Longitudinal ultradeep characterization of HIV type 1 R5 and X4 subpopulations in patients followed from primary infection to coreceptor switch.

In early infection HIV-1 generally uses the CCR5 coreceptor. During disease progression the coreceptor use switches to include CXCR4 in approximately 70% of infected individuals. The primary determinant for coreceptor use is located in the V3 loop of the viral envelope. Here, ultradeep pyrosequencing (UDPS) of the V3 loop was used to investigate if CXCR4-using (X4) virus may be present as a minority population during primary HIV infection (PHI). Three patients with HIV populations that switched coreceptor use, as determined by the MT-2 cell culture assay, were investigated. Longitudinally collected plasma samples (four to nine samples per patient) obtained from PHI until after coreceptor switch were analyzed by UDPS of the V3 loop. From each sample between 279 and 32,094 reads were generated based on template molecule availability. UDPS analysis showed that the X4 virus that emerged after switch was not present during PHI or prior to overt phenotypic switch. In addition, the phylogenetic analyses indicated that the X4 populations originated from R5 variants that had evolved after the previous R5-only sample was obtained. Finally, one to three major variants were found during PHI, supporting the idea that infection is established with one or just a few viral particles.