Replication of Chimeric Human Immunodeficiency Virus Type 1 (HIV-1) Containing HIV-2 Integrase (IN): Naturally Selected Mutations in IN Augment DNA Synthesis

Abstract

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...

Primitive hematopoietic cells resist HIV-1 infection via p21Waf1/Cip1/Sdi1

“…To assess whether the effect of p21 on HIV-1 replication was virus specific, we studied the infectivity of SIVmac-251, a dual tropic (T- and M-tropic) lentivirus related to HIV-1 (29)(30)(31), in both p21 siRNA-treated primary cells and in CMK cells. A low level of viral antigen p27 was detected (0.103 ± 0.001 ng/ml) in culture supernatant of CD34 + cells at day 7, but this dropped to background levels by day 10 (<0.05 ng/ml; Figure 7A).…”

Cooperation between Reverse Transcriptase and Integrase during Reverse Transcription and Formation of the Preintegrative Complex of Ty1

“…It has also been proposed that the active form of the yeast Saccharomyces cerevisiae retrotransposon Ty3 RT is an ␣/␤ heterodimer (27). In other retroviruses, RT and IN are fully separated by proteolytic processing during virion maturation, but mutations or deletions in the nonconserved domain of IN affect several stages of viral replication other than integration, including the initiation of reverse transcription, the level of cDNA, 3Ј-end DNA processing, or nuclear entry (19,28,40,43). Coimmunoprecipitation experiments with antibodies to RT or IN have demonstrated that the human immunodeficiency virus type 1 (HIV-1) or murine leukemia virus proteins interact physically in vitro (16,17,32).…”

“…Increasing evidence suggests that functional and physical interactions between these two enzymes occur during replication (15,28,40,43). In some retroviruses IN is an integral part of the active enzyme; it is an ␣/␤ heterodimer composed of a short RT (␣) polypeptide and an incompletely processed RT-IN (␤) intermediate in the avian leukosis viruses (18,36) and an oligomer of the ␣3/␤ type in human T-cell leukemia virus type 1 (34).…”

“…It has also been proposed that the active form of the yeast Saccharomyces cerevisiae retrotransposon Ty3 RT is an ␣/␤ heterodimer (27). In other retroviruses, RT and IN are fully separated by proteolytic processing during virion maturation, but mutations or deletions in the nonconserved domain of IN affect several stages of viral replication other than integration, including the initiation of reverse transcription, the level of cDNA, 3Ј-end DNA processing, or nuclear entry (19,28,40,43) In the yeast Saccharomyces cerevisiae retrotransposon Ty1, interactions between IN and RT are also important for the function of RT (37, 39). In vitro, an active Ty1 recombinant protein can be obtained only if amino acid residues encoded by the C-terminal region of IN are fused to the N-terminal domain of RT (37).…”

Subunit-Specific Analysis of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase In Vivo

“…Gag-Pol (35,49,50). In this report, we describe a novel trans-complementation approach that enables the function of each RT subunit to be studied in the context of an infectious virus.…”