Genotypic prediction of HIV-1 CRF01-AE tropism.

HIV-1 subtype CRF01-AE predominates in south Asia and has spread throughout the world. The virus tropism must be determined before using CCR5 antagonists. Genotypic methods could be used, but the prediction algorithms may be inaccurate for non-B subtypes like CRF01-AE and the correlation with the phenotypic approach has not been assessed. We analyzed 61 CRF01-AE V3 clonal sequences of known phenotype from the GenBank database. The sensitivity of the Geno2pheno10 genotypic algorithm was 91%, but its specificity was poor (54%). In contrast, the combined 11/25 and net charge rule was highly specific (98%) but rather insensitive (64%). We thus identified subtype CRF01-AE determinants in the V3 region that are associated with CXCR4 use and developed a new simple rule for optimizing the genotypic prediction of CRF01-AE tropism. The concordance between the predicted CRF01-AE genotype and the phenotype was 95% for the clonal data set. We then validated this algorithm by analyzing the data from 44 patients infected with subtype CRF01-AE, whose tropism was determined using a recombinant phenotypic entry assay and V3-loop bulk sequencing. The CRF01-AE genotypic tool was 70% sensitive and 96% specific for predicting CXCR4 use, and the concordance between genotype and phenotype was 84%, approaching the concordance obtained for predicting the tropism of HIV-1 subtype B. Genotypic predictions that use a subtype CRF01-AE-specific algorithm appear to be preferable for characterizing coreceptor usage both in pathophysiological studies and for ensuring the appropriate use of CCR5 antagonists.

Genotypic prediction of HIV-1 subtype D tropism.

BACKGROUND: HIV-1 subtype D infections have been associated with rapid disease progression and phenotypic assays have shown that CXCR4-using viruses are very prevalent. Recent studies indicate that the genotypic algorithms used routinely to assess HIV-1 tropism may lack accuracy for non-B subtypes. Little is known about the genotypic determinants of HIV-1 subtype D tropism. RESULTS: We determined the HIV-1 coreceptor usage for 32 patients infected with subtype D by both a recombinant virus phenotypic entry assay and V3-loop sequencing to determine the correlation between them. The sensitivity of the Geno2pheno10 genotypic algorithm was 75% and that of the combined 11/25 and net charge rule was 100% for predicting subtype D CXCR4 usage, but their specificities were poor (54% and 68%). We have identified subtype D determinants in the V3 region associated with CXCR4 use and built a new simple genotypic rule for optimizing the genotypic prediction of HIV-1 subtype D tropism. We validated this algorithm using an independent GenBank data set of 67 subtype D V3 sequences of viruses of known phenotype. The subtype D genotypic algorithm was 68% sensitive and 95% specific for predicting X4 viruses in this data set, approaching the performance of genotypic prediction of HIV-1 subtype B tropism. CONCLUSION: The genotypic determinants of HIV-1 subtype D coreceptor usage are slightly different from those for subtype B viruses. Genotypic predictions based on a subtype D-specific algorithm appear to be preferable for characterizing coreceptor usage in epidemiological and pathophysiological studies.

CXCR4-using viruses in plasma and peripheral blood mononuclear cells during primary HIV-1 infection and impact on disease progression.

