HIV-1 Replicative Capacity Predicts Disease Progression and is Associated with Early Pathogenesis Open Access

Prince Guerra, Jessica Lee (2014)

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HIV-1 infection is characterized by a gradual decline in peripheral CD4+ T cells and generalized immune dysfunction. While a majority of HIV-infected individuals eventually progress to AIDS, they do so at varying rates. Determining the host and viral factors that shape the trajectory of HIV-1 pathogenesis is key for developing rational prevention strategies. To date, research has focused on elucidating host factors associated with disease progression while viral factors have been relatively understudied. The following study utilizes a well-characterized cohort of subtype C Zambian seroconverters in order to investigate how replicative capacity of the transmitted virus, as defined by Gag, contributes to HIV-1 disease progression and pathogenesis. From over 200 Zambian seroconverters, we created Gag-chimeric HIV-1 viruses in which the patient-derived gag gene was isolated from acute time points and cloned into MJ4. In individuals recently infected with HIV-1 subtype C, low viral replicative capacity as defined by the transmitted Gag sequence, was associated with a delayed loss of CD4+ T cells independent of set point VL and host immunogenetic factors. We hypothesized that early viral replication might initiate crucial events during early infection that influence HIV-1 pathogenesis. Chronic immune activation is a hallmark of HIV-1 infection and predicts disease progression better than viral load. We show that attenuated replicative capacity leads to dampened levels of immune activation. Furthermore, we found that in individuals infected with low replicating viruses, CD8+ T cells were less exhausted and more cytotoxic perhaps leading to a more functional immune response. Moreover, replicative capacity was positively correlated with CD4+ T cell proliferation and with levels of HIV DNA in CD4+ central memory T cells, a population highlighted to be integral for the maintenance of latency and preferentially spared in non-pathogenic SIV infection. Consistent with previous studies, we observed that all of these measures of immune dysfunction were associated with the rate of disease progression in this cohort. Collectively, this provides a mechanistic link between replicative capacity and CD4+ T cell decline. This study highlights the previously unrecognized role that replicative capacity of the transmitted virus plays in defining several facets of HIV-1 immunopathology and disease progression.

Table of Contents

Chapter 1: Introduction

Global burden of HIV/AIDS 1

Origin, classification, and viral diversity of HIV-1 3

HIV-1 transmission 5

HIV-1 genome and viral lifecycle 8

Pathogenesis of HIV-1 infection 10

The cellular immune response to HIV and CTL escape 25

Viral replicative capacity of HIV-1

Chapter 2: Role of transmitted Gag CTL polymorphisms in defining replicative capacity and early HIV-1 pathogenesis 35

Figure 1. Insertion of the gag gene from newly infected individuals dramatically alters the replicative capacity of MJ4 72

Figure 2. Replicative capacity is correlated to viral load in recipients and donors 73

Figure 3. Identification of polymorphisms in Gag that significantly affect RC, several of which can be linked to HLA-class I alleles 74

Figure 4. Rare polymorphisms have a significantly greater impact on RC 76

Figure 5. The balance of fitness increasing and decreasing HLA-associated polymorphisms strongly correlates with RC 78

Figure 6. The balance of HLA-associated fitness increasing and decreasing mutations strongly correlates with set point viral load in newly infected individuals 79

Figure 7. RC affects the rate of CD4 decline in a manner that may be independent of viral load 81

Figure S1. Donor and recipient population gag sequences cluster with one another 83

Figure S2. Gag sequences that are less like the Gag subtype C consensus sequence replicate more efficiently in vitro 85

Table 1. Cohort statistics generated from the 149 transmission pairs selected from the ZEHRP cohort 86

Table 2. Cox proportional hazard models demonstrate the independent effects of RC and VL on CD4 decline 87

Table S1. Amino acids in Gag associated with changes in replicative capacity 88

Chapter 3: Replicative capacity of HIV-1 drives early inflammation T-cell activation and infection of central memory CD4 T cells 102

Figure 1. HIV-1 replicative capacity, when defined by the transmitted gag sequence, predicts CD4+ T cell decline in ART HIV-1 infected individuals 116

Supplementary Figure 1. The effect of log10-increases in early set point VL on longitudinal CD4 T cell decline post seroconversion 118

Supplementary Table 1. Host and viral characteristics independently predict CD4 T cell decline 119

Supplementary Table 2. High vRC significantly increases early inflammatory cytokine levels 120

Figure 2. Low vRC is associated with a distinct cytokine profile early in infection, characterized by muted inflammatory cytokine levels 121

Supplementary Figure 2. The first two principal components are significantly correlated with vRC and set point VL, respectively 124

Figure 3. High vRC is associated with increased CD8 T cell activation and lower cytotoxic potential 125

Supplementary Figure 3. CD8 T cell activation phenotypes early after infection are associated with CD4+ T cell decline 127

Figure 4. vRC is associated with increased cellular activation and proliferation in CD4+ T cell memory subsets 129

Supplementary Figure 4. High vRC is associated with an increased level of activation and turnover in CD4+ T cells that is highly deleterious 131

Figure 5. Inflammatory cytokine profiles associated with vRC correlate with T cell activation 132

Figure 6. vRC correlates with the burden of HIV-1 viral DNA in CD4+ TCM and TN 133

Chapter 4: Discussion 141

Appendix 213

Figure 1: In vitro replication of A*74-associated mutations at residue 12 of Matrix engineered into MJ4 213

Figure 2: Normalized RC values of K12E mutations in several Gag-MJ4 chimeric viruses 216

A restriction enzyme based cloning method to assess the in vitro replication capacity of HIV-1 subtype C Gag-MJ4 chimeric viruses 217

Figure 1. Representative gel images depicting electrophoretic separation of PCR products 241

Figure 2. Representative gel image depicting electrophoretic separation of restriction digests for cloning patient gag genes into MJ4 243

Figure 3. Representative gel image depicting electrophoretic separation of restriction digests of purified Gag-MJ4 chimera plasmid DNA 244

Figure 4. Replication of MJ4 and NL4-3 isolates of HIV-1 in the GXR25 cell line at different multiplicities of infection (MOI) 245

Figure 5. Representative range of replication for different Gag-MJ4 chimeras 246

Figure 6. Comparison of intra-assay variation in the radiolabeled reverse transcriptase (RT) quantification assay 247

Figure 7. Reproducibility of the replication assay over time in the GXR25 cell line 248

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