FIG 9.

Entry of VSV-G-pseudotyped HIV-1 is not affected by Nullbasic. HEK293 cells were treated with 1 μM nevirapine (to prevent reverse transcription and degradation of viral mRNA by RNase H activity) for 2 h at 37°C and then incubated with VSV-G-pseudotyped HIV-1 or HIV-1-Nullbasic viral supernatant containing 500 ng of CA for 2 h at 4°C to facilitate virus attachment. Heat-treated virus was used as a control for attachment and entry. The cells were incubated at 37°C to initiate envelope and cell membrane fusion, followed by incubation for a further 2 h. Total cellular RNA was collected and analyzed by RT-PCR for HIV-1 full-length mRNA and cellular eEF1A mRNA in the presence or absence of RT. The relative copy number of HIV-1 RNA was normalized to levels of eEF1A mRNA in each sample. A negative control without cDNA template was included in every assay. All PCRs were performed in triplicate. The experiment was performed twice with similar results. The results of a representative experiment are shown. NB− and NB+, the absence and presence of Nullbasic, respectively.