Background: Following transmission of HIV-1, the predominant viral quasi-species are those that enter cells exclusively via CCR5 (R5 HIV-1). As disease progresses, in approximately 50% of patients (pts) the viral entry pathway switches to a dual tropic or CXCR4 tropic nature (R5X4 or X4). This switch is rarely seen during the first few years of infection. This may be because resting memory T-cells (RMT) are more susceptible to infection by R5 HIV-1 than by X4 HIV-1. We hypothesize that HIV-1 mediated signaling through CCR5, but not CXCR4, in RMT allows viral replication.

Methods: PBMC were purified from normal donors, stimulated and then cultured without stimulation for 2 wks. These RMT cells were then infected with R5 or X4 HIV-1. Infection was monitored by RT or p24 assay. Quantitative PCR (QPCR) was used to assay completion of each stage of the viral lifecycle. RMTs were stimulated with MIP-1b, R5 HIV-1 or X4 HIV-1 and protein tyrosine phosphorylation was analyzed by Western blot. Signaling pathway inhibitors were added and infection monitored by p24 ELISA. Addition of cellular activators (PMA/Ionomycin) or CCR5 specific ligand (MIP-1b) was used to rescue infection. To determine whether HIV-1 replication is dependent on signaling through CCR5 in RMT, we infected CCR5 D32/D32 PBMC transduced with normal or signal defective CCR5.

Results: Western blots of resting memory T-cells activated with MIP-1b or R5 HIV-1 showed activation of the protein tyrosine kinase, Pyk2. Addition of wortmanin (PI3K inhibitor), or PP2 (c- Src kinase inhibitor) inhibited R5 HIV-1 replication in RMT, but pertussis toxin (Ga_i inhibitor) and NF449 (Ga_s inhibitor) did not. Wortmanin and PP2 inhibition could be rescued by the addition of the cellular activator PMA + ionomycin. CCR5 D32/D32 PBMC transduced with signal defective CCR5 did not support viral replication while cells transduced with normal CCR5 did. Preliminary QPCR results show a decrease in completion of reverse transcription and 2LTR circle production in RMT infected with X4 HIV-1.

Conclusions: Our data implicate R5 HIV-1 mediated signaling via CCR5 in the establishment of infection in normal, resting memory T-cells. In contrast, X4 HIV-1 infection is blocked at a post-entry step in RMT. Signaling through Ga_i and Ga_s do not appear to play a role in the establishment of infection, but the downstream signaling molecules PI3K and c-Src may be important mediators of R5-HIV-1 infection of RMT.