Incorporation of human immunodeficiency virus type 1 Gag proteins into murine leukemia virus virions.

The retroviral Gag polyprotein is necessary and sufficient for assembly and budding of viral particles. However, the exact inter- and intramolecular interactions of the Gag polyproteins during this process are not known. To locate functional domains within Gag, we generated chimeric proviruses between human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MuLV). In these chimeric proviruses, the matrix or capsid proteins of MuLV were precisely replaced with the matrix or capsid proteins of HIV-1. Although the chimeric proviruses were unable to efficiently assemble into mature viral particles by themselves, coexpression of wild-type MuLV Gag rescued the HIV proteins into virions. The specificity of the rescue of HIV proteins into MuLV virions shows that specific interactions involving homologous matrix or capsid regions of Gag are necessary for retroviral particle formation.     

The LEM domain proteins emerin and LAP2alpha are dispensable for human immunodeficiency virus type 1 and murine leukemia virus infections

The human nuclear envelope proteins emerin and lamina-associated polypeptide 2alpha (LAP2alpha) have been proposed to aid in the early replication steps of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). However, whether these factors are essential for HIV-1 or MLV infection has been questioned. Prior studies in which conflicting results were obtained were highly dependent on RNA interference-mediated gene silencing. To shed light on these contradictory results, we examined whether HIV-1 or MLV could infect primary cells from mice deficient for emerin, LAP2alpha, or both emerin and LAP2alpha. We observed HIV-1 and MLV infectivity in mouse embryonic fibroblasts (MEFs) from emerin knockout, LAP2alpha knockout, or emerin and LAP2alpha double knockout mice to be comparable in infectivity to wild-type littermate-derived MEFs, indicating that both emerin and LAP2alpha were dispensable for HIV-1 and MLV infection of dividing, primary mouse cells. Because emerin has been suggested to be important for infection of human macrophages by HIV-1, we also examined HIV-1 transduction of macrophages from wild-type mice or knockout mice, but again we did not observe a difference in susceptibility. These findings prompted us to reexamine the role of human emerin in supporting HIV-1 and MLV infection. Notably, both viruses efficiently infected human cells expressing high levels of dominant-negative emerin. We thus conclude that emerin and LAP2alpha are not required for the early replication of HIV-1 and MLV in mouse or human cells.     

Genetic analysis of the human immunodeficiency virus type 1 integrase protein.

Single-amino-acid changes in a highly conserved central region of the human immunodeficiency virus type 1 (HIV-1) integrase protein were analyzed for their effects on viral protein synthesis, virion morphogenesis, and viral replication. Alteration of two amino acids that are invariant among retroviral integrases, D116 and E152 of HIV-1, as well as a mutation of the highly conserved amino acid S147 blocked viral replication in two CD4+ human T-cell lines. Mutations of four other highly conserved amino acids in the region had no detectable effect on viral replication, whereas mutations at two positions, N117 and Y143, resulted in viruses with a delayed-replication phenotype. Defects in virion precursor polypeptide processing, virion morphology, or viral DNA synthesis were observed for all of the replication-defective mutants, indicating that changes in integrase can have pleiotropic effects on viral replication.     

Efficient magnesium-dependent human immunodeficiency virus type 1 integrase activity.

The integrase protein from human immunodeficiency virus type 1 (HIV-1) has generally been reported to require Mn2+ for efficient in vitro activity. We have reexamined the divalent metal ion requirements of HIV-1 integrase and find that the protein is capable of promoting efficient 3' processing and DNA strand transfer with either Mn2+ or Mg2+. The metal ion preference depended upon the reaction conditions. HIV-1 integrase displayed significantly less nonspecific nuclease activity in reaction mixtures containing Mg2+ than it did under the previously described reaction conditions with mixtures containing Mn2+.     

Mechanism of action of Moloney murine leukemia virus RNA-directed DNA polymerase associated RNase H (RNase H I)

The mechanism of action of the ribonuclease H (RNase H) activity associated with Moloney murine leukemia virus RNA-directed DNA polymerase (RNase H I) and the two-subunit (alpha beta) form of avian myeloblastosis virus DNA polymerase were compared by utilizing the model substrate (A)n.(dT)n and polyacrylamide gel electrophoresis in 7 M urea to analyze digestion products. Examination on 25% polyacrylamide gels revealed that a larger proportion of the RNase H I oligonucleotide products generated by limited digestion of [3H](A)(1100).(dT)n were acid insoluble (15-26 nucleotides long) than acid soluble (less than 15 nucleotides long), while the opposite was true for products generated by alpha beta RNase H. RNase H I was capable of attacking RNA in RNA.DNA in the 5' to 3' and 3' to 5' directions, as demonstrated by the use of [3H,3'- or 5'-32P](A)(380).(dT)n and cellulose--[3H](A)n.(dT)n. Both RNase H I and alpha beta RNase H degraded [3H]-(A)n.(dT)n with a partially processive mechanism, based upon classical substrate competition experiments and analyses of the kinetics of degradation of [3H,3'- or 5'-32P](A)(380).(dT)n. That is, both enzymes remain bound to a RNA.DNA substrate through a finite number of hydrolytic events but dissociate before the RNA is completely degraded. Both RNase H I and alpha beta RNase H were capable of degrading [14C](A)n in [3H](C)n-[14C](A)n-[32P](dA)n.(dT)n, suggesting that retroviral RNase H is capable of removing the tRNA primer at the 5' terminus of minus strand DNA at the appropriate time during retroviral DNA synthesis in vitro.     

