Human immunodeficiency virus type 1 (HIV-1) encodes protease (PR), reverse transcriptase (RT), and integrase (IN) as parts of a large Gag-Pol precursor polyprotein (Pr160 Gag-Pol ). Pr160 Gag-Pol plays an important role in virion assembly and is essential for the formation of infectious virions (for a review, see reference 11). Mutagenesis of the C-terminal region of Pr160Gag-Pol (RT and IN domains) has been associated with defects in virion assembly, release, maturation, and protein composition (2,4,7,8,26,31). Consequently, these defective viruses may appear to be impaired in early steps of the virus life cycle, such as uncoating and viral DNA synthesis.Molecular genetic analysis of IN has revealed pleiotropic effects of mutations among different retroviruses. Mutation of nonconserved amino acids within the IN gene of Ty3 (a retrovirus-like element of Saccharomyces cerevisiae) affects multiple stages of the retrotransposition life cycle, including RT and IN expression, 3Ј-end DNA processing, and nuclear entry (23). These mutations also appear to reduce the level of replicated DNA despite normal levels of exogenous RT activity and capsid maturation (23). Certain point mutations or linker insertion mutations in the Moloney murine leukemia virus (MLV) IN domain impair virion production and proteolytic processing of . Similarly, certain HIV-1 IN mutations can cause defects in virion assembly, production, maturation, and nuclear import of the preintegration complex (2-5, 7, 26, 31). Mutations in the HIV-1 IN coding sequence have been shown to impair viral DNA synthesis in infected cells. Mutations that inhibit translation of the entire IN protein or a small portion (22 amino acids) of its carboxy terminus reduce the amount of early viral DNA products detected by PCR, and viruses containing either point mutations in the N-terminal zinc finger or the central domain (F185) exhibit a similar phenotype (7,8,18,20,37 MATERIALS AND METHODS Cells and antibodies.The TZM-bl (previously named JC53-BL) (36), 293T, and JC53 (25) cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U of penicillin per ml, and 0.1 mg of streptomycin per ml (complete DMEM). SupT1 cells were maintained in RPMI 1640 medium supplemented with 15% FBS and 0.1 mg of gentamicin per ml. The anti-HIV-1 capsid monoclonal antibody 183-H12-5C (contributed by B. Chesebro and K. Wehrly) was obtained through the AIDS Research Reference and Reagent Program, National Institutes of Health. The HIV-2 IN protein was detected by using serum (designated 7312A) from an HIV-2-infected individual that was especially reactive to IN by immunoblot analysis.HIV proviral clones and expression plasmids. The HIV-1 pSG3 proviral clone (GenBank accession no. L02317) contains all of the HIV-1 genes with the exception of vpu (10). To facilitate molecular genetic analysis, unique XmaI and BamHI restriction sites were introduced near the beginning (nucleotide 2136) and end (nucleotide 3760) of the RT coding regio...
The human endogenous retrovirus, type K (HERV-K) represents the most biologically active form of known retroelements present in the human genome. Several HERV-K genomes have transcriptionally active open reading frames and encode their own protease (PR). The HERV-K PR has been shown to authentically cleave human immunodeficiency virus type 1 (HIV-1) matrix-capsid peptide in the presence of HIV-1 PR inhibitors. This raised the possibility that HERV-K PR could complement HIV-1 PR function in HIV-1-infected individuals. To investigate this possibility, we fused the HIV-1 vpr gene to the HERV-K PR gene (vpr-PR). The vpr-PR expression plasmid and a PR-defective HIV-1 clone were cotransfected into 293T cells. Progeny virions were assayed for processing of the HIV-1 polyproteins by Western blot and for changes in infectivity. HERV-K PR fused to Vpr was incorporated into HIV-1 virions at a high concentration and cleaved the Gag and Pol precursor proteins. However, neither Gag nor Pol polyproteins were correctly processed. Moreover, the HERV-K PR did not restore virus infectivity. While these results do not exclude the possibility that the HERV-K PR could complement an HIV-1 PR whose function is impaired due to drugs or drug-resistant mutations, they clearly demonstrate that the HERV-K PR cannot substitute for the function of the wild-type HIV-1 PR.
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