DNA was extracted at 2, 6, 12, and 24 h post-infection. Early reverse transcripts and late reverse transcripts were amplified by PCR with specific pairs of primers. At two hours postinfection, amounts of early and late reverse transcripts were similar in DMSO- and CypItreated cells, suggesting that CypI did not affect HIV-1 entry into cells nor the earliest stages of reverse transcription. However, at six hours post-infection and beyond, early and late RT products were very much lower in CypI-treated cells, suggesting impairment in reverse transcription or degradation of newly made cDNA. CypI-treated cells that did not contain late reverse transcripts also did not contain long terminal repeat circles, a hallmark of Potassium clavulanate:cellulose (1:1) nuclear import. These results are in accordance with the hypothesis that CypA plays a critical role in reverse transcription. To further elucidate the effects of CypA-capsid disruption, we analyzed how CPI-431-32 affects the distribution of incoming HIV-1 cores within the first 6 hours of infection as determined by the presence of the HIV-1 integrase ROR gama modulator 1 biological activity protein. TZM cells were infected with HIV-1 NL4.3 for 6 h in the absence or presence of CPI-431-32, then fractionated into cytosolic and nuclear extracts for quantitation of HIV-1 integrase byWestern blotting. Purity of the fractions was confirmed by identifying CypA exclusively in cytosolic extracts and histone 2 exclusively in nuclear extracts. Similar amounts of HIV-1 integrase were found in the cytosol of DMSOtreated and CPI-431-32-treated cells, which is consistent with our previous interpretation that cyclophilin inhibition does not affect viral entry into cells. In contrast, we found that CPI-431-32 completely prevented the nuclear accumulation of integrase. Together these results strongly suggest that CPI-431-32 inhibited HIV-1 infection by interfering with reverse transcription and nuclear import of the viral integration complex. We then investigated the mechanism by which CPI-431-32 inhibits HCV replication. HCV is well known for its ability to reshape intracellular membranes to optimize RNA synthesis, especially by re-organizing the ER membrane close to the nucleus��the so-called membrano