Posttranslational acetylation of the human immunodeficiency virus type 1 integrase carboxyl-terminal domain is dispensable for viral replication

A recent report sought to demonstrate that acetylation of specific lysines within integrase (IN) by the histone acetyltransferase (HAT) p300 regulates human immunodeficiency virus type 1 (HIV-1) integration and is essential for viral replication (A. Cereseto, L. Manganaro, M. I. Gutierrez, M. Terreni, A. Fittipaldi, M. Lusic, A. Marcello, and M. Giacca, EMBO J. 24:3070-3081, 2005). We can corroborate the efficient and specific acetylation of the IN carboxyl-terminal domain (CTD) (amino acids 212 to 288) by p300 using purified recombinant components. Although arginine substitution mutagenesis of the isolated CTD confirms that the majority of p300 acetylation occurs at lysine residues 264, 266, and 273, the pattern of acetylation is not uniform and a hierarchy of reactivity can be established. Several combinatorial mutations of the CTD lysines modified by p300 in vitro were reconstructed into an otherwise infectious proviral plasmid clone and examined for viral growth and frequency of productive chromosomal integration. In contrast to the findings of Cereseto and coworkers, who used epitope-tagged viruses for their experiments, we find that an untagged mutant virus, IN K(264/266/273)R, is fully replication competent. This discrepancy may be explained by the use of an acidic epitope tag placed at the extreme carboxyl terminus of integrase, near the target site for acetylation. Although the tagged, wild-type virus is viable, the combination of this epitope tag with the RRR substitution mutation results in a replication-defective phenotype. Although IN belongs to the very small set of nonhistone proteins modified by HAT-mediated activity, an obligate role for acetylation at the reactive CTD lysines in HIV-1 IN cannot be confirmed.     

Monoclonal antibodies against the minimal DNA-binding domain in the carboxyl-terminal region of human immunodeficiency virus type 1 integrase

Integrase of human immunodeficiency virus type 1 (HIVIN) consists of 288 amino acids, and its minimum DNA-binding domain (MDBD) (amino acids [aa] 220 to 270) is required for the integration reaction. We produced and characterized four murine monoclonal antibodies (MAbs) to the MDBD of HIVIN (strain LAI). Immunoblot and enzyme-linked immunosorbent assays with truncated HIVINs showed that those MAbs recognized sequential epitopes within the MDBD (aa 228 to 236, 237 to 252, 253 to 261, and 262 to 270). Their binding to HIVIN inhibited terminal cleavage and strand transfer activities but not disintegration activity in vitro. This collection of MAbs is useful for studying the structure and function of the MDBD by complementing mutational analyses and other biochemical studies.     

Dissecting the role of the N-terminal domain of human immunodeficiency virus integrase by trans-complementation analysis

The human immunodeficiency virus (HIV) integrase protein (IN) catalyzes two reactions required to integrate HIV DNA into the human genome: 3' processing of the viral DNA ends and integration. IN has three domains, the N-terminal zinc-binding domain, the catalytic core, and the C-terminal SH3 domain. Previously, it was shown that IN proteins mutated in different domains could complement each other. We now report that this does not require any overlap between the two complementing proteins; an N-terminal domain, provided in trans, can restore IN activity of a mutant lacking this domain. Only the zinc-coordinating form of the N-terminal domain can efficiently restore IN activity of an N-terminal deletion mutant. This suggests that interaction between different domains of IN is needed for functional multimerization. We find that the N-terminal domain of feline immunodeficiency virus IN can support IN activity of an N-terminal deletion mutant of HIV type 2 IN. These cross-complementation experiments indicate that the N-terminal domain contributes to the recognition of specific viral DNA ends.     

Posttranscriptional regulation by the human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex proteins through a heterologous RNA binding site.

The human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex proteins induce cytoplasmic expression of incompletely spliced viral mRNAs by binding to these mRNAs in the nucleus. Each protein binds a specific cis-acting element in its target RNAs. Both proteins also associated with nucleoli, but the significance of this association is uncertain because mutations that inactivate nucleolar localization signals in Rev or Rex also prevent RNA binding. Here we demonstrate that Rev and Rex can function when tethered to a heterologous RNA binding site by a bacteriophage protein. Under these conditions, cytoplasmic accumulation of unspliced RNA occurs without the viral response elements, mutations in the RNA binding domain of Rev do not inhibit function, and nucleolar localization can be shown to be unnecessary for the biological response.     

Irreversible inhibition of human immunodeficiency virus type 1 integrase by dicaffeoylquinic acids

Human immunodeficiency virus type 1 (HIV-1) and other retroviruses require integration of a double-stranded DNA copy of the RNA genome into the host cell chromosome for productive infection. The viral enzyme, integrase, catalyzes the integration of retroviral DNA and represents an attractive target for developing antiretroviral agents. We identified several derivatives of dicaffeoylquinic acids (DCQAs) that inhibit HIV-1 replication in tissue culture and catalytic activities of HIV-1 integrase in vitro. The specific step at which DCQAs inhibit the integration in vitro and the mechanism of inhibition were examined in the present study. Titration experiments with different concentrations of HIV-1 integrase or DNA substrate found that the effect of DCQAs was exerted on the enzyme and not the DNA. In addition to HIV-1, DCQAs also inhibited the in vitro activities of MLV integrase and truncated variants of feline immunodeficiency virus integrase, suggesting that these compounds interacted with the central core domain of integrase. The inhibition on retroviral integrases was relatively specific, and DCQAs had no effect on several other DNA-modifying enzymes and phosphoryltransferases. Kinetic analysis and dialysis experiments showed that the inhibition of integrase by DCQAs was irreversible. The inhibition did not require the presence of a divalent cation and was unaffected by preassembling integrase onto viral DNA. The results suggest that the irreversible inhibition by DCQAs on integrase is directed toward conserved amino acid residues in the central core domain during catalysis.