We found no expression of CD4, CXCR4, or CCR5 on any of the cell lines (Fig. (Fig.2A2A [data are from HepG2 cells only]). Expression of CD4, CXCR4, and CCR5 was clearly demonstrated in TZM-bl cells (Fig. (Fig.2A,2A, lower panels). To determine if these coreceptors mediated infection despite lack of detectable surface expression, we infected AD38 cells with either under NL4-3 or AD8 in the presence or absence of a CXCR4 antagonist, AMD3100, or a CCR5 antagonist, maraviroc (Fig. (Fig.2B).2B). Following infection with NL4-3, infection was inhibited with AMD3100 but not maraviroc, demonstrating entry via CXCR4. Similarly, following infection with AD8, infection was inhibited with maraviroc but not AMD3100. These data demonstrate that HIV enters AD38 hepatic cell lines via CXCR4 or CCR5, despite the inability to detect surface expression of each coreceptor.

High-level HIV infection with VSV-NLNE-pseudotyped virus. We then wanted to determine what effects HIV replication within a hepatocyte would have on the HBV life cycle. In order to increase the level of HIV infection and to directly visualize HIV-infected hepatic cells, we infected hepatic cell lines with VSV-NLNE-pseudotyped virus. High-level infection was determined by measurement of HIV RT in addition to detection of EGFP by flow cytometry and fluorescence microscopy (peak = 51%) (Fig. (Fig.3A)3A) and was achieved at levels similar to that seen in the TZM-bl cell line (data not shown). EGFP expression in VSV-NLNE-infected AD38 cells also expressing HBcAg was demonstrated using fluorescence microscopy (Fig. (Fig.3B).3B).

Following infection of the AD38 cell line (n = 3) with VSV-NLNE, the peak RT level (mean �� standard error [SE]) was 6,366 �� 534 cpm (Fig. (Fig.3C).3C). Despite this high-level infection, cell proliferation determined using the MTS assay was similar to that in uninfected AD38 cells (Fig. (Fig.3D),3D), although we did not measure apoptosis specifically. In contrast, we observed toxicity in TZM-bl Batimastat cells at higher concentrations of VSV-NLNE (Fig. (Fig.3D).3D). Therefore, the use of VSV-NLNE allowed for high-level infection of HBV-expressing hepatic cell lines without significant cell death. This model was then used to evaluate any changes in the HBV life cycle following HIV coinfection. FIG. 3. Infection of hepatic cell lines with VSV-NLNE. (A and B) AD38 and TZM-bl cells were infected with VSV-NLNE, and infection was quantified by detection of EGFP using flow cytometry (A) or fluorescence microscopy (B). An AD38 cell is shown. Hoechst staining … Effect of high-level HIV infection on HBV expression.