Angélique B. van 't Wout

Improved coreceptor usage prediction and genotypic monitoring of R5-to-X4 transition by motif analysis of human immunodeficiency virus type 1 env V3 loop sequences.

ABSTRACT Early in infection, human immunodeficiency virus type 1 (HIV-1) generally uses the CCR5 chemokine receptor (along with CD4) for cellular entry. In many HIV-1-infected individuals, viral genotypic changes arise that allow the virus to use CXCR4 (either in addition to CCR5 or alone) as an entry coreceptor. This switch has been associated with an acceleration of both CD3+ T-cell decline and progression to AIDS. While it is well known that the V3 loop of gp120 largely determines coreceptor usage and that positively charged residues in V3 play an important role, the process of genetic change in V3 leading to altered coreceptor usage is not well understood. Further, the methods for biological phenotyping of virus for research or clinical purposes are laborious, depend on sample availability, and present biosafety concerns, so reliable methods for sequence-based“ virtual phenotyping” are desirable. We introduce a simple bioinformatic method of scoring V3 amino acid sequences that reliably predicts CXCR4 usage (sensitivity, 84%; specificity, 96%). This score (as determined on the basis of position-specific scoring matrices [PSSM]) can be interpreted as revealing a propensity to use CXCR4 as follows: known R5 viruses had low scores, R5X4 viruses had intermediate scores, and X4 viruses had high scores. Application of the PSSM scoring method to reconstructed virus phylogenies of 11 longitudinally sampled individuals revealed that the development of X4 viruses was generally gradual and involved the accumulation of multiple amino acid changes in V3. We found that X4 viruses were lost in two ways: by the dying off of an established X4 lineage or by mutation back to low-scoring V3 loops.

Cellular Gene Expression upon Human Immunodeficiency Virus Type 1 Infection of CD4+-T-Cell Lines

ABSTRACT The expression levels of ∼4,600 cellular RNA transcripts were assessed in CD4+-T-cell lines at different times after infection with human immunodeficiency virus type 1 strain BRU (HIV-1BRU) using DNA microarrays. We found that several classes of genes were inhibited by HIV-1BRU infection, consistent with the G2 arrest of HIV-1-infected cells induced by Vpr. These included genes involved in cell division and transcription, a family of DEAD-box proteins (RNA helicases), and all genes involved in translation and splicing. However, the overall level of cell activation and signaling was increased in infected cells, consistent with strong virus production. These included a subgroup of transcription factors, including EGR1 and JUN, suggesting they play a specific role in the HIV-1 life cycle. Some regulatory changes were cell line specific; however, the majority, including enzymes involved in cholesterol biosynthesis, of changes were regulated in most infected cell lines. Compendium analysis comparing gene expression profiles of our HIV-1 infection experiments to those of cells exposed to heat shock, interferon, or influenza A virus indicated that HIV-1 infection largely induced specific changes rather than simply activating stress response or cytokine response pathways. Thus, microarray analysis confirmed several known HIV-1 host cell interactions and permitted identification of specific cellular pathways not previously implicated in HIV-1 infection. Continuing analyses are expected to suggest strategies for impacting HIV-1 replication in vivo by targeting these pathways.

A Reliable Phenotype Predictor for Human Immunodeficiency Virus Type 1 Subtype C Based on Envelope V3 Sequences

ABSTRACT In human immunodeficiency virus type 1 (HIV-1) subtype B infections, the emergence of viruses able to use CXCR4 as a coreceptor is well documented and associated with accelerated CD4 decline and disease progression. However, in HIV-1 subtype C infections, responsible for more than 50% of global infections, CXCR4 usage is less common, even in individuals with advanced disease. A reliable phenotype prediction method based on genetic sequence analysis could provide a rapid and less expensive approach to identify possible CXCR4 variants and thus increase our understanding of subtype C coreceptor usage. For subtype B V3 loop sequences, genotypic predictors have been developed based on position-specific scoring matrices (PSSM). In this study, we apply this methodology to a training set of 279 subtype C sequences of known phenotypes (228 non-syncytium-inducing [NSI] CCR5+ and 51 SI CXCR4+ sequences) to derive a C-PSSM predictor. Specificity and sensitivity distributions were estimated by combining data set bootstrapping with leave-one-out cross-validation, with random sampling of single sequences from individuals on each bootstrap iteration. The C-PSSM had an estimated specificity of 94% (confidence interval [CI], 92% to 96%) and a sensitivity of 75% (CI, 68% to 82%), which is significantly more sensitive than predictions based on other methods, including a commonly used method based on the presence of positively charged residues (sensitivity, 47.8%). A specificity of 83% and a sensitivity of 83% were achieved with a validation set of 24 SI and 47 NSI unique subtype C sequences. The C-PSSM performs as well on subtype C V3 loops as existing subtype B-specific methods do on subtype B V3 loops. We present bioinformatic evidence that particular sites may influence coreceptor usage differently, depending on the subtype.

