Improved Coreceptor Usage Prediction and GenotypicMonitoring of R5-to-X4 Transition by Motif Analysis of HumanImmunodeficiency Virus Type 1 env V3 LoopSequences

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 sequencebased "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.Early studies of the biological properties of human immunodeficiency virus type 1 (HIV-1) found that virus isolates could be placed into as few as two phenotypic categories (defined in vitro as either non-syncytium-inducing [NSI] or syncytium-inducing [SI]) in certain CD4 ϩ T-cell lines. These phenotypes were often found to be associated with differences in growth properties and cytopathicity on peripheral blood mononuclear cells (PBMC) (1,14,46) and in cellular host range (3,48). Ultimately, the difference between the NSI and SI phenotypes was shown to be due largely to the differential use of chemokine receptors as coreceptors for viral entry: NSI viruses predominantly use CCR5, while SI viruses can use CCR5 and CXCR4 or CXCR4 exclusively (2, 29, 31, 52, 54). Results determined on the basis of SI phenotype and/or coreceptor usage typing showed that although HIV-1 present at primary infections used the CCR5 coreceptor (R5 virus) ϳ90% of the time (63, 67, 68), a substantial proportion of individuals eventually developed virus that used the CXCR4 coreceptor (X4 virus). These X4/SI viruses are associated with accelerated CD4 decline and more rapid progression of HIV-1 disease (8,28,33,43,47). Little is known about the mechanisms by which these viruses come to predominate among the HIV-1 strains present in an infected person. For example, it is no...

R5 and X4 HIV envelopes induce distinct gene expression profiles in primary peripheral blood mononuclear cells

“…Treatment of resting CD4 ϩ T cells with gp120 induces the dephosphorylation and nuclear translocation of NFAT (23). These observations are consistent with gene expression profiles obtained from cell lines, which suggest HIV infection influences cellular pathways that facilitate viral replication (24,25).Here, we further investigated envelope-mediated signaling by comparing the response of PBMCs to gp120s derived from both R5 and X4 strains of HIV. R5 and X4 viruses exhibit markedly different properties in vivo.…”

Variability of the conserved V3 loop tip motif in HIV-1 subtype B isolates collected from Brazilian and French patients

“…Different coreceptor usage and switch from NSI to SI phenotype have been associated with amino acid substitutions in the V3 loop. On the basis of the V3 loop sequence, a bioinformatic tool, the position-specific scoring matrix (PSSM) was created to predict the viral phenotype (4,18,19). This tool demonstrated a high sensitivity to identify X4 viruses in HIV-1 subtype B isolates (17).…”

Development of a Novel Codon-Specific Polymerase Chain Reaction for the Detection of CXCR4-Utilizing HIV Type 1 Subtype B

“…When background was high, usually due to subpopulations with insertions/deletions, only the highest peaks were recorded. All possible amino acid sequences were subjected to webPSSM (http://indra.mullins.microbiol.washington.edu/ webpssm/) 2 and Geno2pheno (http://coreceptor.bioinf.mpiinf.mpg.de/index.php). 3 CS-PCR data were analyzed using the iCycler software.…”

“…10 The identification of basic amino acids at positions 11 and 25 has > 90% specificity for identifying HIV-1 subtype B X4 variants. 2,3 Multiple three-base combinations encode the many amino acids that can be found at positions 11 and 25, including the eight that can encode lysine or arginine. Therefore, no single base can be used to differentiate X4-associated versus R5-associated codons.…”

“…X4 subpopulations are often at low levels-particularly early during disease progression-and even though there are multiple, wellestablished genotypic tools, [2][3][4][5] population-based sequencing can generally detect only variants that are greater than 20%…”