Gag-Pol supplied in trans is efficiently packaged and supports viral function in human immunodeficiency virus type 1.

The intracellular trafficking and subsequent incorporation of Gag-Pol into human immunodeficiency virus type 1 (HIV-1) remains poorly defined. Gag-Pol is encoded by the same mRNA as Gag and is generated by ribosomal frameshifting. The multimerization of Gag and Gag-Pol is an essential step in the formation of infectious viral particles. In this study, we examined whether the interaction between Gag and Gag-Pol is initiated during protein translation in order to facilitate the trafficking and subsequent packaging of Gag-Pol into the virion. A conditional cotransfection system was developed in which virion formation required the coexpression of two HIV-1-based plasmids, one that produces both Gag and Gag-Pol and one that only produces Gag-Pol. The Gag-Pol proteins were either immunotagged with a His epitope or functionally tagged with a mutation (K65R) in reverse transcriptase that is associated with drug resistance. Gag-Pol packaging was assessed to determine whether the Gag-Pol incorporated into the virion was preferentially packaged from the plasmid that expressed both Gag and Gag-Pol or whether it could be packaged from either plasmid. Our data show that translation of Gag and Gag-Pol from the same mRNA is not critical for virion packaging of the Gag-Pol polyprotein or for viral function.

Inhibitors of human immunodeficiency virus type 1 reverse transcriptase target distinct phases of early reverse transcription.

Early HIV-1 reverse transcription can be separated into initiation and elongation phases. Here we show, using PCR analysis of negative-strand strong-stop DNA [(-)ssDNA] synthesis in intact virus, that different reverse transcriptase (RT) inhibitors affect distinct phases of early natural endogenous reverse transcription (NERT). The effects of nevirapine on NERT were consistent with a mechanism of action including both specific and nonspecific binding events. The nonspecific component of this inhibition targeted the elongation reaction, whereas the specific effect seemed principally to be directed at very early events (initiation or the initiation-elongation switch). In contrast, foscarnet and the nucleoside analog ddATP inhibited both early and late (-)ssDNA synthesis in a similar manner. We also examined compounds that targeted other viral proteins and found that Ro24-7429 (a Tat antagonist) and rosmarinic acid (an integrase inhibitor) also directly inhibited RT. Our results indicate that NERT can be used to identify and evaluate compounds that directly target the reverse transcription complex.

The human immunodeficiency virus type 1 TAR RNA upper stem-loop plays distinct roles in reverse transcription and RNA packaging.

The human immunodeficiency virus type 1 (HIV-1) RNA genome is flanked by a repeated sequence (R) that is required for HIV-1 replication. The first 57 nucleotides of R form a stable stem-loop structure called the transactivation response element (TAR) that can interact with the virally encoded transcription activator protein, Tat, to promote high levels of gene expression. Recently, we demonstrated that TAR is also important for efficient HIV-1 reverse transcription, since HIV-1 mutated in the upper stem-loop of TAR showed a reduced ability both to initiate and to complete reverse transcription. We have analyzed a series of HIV-1 mutant viruses to better defined the structural or sequence elements required for natural endogenous reverse transcription and packaging of virion RNA. Our results indicate that the requirement for TAR in reverse transcription is conformation dependent, since mutants with mutations that alter the upper stem-loop orientation are defective for reverse transcription initiation and have minor defects in RNA packaging. In contrast, TAR mutations that allowed the formation of alternative upper stem-loop structure greatly reduced RNA packaging but did not affect reverse transcription efficiency. These results are consistent with direct involvement of the upper stem-loop structure in packaging of genomic RNA and suggest that the TAR RNA stem-loop from nucleotide +18 to +42 interacts with other components of the reverse transcription initiation complex to promote efficient reverse transcription.

Functional domains of Tat required for efficient human immunodeficiency virus type 1 reverse transcription.

Tat expression is required for efficient human immunodeficiency virus type 1 (HIV-1) reverse transcription. In the present study, we generated a series of 293 cell lines that contained a provirus with a tat gene deletion (Deltatat). Cell lines that contained Deltatat and stably transfected vectors containing either wild-type tat or a number of tat mutants were obtained so that the abilities of these tat genes to stimulate HIV-1 gene expression and reverse transcription could be compared. tat genes with mutations in the amino terminus did not stimulate either viral gene expression or HIV-1 reverse transcription. In contrast, tat mutants in the activation, core, and basic domains of Tat did not stimulate HIV-1 gene expression but markedly stimulated HIV-1 reverse transcription. No differences in the levels of virion genomic RNA or tRNA3Lys were seen in the HIV-1 Deltatat viruses complemented with either mutant or wild-type tat. Finally, overexpression of the Tat-associated kinases CDK7 and CDK9, which are involved in Tat activation of HIV-1 transcription, was not able to complement the reverse transcription defects associated with the lack of a functional tat gene. These results indicate that the mechanism by which tat modulates HIV-1 reverse transcription is distinct from its ability to activate HIV-1 gene expression.