Nef Induces Multiple Genes Involved in Cholesterol Synthesis and Uptake in Human Immunodeficiency Virus Type 1-Infected T Cells

ABSTRACT Several recent reports indicate that cholesterol might play an important role in human immunodeficiency virus type 1 (HIV-1) replication. We investigated the effects of HIV-1 infection on cholesterol biosynthesis and uptake using microarrays. HIV-1 increased gene expression of cholesterol genes in both transformed T-cell lines and primary CD4+ T cells. Consistent with our microarray data, 14C-labeled mevalonate and acetate incorporation was increased in HIV-1-infected cells. Our data also demonstrate that changes in cholesterol biosynthesis and uptake are only observed in the presence of functional Nef, suggesting that increased cholesterol synthesis may contribute to Nef-mediated enhancement of virion infectivity and viral replication.

Association of HLA-C and HCP5 gene regions with the clinical course of HIV-1 infection

Background:Recently, a genome-wide association analysis revealed single-nucleotide polymorphisms (SNPs) in the gene regions of HLA-C and HCP5 to be associated with viral load at set point and SNPs in the RNF39/ZNRD1 gene region to be associated with HIV-1 disease course. Methods:We studied whether the association of these SNPs with viral load at set point could be replicated and whether these SNPs also associated with other clinical outcomes of HIV-1 infection in 335 HIV-1-infected homosexual participants from the Amsterdam Cohort Studies on HIV infection and AIDS (ACS). Results:Significant associations between the minor allele variants of SNPs HLA-C rs9264942 and HCP5 rs2395029 and a lower viral load at set point could be replicated in the ACS. Moreover, these SNPs were significantly associated with delayed progression to AIDS, AIDS-related death, and a CD4+ T-cell count below 400 cells/μl. Both minor allele variants were independent predictors of disease progression, also when a CCR5 Δ32 heterozygous genotype was included in the analysis. However, predictive value was not independent from viral load and CD4+ T-cell count at set point. The SNPs in the RNF39/ZNRD1 gene region were associated with set point CD4+ T-cell count but not with disease course in the ACS. Conclusion:The minor allele variants of SNPs in the HLA-C and HCP5 gene regions are also in the ACS associated with a lower viral load at set point and additionally with delayed HIV-1 disease progression. The association of these SNPs with the relatively early course of infection may help unravel their mode of action.

Clinical significance of HIV-1 coreceptor usage

The identification of phenotypically distinct HIV-1 variants with different prevalence during the progression of the disease has been one of the earliest discoveries in HIV-1 biology, but its relevance to AIDS pathogenesis remains only partially understood. The physiological basis for the phenotypic variability of HIV-1 was elucidated with the discovery of distinct coreceptors employed by the virus to infect susceptible cells. The role of the viral phenotype in the variable clinical course and treatment outcome of HIV-1 infection has been extensively investigated over the past two decades. In this review, we summarize the major findings on the clinical significance of the HIV-1 coreceptor usage.

HIV-1 Variation before Seroconversion in Men Who Have Sex with Men: Analysis of Acute/Early HIV Infection in the Multicenter AIDS Cohort Study

Understanding the characteristics of human immunodeficiency virus (HIV) necessary for infection in a new host is a critical goal for acquired immunodeficiency syndrome (AIDS) research. We studied the characteristics of HIV-1 envelope genes in 38 men in the Multicenter AIDS Cohort Study cohort before seroconversion. We found a range of diversity (0.2%-5.6% [median, 0.86%]), V1-V2 loop length (58-93 aa), and potential N-linked glycosylation sites (n = 2-9). However, at least 46% of the men had replicating virus that appeared to have been derived from a single viral variant. Nearly all variants were predicted to be CCR5 tropic. We found no correlation between these viral characteristics and the HIV outcomes of time to clinical AIDS or death and/or a CD4 cell count <200 cells/microL.

