Summary The cellular and viral determinants required for HIV-1 infection of nondividing cells have been a subject of intense scrutiny. Here we identify the 68 kDa subunit of cleavage factor Im, CPSF6, as an inhibitor of HIV-1 infection. When enriched in the cytoplasm by high level expression or mutation, CPSF6 prevents nuclear entry of the virus. Similar to TRIM5 and Fv1 type restrictions, CPSF6 targets the viral capsid (CA). N74D mutation of the HIV-1 CA leads to a loss of interaction with CPSF6 and evasion of the nuclear import restriction. Interestingly, N74D mutation of CA changes HIV-1 nucleoporin (NUP) requirements. Whereas wild-type HIV-1 requires NUP153, N74D HIV-1 mimics the NUP requirements of feline immunodeficiency virus (FIV) and is more sensitive to NUP155 depletion. These findings reveal a remarkable flexibility in HIV-1 nuclear transport and highlight a single residue in CA as essential in regulating interactions with NUPs.

The rapid emergence of drug-resistant variants of human immunodeficiency virus, type 1 (HIV-1), has limited the efficacy of anti-acquired immune deficiency syndrome (AIDS) treatments, and new lead compounds that target novel binding sites are needed. We have determined the 3.15 Å resolution crystal structure of HIV-1 reverse transcriptase (RT) complexed with dihydroxy benzoyl naphthyl hydrazone (DHBNH), an HIV-1 RT RNase H (RNH) inhibitor (RNHI). DHBNH is effective against a variety of drug-resistant HIV-1 RT mutants. While DHBNH has little effect on most aspects of RT-catalyzed DNA synthesis, at relatively high concentrations it does inhibit the initiation of RNAprimed DNA synthesis. Although primarily an RNHI, DHBNH binds >50 Å away from the RNH active site, at a novel site near both the polymerase active site and the non-nucleoside RT inhibitor (NNRTI) binding pocket. When DHBNH binds, both Tyr181 and Tyr188 remain in the conformations seen in unliganded HIV-1 RT. DHBNH interacts with conserved residues (Asp186, Trp229) and has substantial interactions with the backbones of several less well-conserved residues. On the basis of this structure, we designed substituted DHBNH derivatives that interact with the NNRTI-binding pocket. These compounds inhibit both the polymerase and RNH activities of RT.Human immunodeficiency virus, type 1 (HIV-1), reverse transcriptase (RT) is essential for HIV replication. RT converts the single-stranded viral genomic RNA into a linear doublestranded DNA that can be integrated into the host chromosomes (reviewed in ref 1). The enzyme has two activities, (i) a DNA polymerase that can use either RNA or DNA as a template and (ii) an RNase H (RNH) that selectively degrades the RNA strand of an RNA-DNA heteroduplex. The RNH activity of RT is required for virus replication; cellular RNH cannot substitute for the retroviral enzyme (2). The RNH activity degrades the genomic RNA during first-strand ("minus-strand") DNA synthesis, which allows the newly synthesized DNA to be used as a template for second-strand ("plus-strand") DNA synthesis.HIV-1 RT is a heterodimer consisting of 66 kDa (p66) and 51 kDa (p51) subunits. The two polypeptide chains have 440 N-terminal amino acid residues in common. These comprise four polymerase subdomains: the thumb, palm, fingers, and connection (3,4). The C-terminus of p66 contains an additional 120 amino acid residues that form the bulk of the RNH domain. Despite having identical N-terminal sequences, the arrangement of the subdomains in the two subunits differs dramatically. The p66 subunit contains a large cleft formed by the fingers, palm, and thumb subdo-mains that can accommodate double-stranded nucleic acid templateprimers (3-6). Although the p51 subunit contains the same four subdomains, it does not form a nucleic acid binding cleft.Because of its pivotal role in the HIV life cycle, HIV RT is a primary target for antiretroviral agents. All RT inhibitors currently approved for the treatment of acquired immune deficiency syndrome (AIDS) inhibit...

