title: Characterization of CXCR4-using HIV-1 during primary infection by ultra-deep pyrosequencing.
abstract: OBJECTIVE
  R5 viruses have long been thought to account for almost all strains present in primary HIV-1 infections (PHIs), but recent studies using sensitive phenotypic assays have revealed that 3%-6.4% of subjects also harbour CXCR4-using viruses. Phenotypic assays provide only qualitative results: the presence or absence of CXCR4-using viruses. We have therefore used ultra-deep pyrosequencing (UDS) to determine the frequency of CXCR4-using viruses among HIV-1 quasispecies.
  
  
  METHODS
  We first screened 200 patients for HIV-1 tropism using a sensitive phenotypic assay during PHI and identified 11 infected with an R5X4 dual/mixed (D/M) virus population. We then used UDS of the V3 env region with the geno2pheno algorithm (false positive rate = 5.75) to identify the HIV-1 quasispecies.
  
  
  RESULTS
  CXCR4-using viruses were detected in all but 1 of the 11 patients by UDS, and accounted for 0.2%-100% of the virus populations. The frequency of CXCR4-using viruses was <20% in six subjects and 100% in four subjects. Virus populations containing 100% CXCR4-using variants during PHI persisted for at least 1-2 years after the acute phase. The CCR5 Δ32 heterozygous genotype was similarly prevalent in patients infected with D/M (27%) and R5 (15%) viruses.
  
  
  CONCLUSIONS
  UDS and the phenotype were concordant for determining HIV-1 coreceptor usage. UDS analysis indicated large differences in the percentage of CXCR4-using viruses in the HIV-1 quasispecies during PHI. Further studies should examine the impact of the proportion of CXCR4-using viruses on disease prognosis.
authors.affiliation: INSERM, UMR1043, Toulouse, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Raymond
authors.forename: Adrien
authors.initials: A
authors.lastname: Saliou
authors.forename: Florence
authors.initials: F
authors.lastname: Nicot
authors.forename: Pierre
authors.initials: P
authors.lastname: Delobel
authors.forename: Martine
authors.initials: M
authors.lastname: Dubois
authors.forename: Romain
authors.initials: R
authors.lastname: Carcenac
authors.forename: Karine
authors.initials: K
authors.lastname: Sauné
authors.forename: Bruno
authors.initials: B
authors.lastname: Marchou
authors.forename: Patrice
authors.initials: P
authors.lastname: Massip
authors.forename: Jacques
authors.initials: J
authors.lastname: Izopet
mesh_terms: D000328:Adult; D005260:Female; D014644:Genetic Variation; D015699:HIV Envelope Protein gp120 / Q000235:genetics; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000145:classification / Q000235:genetics* / Q000502:physiology*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D012367:RNA, Viral / Q000235:genetics*; D019718:Receptors, CXCR4 / Q000378:metabolism*; D015728:Receptors, HIV / Q000378:metabolism; D056189:Viral Tropism; D055815:Young Adult
chemical_list: C527276:CXCR4 protein, human; D015699:HIV Envelope Protein gp120; D012367:RNA, Viral; D019718:Receptors, CXCR4; D015728:Receptors, HIV; C514793:gp120 protein, Human immunodeficiency virus 1
keywords: HIV envelope protein gp120; HIV quasispecies; HIV tropism; V3 genotype; chemokine receptor