Cloning of a cDNA encoding SjIrV1, a Schistosoma japonicum calcium-binding protein similar to calnexin, and expression of the recombinant protein in Escherichia coli.

Membrane-associated proteins were isolated from adult Philippine strain Schistosoma japonicum by partitioning into the detergent phase of Triton X-114. A rabbit polyclonal antiserum raised against these proteins was used to screen an S. japonicum expression cDNA library. Positive clones were identified which encoded the species orthologue of SmIrV1, a Schistosoma mansoni protein which was initially identified by screening with sera from mice protectively vaccinated with irradiated cercariae [Hawn et al., J. Biol. Chem. 268 (1993) 7692-7698]. The S. japonicum molecule, which we term SjIrV1, is 83% identical to SmIrV1 at the predicted amino acid level and is a member of the calreticulin family of non-EF-hand, calcium-binding proteins. The Chinese strain S. japonicum orthologue of SjIrV1 was obtained by screening with the radiolabelled insert of the Philippine strain clone. Northern blot analysis revealed a single message of around 2.4 kb and gave no indication of alternative splicing. Southern blot analysis gave a simple pattern, indicating a single-copy gene, and showed a single restriction fragment length polymorphism between the genomes of Chinese and Philippine strains of S. japonicum. Recombinant, full-length SjIrV1 was expressed with a hexahistidine tag in Escherichia coli and the recombinant protein isolated by nickel-chelate chromatography. Recombinant SjIrV1 was shown to exhibit calcium-dependent, differential electrophoretic migration and to bind ruthenium red in the absence but not in the presence of calcium ions. The presence of conserved Ca(2+)-binding motifs predicted from the primary sequence, together with the Ca(2+)-dependent electrophoretic mobility of recombinant SjIrV1, confirmed that SjIrV1 was a functional calcium-binding protein.

A new member of the transmembrane 4 superfamily (TM4SF) of proteins from schistosomes, expressed by larval and adult Schistosoma japonicum.

The transmembrane 4 superfamily (TM4SF) comprises an assemblage of surface antigens from mammalian cells and from the human blood flukes. Member proteins of the TM4SF are characterized by the presence of four hydrophobic domains, which are presumed to be membrane-spanning, and specific conserved motifs. The Sm23 group of TM4SF, which includes Sm23, Sj23, and Sh23 from blood flukes, shows potential as immunodiagnostic and vaccine target antigens for use in controlling human schistosomiasis. Here we describe a cDNA from miracidia and adult Schistosoma japonicum parasites which apparently encodes a new member of the TM4SF. The deduced polypeptide, termed Sj25/TM4, has substantial amino acid homology to Sm23 from Schistosoma mansoni although it is not a species homologue of Sm23. Sj25/TM4 is predicted to span the cell membrane four times, with its NH2- and COOH-termini embedded in the cytoplasm, and to have two extracellular hydrophilic loops, one of which may be N-glycosylated. This topology is characteristic of TM4SF proteins; in addition, Sj25/TM4 contains the sequence motifs conserved in the TM4SF. Southern hybridization analysis demonstrated that Sj25/TM4 and Sj23 are encoded by genes at separate loci and, further, showed interstrain variation at the locus encoding Sj25/TM4 in Chinese and Philippine isolates of S. japonicum.

Schistosoma japonicum: heterogeneity in paramyosin genes.

Paramyosin is an integral muscle protein found in many invertebrates including schistosomes, and is considered an important candidate vaccine antigen in schistosomiasis. The characterisation of natural molecular variation in vaccine antigens including paramyosin is important because strain-specific vaccination may be necessary against schistosomiasis japonica. We have isolated partial cDNAs encoding paramyosin from an adult, Chinese strain Schistosoma japonicum gene library. Two of these cDNAs (B6 and Y6) encode the same region of paramyosin but their nucleotide sequences differ at eight positions and their deduced amino acid sequences differ by an arginine/cysteine substitution, demonstrating intrastrain variation in paramyosin. Southern blot analysis of genomic DNA from the Chinese and Philippine strains of S. japonicum demonstrated strain-related RFLPs at the paramyosin locus, and suggested that more than one copy of the paramyosin gene was present in the S. japonicum genome. PCR-based RFLP analysis which exploited restriction site differences between B6 and Y6 showed that paramyosin genotype B6 was much more common in schistosome populations and verified the existence of introns in the paramyosin gene(s) of S. japonicum.