The amount of excess polymerase and RNase H activity in human immunodeficiency virus type 1 virions was measured by using vectors that undergo a single round of replication. Vectors containing wild-type reverse transcriptase (RT), vectors encoding the D110E mutation to inactivate polymerase, and vectors encoding mutations D443A and E478Q to inactivate RNase H were constructed. 293 cells were cotransfected with different proportions of plasmids encoding these vectors to generate phenotypically mixed virions. The resulting viruses were used to infect human osteosarcoma cells, and the relative infectivity of the viruses was determined by measuring transduction of the murine cell surface marker CD24, which is encoded by the vectors. The results indicated that there is an excess of both polymerase and RNase H activities in virions. Viral replication was reduced to 42% of wild-type levels in virions with where half of the RT molecules were predicted to be catalytically active but dropped to 3% of wild-type levels when 25% of the RT molecules were active. However, reducing RNase H activity had a lesser effect on viral replication. As expected, based on previous work with murine leukemia virus, there was relatively inefficient virus replication when the RNase H and polymerase activities were encoded on separate vectors (D110E plus E478Q and D110E plus D443A). To determine how virus replication failed when polymerase and RNase H activities were reduced, reverse transcription intermediates were measured in vector-infected cells by using quantitative real-time PCR. The results indicated that using the D11OE mutation to reduce the amount of active polymerase reduced the number of reverse transcripts that were initiated and also reduced the amounts of products from the late stages of reverse transcription. If the E478Q mutation was used to reduce RNase H activity, the number of reverse transcripts that were initiated was reduced; there was also a strong effect on minus-strand transfer.The viral enzyme reverse transcriptase (RT) converts the single-stranded genomic RNA found in retroviruses into double-stranded DNA. RT contains two enzymatic activities that collaborate in this conversion: a DNA polymerase that can copy either an RNA or a DNA template, and RNase H, which cleaves RNA if (and only if) it is part of an RNA-DNA hybrid (see references 6, 25, and 66 for reviews). Both the polymerase and RNase H activities are necessary for retroviral replication; mutations that block RNase H activity also block virus replication (47,49,56,61). RNase H activity is required during several steps of reverse transcription. RNase H degradation is required for the first-strand transfer reaction and generates the RNA primer for plus-strand DNA synthesis. RNase H also removes the RNA primers used to initiate plus-and minusstrand DNA synthesis (15, 18, 37, 38, 44-46, 51, 58, 62).Human immunodeficiency virus type 1 (HIV-1) RT is a heterodimeric protein; the two subunits are p66 and p51 (10). The p66 subunit contains 560 amino acids, and the p5...

Retroviral reverse transcriptases contain a DNA polymerase activity that can copy an RNA or DNA template and an RNase H activity that degrades the viral RNA genome during reverse transcription. RNase H makes both specific and nonspecific cleavages; specific cleavages are used to generate and remove the polypurine tract primer used for plus-strand DNA synthesis and to remove the tRNA primer used for minus-strand DNA synthesis. We generated mutations in an HIV-1-based vector to change amino acids in the RNase H domain that contact either the RNA and DNA strands. Some of these mutations affected the initiation of DNA synthesis, demonstrating an interdependence of the polymerase and RNase H activities of HIV-1 reverse transcription during viral DNA synthesis. The ends of the linear DNA form of the HIV-1 genome are defined by the specific RNase H cleavages that remove the plus-and minus-strand primers; these ends can be joined to form two-longterminal repeat circles. Analysis of two-long-terminal repeat circle junctions showed that mutations in the RNase H domain affect the specificity of RNase H cleavage.H IV-1 reverse transcriptase (RT) is the virally encoded enzyme that converts the single-stranded RNA genome found in virions into double-stranded DNA. The conversion of the RNA genome into DNA is accomplished through the collaboration of the two enzymatic activities of RT: a DNA polymerase that can use either RNA or DNA as a template and an RNase H that cleaves RNA if (and only if) it is present in an RNA⅐DNA duplex (reviewed in refs. 1-3). Both polymerase and RNase H activities are required for the conversion of the RNA genome into double-stranded DNA; mutations that inactivate either the polymerase or RNase H block viral replication (4-7).In vitro assays showed that polymerase and RNase H activities of HIV-1 RT are interdependent (8-13). Both the polymerase and RNase H domains of HIV-1 RT simultaneously contact the nucleic acid substrate and contribute to the binding (and proper positioning) of the nucleic acid. In in vitro assays, mutations in the RNase H domain can affect polymerase activity (and vice versa). We found that reducing RNase H activity by cotransfecting a mixture of wild-type HIV-1 vector DNA and DNA encoding HIV-1 vectors with mutations in the RNase H active site reduced the efficiency of initiation of DNA synthesis (14). On the basis of these observations, we wanted to determine whether mutating amino acids that make specific contacts with the nucleic acid substrate in the vicinity of the RNase H active site would also affect the initiation of DNA synthesis and͞or the specificity of RNase H cleavage.Reverse transcription requires that RNase H make both specific and nonspecific cleavages. The process of reverse transcription is shown schematically in Fig. 1 (reviewed in refs. 1 and 3). The RNase H cleavages that remove the tRNA primer, and the cleavages that generate and remove the polypurine tract (ppt) primer define the ends of the unintegrated linear viral DNA that is the substrate for the int...