Even though a 2-fold decrease in nuclear entry by PF74 (Fig. infected in the presence of PF74 exhibited elevated integration activity. PF74s effect on PIC activity is CA specific since the compound did not increase the integration activity of PICs of a PF74-resistant HIV-1 CA mutant. Sucrose gradient-based fractionation studies revealed that PICs assembled in the presence of PF74 contained lower levels of CA, suggesting a negative association between CA and PIC-associated integration activity. Finally, the addition of a CA-specific antibody or PF74 inhibited PIC-associated integration activity. Collectively, our results demonstrate that PF74s targeting of PIC-associated CA results in impaired HIV-1 integration. IMPORTANCE Antiretroviral therapy (ART) that uses various combinations of small molecule inhibitors has been highly effective in controlling HIV. However, the drugs used in the ART regimen are expensive, cause side effects, and face viral resistance. The HIV-1 CA plays critical roles in the virus life cycle and is an attractive therapeutic target. While currently there is no CA-based therapy, highly potent CA-specific inhibitors are being developed as a new class of antivirals. Efforts to develop a CA-targeted therapy can be aided through a clear understanding of the role of CA in HIV-1 infection. CA is well established to coordinate reverse transcription and nuclear entry of the virus. However, the role of CA in post-nuclear entry steps of HIV-1 infection is poorly understood. We show that a CA-specific drug PF74 inhibits HIV-1 integration revealing a novel role of this multifunctional viral protein in a post-nuclear entry step of HIV-1 infection. (16). In addition, the infectivity defect of the CA mutants A92E and G94D was attributed to impaired chromosomal integration (44, 45). The altered integration efficiency and integration site preferences rendered by the N74D mutation in CA (46, 47) and the retargeted integration by the CypA/RanBP2-independent CA mutant P90A (47, 48) suggest an intranuclear role for CA (38, 46, 47). The recent report that the CA-binding cellular cofactor CPSF6 enables targeting of the vDNA into gene dense regions further supports a role of CA in post-nuclear entry steps of HIV-1 infection (49, 50). Studies of CA-targeting small molecule inhibitors also support CAs role in post-nuclear entry steps of HIV-1 (51, 52). The CA-specific inhibitor PF74 inhibits HIV-1 replication and exhibits no inhibitory effect on HIV-1 protease or reverse transcriptase (RT) (53). Selection for drug resistance has identified substitutions AM-2099 in CA that confer resistance to the antiviral effects AM-2099 of PF74 (53,C55). PF74 binds at the interface of CA between the N-terminal domain (NTD) and the C-terminal domain (CTD) and inhibits HIV-1 infection by blocking reverse transcription and nuclear entry (53, 54, 56). A recent report shows that PF74 treatment affects distribution of viral DNA integration into the host genome (57). The less potent antiviral compound BI-2 also binds to the same pocket within the CA targeted by PF74 (58). Interestingly, BI-2 does not affect reverse transcription but reduces nuclear entry of HIV-1 (58). Another study attributed that the antiviral effect of coumermycin A1 (C-A1) is linked to integration and Rabbit polyclonal to TRAP1 the inhibitory mechanism may depend on the binding of the compound to CA (59). Although these studies suggest an intranuclear role for CA, AM-2099 biochemical data demonstrating a direct role of CA in HIV-1 integration are lacking. We probed a direct link between CA and integration by combining the use of PF74 as a pharmacologic probe with the measurement of integration activity of HIV-1 PICs isolated from acutely infected cells. At 2?M, PF74 (54) markedly inhibited HIV-1 infection without affecting reverse transcription. Although PF74 reduced nuclear entry, the level of reduction in nuclear entry did not account for the 95% inhibition in infection, indicating that PF74 also blocks a post-nuclear entry step(s). Interestingly, PF74 reduced proviral integration, and this reduction correlated quantitatively with antiviral activity. Unexpectedly, PICs extracted from cells cultured in the presence of PF74 retained higher integration activity (72, 75). We predicted that PICs recovered from infected cells treated with 2?M PF74 would be impaired for integration activity since the inhibitor did not.