Infectivity was determined through quantification of HIV-1 p24, an HIV capsid protein, in culture supernatants after infection. With progressive time in culture, there was a significant increase in the p24 concentration in culture supernatants of HSCs challenged with both strains of HIV-1, suggesting that HSCs are permissive to HIV infection in vitro (Fig. 1). HIV-IIIB infection of primary HSCs resulted in a four- to eight-fold increase in p24 levels compared with infection with HIV-BaL. Although this observed difference may result from
a specific donor susceptibility to X4, HIV-IIIB is a laboratory X4 isolate recognized by its increased efficiency relative to BaL and other R5 strains in vitro. Therefore, primary HSCs were also challenged with HIV-IIIB at a lower moi of 0.1, with resulting p24 levels comparable to HIV-BaL at a moi of 0.5 (data Small molecule library not shown). To support the physiologic relevance of these findings, primary HSCs were also challenged with HIV primary isolates, both X4-tropic and R5-tropic (Fig. 1E,F). Although a higher p24 level was observed with the X4-tropic primary isolate compared with the R5-tropic virus, a much
larger panel of isolates would be needed to determine whether primary HSCs are more permissive to infection by X4-tropic viruses. To further confirm HIV entry and explore whether HSCs could Pirfenidone mw support HIV gene expression, LX-2 and primary HSCs were exposed to HIV-X4 and HIV-R5 expressing GFP.12 GFP expression indicates different stages of the infectious viral cycle (Fig. 2A). HIV NL-GI is a replication-competent virus carrying an enhanced 上海皓元 GFP gene in place of the nef start codon that reflects early gene expression. HIV Gag-iGFP is an infectious clone that carries the GFP gene in subdomains of Gag; expression of its Gag-GFP fusion during later stages of the HIV replication cycle are indicative of late viral gene expression.12 GFP expression was observed in primary HSCs 48-72 hours after exposure to both viral constructs, indicating viral entry and early and late gene expression. Furthermore, preincubation with AZT, a reverse-transcriptase
inhibitor, blocked HIV-GFP gene expression in HSCs (Fig. 2B). R5-tropic GFP viruses did not show efficient viral entry into HSCs though the available clones are less infectious, making direct comparisons difficult (data not shown). To quantify the percentage of HSCs infected by HIV NL-GI GFP, LX-2 and primary HSCs were analyzed by way of flow cytometry 72 hours after viral exposure. In three independent experiments, 22%-28% of infected cells were positive for GFP expression (Fig. 2C). AZT almost completely abolished GFP expression in HSCs, with levels comparable to noninfected cells by both fluorescence microscopy and flow cytometry (0%-2% positive cells). Cell viability in the presence of AZT was confirmed by bright field microscopy (Fig. 2E) and cell viability assay (Fig. 2F).