Isolation and propagation of HIV-1 on peripheral blood mononuclear cells

Human immunodeficiency virus type 1 (HIV-1) infection is characterized by a gradual loss of CD4+ T cells and T-cell function and an ongoing high level of virus replication. The high replication rate and the error-prone nature of HIV-1 reverse transcriptase create a diverse viral quasispecies throughout infection. To study biological properties of HIV-1 quasispecies in relation to the clinical course of infection, the in vitro preservation of phenotypical characteristics of the virus is essential. Here, we describe the method for bulk isolation of the HIV-1 quasispecies and a limiting dilution virus isolation protocol by which single coexisting HIV-1 variants can be obtained using peripheral blood mononuclear cells from a healthy donor as target cells. In addition, methods for propagation and titration of HIV-1 are provided.

Detection of inferred CCR5- and CXCR4-using HIV-1 variants and evolutionary intermediates using ultra-deep pyrosequencing.

The emergence of CXCR4-using human immunodeficiency virus type 1 (HIV-1) variants is associated with accelerated disease progression. CXCR4-using variants are believed to evolve from CCR5-using variants, but due to the extremely low frequency at which transitional intermediate variants are often present, the kinetics and mutational pathways involved in this process have been difficult to study and are therefore poorly understood. Here, we used ultra-deep sequencing of the V3 loop of the viral envelope in combination with the V3-based coreceptor prediction tools PSSMNSI/SI and geno2pheno[coreceptor] to detect HIV-1 variants during the transition from CCR5- to CXCR4-usage. We analyzed PBMC and serum samples obtained from eight HIV-1-infected individuals at three-month intervals up to one year prior to the first phenotypic detection of CXCR4-using variants in the MT-2 assay. Between 3,482 and 10,521 reads were generated from each sample. In all individuals, V3 sequences of predicted CXCR4-using HIV-1 were detected at least three months prior to phenotypic detection of CXCR4-using variants in the MT-2 assay. Subsequent analysis of the genetic relationships of these V3 sequences using minimum spanning trees revealed that the transition in coreceptor usage followed a stepwise mutational pathway involving sequential intermediate variants, which were generally present at relatively low frequencies compared to the major predicted CCR5- and CXCR4-using variants. In addition, we observed differences between individuals with respect to the number of predicted CXCR4-using variants, the diversity among major predicted CCR5-using variants, and the presence or absence of intermediate variants with discordant phenotype predictions. These results provide the first detailed description of the mutational pathways in V3 during the transition from CCR5- to CXCR4-usage in natural HIV-1 infection.

Reduced Prevalence of the CCR5 Δ32 Heterozygous Genotype in Human Immunodeficiency Virus-Infected Individuals with AIDS Dementia Complex

Heterozygosity for a 32-bp deletion in the CCR5 gene (CCR5 Delta32), which encodes the coreceptor for macrophage-tropic non-syncytium-inducing (NSI) human immunodeficiency virus type 1 (HIV-1) variants, results in a lower CCR5 expression and reduced NSI HIV-1 replication. Because infection of macrophages and microglial cells by NSI HIV-1 is considered to be instrumental for the development of AIDS dementia complex (ADC), we studied whether the CCR5 Delta32 heterozygous genotype correlated with a reduced frequency of ADC. Two (4.1%) of 49 patients with ADC versus 27 (14. 5%) of 186 AIDS patients without ADC were heterozygous for CCR5 Delta32 (P=.05). In contrast, a point mutation in the first transmembrane domain of CCR2 (CCR2 64I) did not show this protective effect (P=.57). The reduced prevalence of the CCR5 Delta32 allele among patients with ADC may indicate a reduced or absent reservoir of macrophage-tropic NSI HIV-1 in the brain of CCR5 Delta32 heterozygotes.