The complete nucleotide sequence of two human T-cell leukaemia type III (HTLV-III) proviral DNAs each have four long open reading frames, the first two corresponding to the gag and pol genes. The fourth open reading frame encodes two functional polypeptides, a large precursor of the major envelope glycoprotein and a smaller protein derived from the 3'-terminus long open reading frame analogous to the long open reading frame (lor) product of HTLV-I and -II.

The genome of the human immunodeficiency virus HIV-1 contains at least eight genes, of which three (sor, R, and 3' orf) have no known function. In this study, the role of the sor gene was examined by constructing a series of proviral genomes of HIV-1 that either lacked the coding sequences for sor or contained point mutations in sor. Analysis of four such mutants revealed that although each clone could generate morphologically normal virus particles upon transfection, the mutant viruses were limited in their capacity to establish stable infection. Virus derived from transfection of Cos-1 cells (OKT4-) with sor mutant proviral DNA's was resistant to transmission to OKT4+ "susceptible" cells under cell-free conditions, and was transmitted poorly by coculture. In contrast, virus derived from clones with an intact sor frame was readily propagated by either approach. Normal amounts of gag-, env-, and pol-derived proteins were produced by all four mutants and assays in both lymphoid and nonlymphoid cells indicated that their trans-activating capacity was intact and comparable with wild type. Thus the sor gene, although not absolutely required in HIV virion formation, influences virus transmission in vitro and is crucial in the efficient generation of infectious virus. The data also suggest that sor influences virus replication at a novel, post-translational stage and that its action is independent of the regulatory genes tat and trs.

We have engineered a segment of the poliovirus genome (nucleotides 5438-6061) that encodes the 183 amino acid residues of the 3C region and 25 residues of the 3D region of the viral polyprotein into an Escherichia coli expression vector. The 3C region is a virus-specific protease, which, when expressed in E. coli, is shown to be active and autocatalytic. In our system, three poliovirus-specific proteins are produced: a precursor polyprotein (3C-3D), an internal initiation product, and the mature protease (3C). Mutants in the 3C region have been constructed by oligonucleotidedirected mutagenesis and their effect on the proteolytic activity has been assayed by the in vivo production of the mature protease. The mutation of highly conserved residues (cysteine-47 or histide-161) produced an inactive enzyme, while the mutation of a nonconserved residue (cysteine-153) had a negligible effect on the proteolytic activity.Poliovirus, a small RNA virus, is a member of the picornavirus family, which includes rhinovirus, encephalomyocarditis virus, and foot and mouth disease virus. During the viral life cycle, the genomic RNA is initially translated into a >200-kDa polyprotein, which, while nascent, is cleaved to a series of polypeptide intermediates, termed P1, P2, and P3. Further processing yields the majority of mature viral products (structural and nonstructural) and is dependent on a viral protease(s). The poliovirus P3 intermediate consists of three nonstructural proteins-the genome-linked virion protein (VPg), the protease (3C), and the replicase (3D)-and two proteins of unknown function (3C' and 3D') (1, 2). Hanecak et al. (3) have demonstrated that the 3C region of the poliovirus genome represents the core sequence responsible for the proteolytic processing (at Gln-Gly) of the precursor polypeptides. In addition, the poliovirus 3C protease has been reported to be autocatalytic (4), in agreement with results for encephalomyocarditis virus (5) and foot and mouth disease virus (6).The enzymatic mechanism of these viral proteases has not been well characterized. Inhibitors such as iodoacetamide, N-ethyl maleimide, and para-chloromercuribenzoate have implicated a cysteine residue in the active site (7,8). However, diisopropylphosphofluoridate, an inhibitor of serine proteases, has also been reported as an effective inhibitor of poliovirus protein processing (9). A comparative study (10) of the derived protease proteins from several picornaviruses as well as the related plant virus cowpea mosaic virus (11) revealed one region of significant homology located in the carboxyl third ofthe protein. Within this region a cysteine and a histidine residue have been strictly conserved. These observations suggest that the picornavirus proteases most probably belong to the class of cysteine proteases.To obtain sufficient quantities of the protease proteins for study, we have engineered a cDNA clone of the poliovirus 3C-3D region into an Escherichia coli expression vector, which produced the protease polypeptides in high yield...