate of 5.75%. Together, our results provide further mechanistic insights into the intra-molecular interactions within Env that contribute to coreceptor specificity of subtype B HIV-1 strains, and demonstrate that incorporation of Env determinants outside V3 can improve the reliability of coreceptor usage prediction algorithms.

Genotypic Prediction of Co-receptor Tropism of HIV-1 Subtypes A and C.

Antiretroviral treatment of Human Immunodeficiency Virus type-1 (HIV-1) infections with CCR5-antagonists requires the co-receptor usage prediction of viral strains. Currently available tools are mostly designed based on subtype B strains and thus are in general not applicable to non-B subtypes. However, HIV-1 infections caused by subtype B only account for approximately 11% of infections worldwide. We evaluated the performance of several sequence-based algorithms for co-receptor usage prediction employed on subtype A V3 sequences including circulating recombinant forms (CRFs) and subtype C strains. We further analysed sequence profiles of gp120 regions of subtype A, B and C to explore functional relationships to entry phenotypes. Our analyses clearly demonstrate that state-of-the-art algorithms are not useful for predicting co-receptor tropism of subtype A and its CRFs. Sequence profile analysis of gp120 revealed molecular variability in subtype A viruses. Especially, the V2 loop region could be associated with co-receptor tropism, which might indicate a unique pattern that determines co-receptor tropism in subtype A strains compared to subtype B and C strains. Thus, our study demonstrates that there is a need for the development of novel algorithms facilitating tropism prediction of HIV-1 subtype A to improve effective antiretroviral treatment in patients.

HIV-1 and SIV Predominantly Use CCR5 Expressed on a Precursor Population to Establish Infection in T Follicular Helper Cells

Background T follicular helper (Tfh) cells are increasingly recognized as a major reservoir of HIV infection that will likely need to be addressed in approaches to curing HIV. However, Tfh express minimal CCR5, the major coreceptor for HIV-1, and the mechanism by which they are infected is unclear. We have previously shown that macaque Tfh lack CCR5, but are infected in vivo with CCR5-using SIV at levels comparable to other memory CD4+ T cells. Similarly, human splenic Tfh cells are highly infected with HIV-1 DNA. Therefore, we set out to examine the mechanism of infection of Tfh cells. Methodology Tfh and other CD4+ T cell subsets from macaque lymph nodes and spleens, splenic Tfh from HIV+ subjects, and tonsillar Tfh from HIV-uninfected subjects were isolated by cell sorting prior to cell surface and molecular characterization. HIV proviral gp120 sequences were submitted to genotypic and phenotypic tropism assays. Entry of CCR5- and CXCR4-using viruses into Tfh from uninfected tonsillar tissue was measured using a fusion assay. Results Phylogenetic analysis, genotypic, and phenotypic analysis showed that splenic Tfh cells from chronic HIV+ subjects were predominantly infected with CCR5-using viruses. In macaques, purified CCR5+PD-1intermediate(int)+ memory CD4+ T cells were shown to include pre-Tfh cells capable of differentiating in vitro to Tfh by upregulation of PD-1 and Bcl6, confirmed by qRT-PCR and single-cell multiplex PCR. Infected PD-1int cells survive, carry SIV provirus, and differentiate into PD-1hi Tfh after T cell receptor stimulation, suggesting a pathway for SIV infection of Tfh. In addition, a small subset of macaque and human PD-1hi Tfh can express low levels of CCR5, which makes them susceptible to infection. Fusion assays demonstrated CCR5-using HIV-1 entry into CCR5+ Tfh and pre-Tfh cells from human tonsils. Conclusion The major route of infection of Tfh in macaques and humans appears to be via a CCR5-expressing pre-Tfh population. As the generation of Tfh are important for establishing effective immune responses during primary infections, Tfh are likely to be an early target of HIV-1 following transmission, creating an important component of the reservoir that has the potential to expand over time.