OBJECTIVE: Cysteine-cysteine receptor 5 (CCR5)-using viruses classically predominate during HIV-1 primary infection but the frequency of cysteine-X-cysteine receptor 4 (CXCR4)-using viruses varies between studies and could be different between plasma and peripheral blood mononuclear cells (PBMCs). Thus, we determined HIV-1 tropism in both these compartments during primary infection and evaluated the impact of CXCR4-using viruses on disease progression. DESIGN: One hundred and thirty-three patients with primary HIV-1 infection were screened for HIV-1 coreceptor usage in plasma and PBMCs using both genotypic and phenotypic methods. The impact of CXCR4-using viruses' transmission on subsequent disease progression was assessed in a case-control study. METHODS: HIV-1 coreceptor usage was determined using a recombinant virus phenotypic entry assay and V3-based genotypic algorithms. We also monitored CD4(+) T-cell count, clinical events and therapeutic intervention. RESULTS: There was 6.4% of CXCR4-using HIV-1 in plasma during primary infection as measured by a phenotypic assay and combined criteria from the 11/25 and net charge genotypic rules. Geno2pheno10 overestimated the prevalence of CXCR4-using viruses (12%). HIV-1 tropism in plasma and PBMCs was 98% concordant. The HIV-1 RNA load and CD4(+) T-cell count during primary infection were not related to virus tropism. Primary infection with CXCR4-using viruses was associated with an accelerated rate of disease progression, estimated by a faster decline of CD4 T-cell count under 350 cells/microl and by a reduced delay in initiating a first antiretroviral treatment. CONCLUSIONS: Plasma or PBMC samples can be used for determining HIV-1 tropism during primary infection. CXCR4-using viruses are rare during primary infection but increase the risk of disease progression.

Development and performance of a new recombinant virus phenotypic entry assay to determine HIV-1 coreceptor usage.

BACKGROUND: Clinical trials of CCR5 antagonists have relied on the phenotypic determination of HIV-1 coreceptor usage. Few phenotypic assays are available, with few data on their concordance, and none has been designed to determine tropism from cell-associated HIV-1 DNA. OBJECTIVES: To assess the performance of the new Toulouse Tropism Test (TTT) phenotypic assay to characterize HIV-1 tropism using blood plasma and peripheral blood mononuclear cells (PBMC). STUDY DESIGN: 434 plasma and 168 PBMC samples were tested with the TTT assay. We determined the correlation between our assay results on plasma samples and those of the commercial Trofile assay. RESULTS: The TTT assay determined the tropism of 97% of samples after successful amplification of the env gene. It performed well on both cell samples and plasma samples with various HIV-1 loads and subtypes. It detected 0.5% of minor CXCR4-using variants in the virus population. The TTT and the Trofile assays were >90% concordant for predicting HIV-1 tropism. CONCLUSION: We have validated a new recombinant virus phenotypic assay for determining HIV-1 tropism using both plasma and cell samples from patients who are candidates for treatment with CCR5 antagonists.

The polyadenylation site of Mimivirus transcripts obeys a stringent 'hairpin rule'.

Mimivirus, a giant DNA virus infecting Acanthamoeba, is revealing an increasing list of unique features such as a 1.2-Mb genome with numerous genes not found in other viruses, a uniquely conserved promoter signal, and a particle of unmatched complexity using two distinct portals for genome delivery and packaging. Herein, we contribute a further Mimivirus distinctive feature discovered by sequencing a panel of viral cDNAs produced for probing the structure of Mimivirus transcripts. All Mimivirus mRNAs are polyadenylated at a site coinciding exactly with unrelated, but strongly palindromic, genomic sequences. The analysis of 454 Life Sciences (Roche) FLX cDNA tags (150,651) confirmed this finding for all Mimivirus genes independent of their transcription timings and expression levels. The absence of a suitable palindromic signal between adjacent genes results in transcripts encompassing multiple ORFs in the same or even in opposite orientations. Surprisingly, Mimivirus tRNAs are expressed as polyadenylated messengers, including an ORF/tRNA composite mRNA. To our knowledge, both the nature and the stringency of the "hairpin rule" defining the location of polyadenylation sites are unique, raising once more the question of Mimivirus's evolutionary origin. The precise molecular mechanisms implementing the hairpin rule into the 3'-end processing of Mimivirus pre-mRNAs remain to be elucidated.

Genotypic prediction of human immunodeficiency virus type 1 CRF02-AG tropism.

We assessed the performance of genotypic algorithms for predicting the tropism of human immunodeficiency virus type 1 coreceptor usage in 52 patients infected with the CRF02-AG subtype. The combined criteria of the 11/25 and net charge rules accurately detected CXCR4-using CRF02-AG viruses, whereas the Geno2pheno tool lacked sensitivity and the position-specific scoring matrix (PSSM) tool WebPSSM lacked specificity.