Cloning and characterization of hIF2, a human homologue of bacterial translation initiation factor 2, and its interaction with HIV-1 matrix.

The cDNA for a human homologue (hIF2) of bacterial (bIF2) and yeast (yIF2) translation initiation factor two (IF2) has been identified during a screen for proteins which interact with HIV-1 matrix. The hIF2 cDNA encodes a 1220-amino-acid protein with a predicted relative molecular mass of 139 kDa, though endogeneous hIF2 migrates anomalously on SDS/PAGE at 180 kDa. hIF2 has an extended N-terminus compared with its homologues, although its central GTP-binding domain and C-terminus are highly conserved, with 58% sequence identity with yIF2. We have confirmed that hIF2 is required for general translation in human cells by generation of a point mutation in the P-loop of the GTP-binding domain. This mutant protein behaves in a transdominant manner in transient transfections and leads to a significant decrease in the translation of a reporter gene. hIF2 interacts directly with HIV-1 matrix and Gag in vitro, and the protein complex can be immunoprecipitated from human cells. This interaction appears to block hIF2 function, since purified matrix protein inhibits translation in a reticulocyte lysate. hIF2 does not correspond to any of the previously characterized translation initiation factors identified in mammals, but its essential role in translation appears to have been conserved from bacteria to humans.

Structure-function studies of the human immunodeficiency virus type 1 matrix protein, p17.

The human immunodeficiency virus type 1 (HIV-1) matrix protein, p17, plays important roles in both the early and late stages of the viral life cycle. Using our previously determined solution structure of p17, we have undertaken a rational mutagenesis program aimed at mapping structure-function relationships within the molecule. Amino acids hypothesized to be important for p17 function were mutated and examined for effect in an infectious proviral clone of HIV-1. In parallel, we analyzed by nuclear magnetic resonance spectroscopy the structure of recombinant p17 protein containing such substitutions. These analyses identified three classes of mutants that were defective in viral replication: (i) proteins containing substitutions at internal residues that grossly distorted the structure of recombinant p17 and prevented viral particle formation, (ii) mutations at putative p17 trimer interfaces that allowed correct folding of recombinant protein but produced virus that was defective in particle assembly, and (iii) substitution of basic residues in helix A that caused some relocation of virus assembly to intracellular locations and produced normally budded virions that were completely noninfectious.

Conserved sequences in the carboxyl terminus of integrase that are essential for human immunodeficiency virus type 1 replication.

We have previously identified a residue in the carboxyl terminus of human immunodeficiency virus type 1 integrase (HIV-1 IN), W-235, the requirement for which is only revealed in viral assays for integrase function (P. M. Cannon, W. Wilson, E. Byles, S. M. Kingsman, and A. J. Kingsman, J. Virol. 68:4768-4775, 1994). Our further analysis of this region of retroviral IN has now identified several sequence motifs which are conserved in all the retroviruses we examined, apart from human spumaretrovirus. We have made mutations within these motifs in HIV-1 IN and examined their phenotypes when reintroduced into an infectious proviral clone. The deleterious effects of several of these mutations demonstrate the importance of these regions for IN function in vivo. We observed a further discrepancy, at a motif that is only conserved in the lentiviruses, in the ability of mutants to function in in vitro and in vivo assays. Substitutions both in this region and at W-235 abolish HIV-1 infectivity but do not affect particle production, morphology, reverse transcription, or nuclear import in T-cell lines. Taken together with the in vitro data suggesting that neither of these residues is directly involved in the catalytic reactions of IN, it seems likely that we have identified regions of IN that are essential for interactions with other components of the integration machinery.

Human immunodeficiency virus type 1 integrase: effect on viral replication of mutations at highly conserved residues.

Sequence comparisons of the integrase (IN) proteins from different retroviruses have identified several highly conserved residues. We have introduced mutations at 16 of these sites into the integrase gene of human immunodeficiency virus type 1 and analyzed the phenotypes of the resulting viruses. The viruses were all normal for p24 content and reverse transcriptase activity. In addition, all of the mutants could infect T-cell lines and undergo reverse transcription, as assessed by PCR analysis. Most of the mutant viruses also had normal Western blot (immunoblot) profiles, although three of the mutations resulted in reduced signals for IN relative to the wild type on the immunoblots and mutation of residue W235 completely abolished recognition of the protein by pooled sera from human immunodeficiency virus type 1-positive patients. Mutations that have previously been shown to abolish activity in in vitro studies produced noninfectious viruses. The substitution of W235 was notable in producing a noninfectious virus, despite previous reports of this residue being nonessential for IN activity in vitro (A.D. Leavitt, L. Shiue, and H.E. Varmus, J. Biol. Chem. 268:2113-2119, 1993). In addition, we have identified four highly conserved residues that can be mutated without any affect on viral replication in T-cell lines.