The hepatitis C virus (HCV) H strain polyprotein is cleaved to produce at least nine distinct products: NH2-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. In this report, a series of C-terminal truncations and fusion with a human c-myc epitope tag allowed identification of a tenth HCV-encoded cleavage product, p7, which is located between the E2 and NS2 proteins. As determined by N-terminal sequence analysis, p7 begins with position 747 of the HCV H strain polyprotein. p7 is preceded by a hydrophobic sequence at the C terminus of E2 which may direct its translocation into the endoplasmic reticulum, allowing cleavage at the E2/p7 site by host signal peptidase. This hypothesis is supported by the observation that cleavage at the E2/p7 and p7/NS2 sites in cell-free translation studies was dependent upon the addition of microsomal membranes. However, unlike typical cotranslational signal peptidase cleavages, pulse-chase experiments indicate that cleavage at the E2/p7 site is incomplete, leading to the production of two E2-specific species, E2 and E2-p7. Possible roles of p7 and E2-p7 in the HCV life cycle are discussed.

Infection of vertebrate cells with alphaviruses normally leads to prodigious expression of virus-encoded genes

Alphaviruses are positive-strand RNA viruses that can mediate efficent cytoplasmic gene expression in insect and vertebrate cells. Through recombinant DNA technology, the alphavirus RNA replication machinery has been engineered for high-level expression of heterologous RNAs 1 and 2). Over the past 10 years, the alphavirus RNA replication and packaging machinery has been adapted for expression of heterologous RNAs and proteins in animal cells (for reviews, see refs. 3-6). As transient expression systems, alphaviruses offer several advantages. These include (i) a broad range of susceptible host cells including those of insect, avian, and mammalian origin; (ii) high levels of cytoplasmic RNA and protein expression without splicing; and (iii) the facile construction and manipulation of recombinant RNA molecules using full-length cDNA clones from which infectious RNA transcripts can be generated by in vitro transcription. Two principal strategies are being employed for expression of heterologous sequences: (i) engineering infectious recombinant RNAs that express additional subgenomic RNAs and (ii) replacement of the structural genes to produce self-replicating RNA "replicons" that can be packaged into infectious particles using defective helper RNAs or packaging cell lines. In addition, incorporation of heterologous ligands or receptors into the virion envelope may eventually allow targeting of engineered alphavirus RNAs to specific cell types. This overview briefly discusses the background, methodology, and applications of these alphavirus vector systems, which range from high-level protein production in cell culture to the induction of protective immunity in animals. (Fig; 1). This subgenomic RNA, which can accumulate to levels approaching 106 molecules per cell, is the mRNA for translation of the structural proteins. The synthesis of minus, plus, and subgenomic RNAs is temporally regulated via proteolytic processing of nonstructural polyprotein replicase components by a virus-encoded protease residing in the C-terminal region of nsP2 (8, 9).The structural proteins are initially translated as a polyprotein (NH2-C-E3-E2-6K-E1-COOH) that is processed coand posttranslationally to produce the mature products. Cleavage at the C-E3 site is mediated by a chymotrypsin-like protease activity residing in the C-terminal portion of the C protein. E3 and E2 are initially made as a precursor (called PE2 or P62) that is processed by a furin-like activity late during release of the virus from infected cells. Envelope glycoproteins El and PE2, separated by signal peptidase cleavages, form a heterodimer that migrates through the secretory pathway to the plasma membrane. In the cytoplasm, C-protein subunits complex with the genome RNA to form a nucleocapsid that matures by budding through the plasma membrane, acquiring a lipid bilayer envelope with embedded viral glycoproteins. Infectious Alphavirus cDNA ClonesStudies on the use of alphaviruses as vectors have required the recovery of infectious replication-competent ...

The hepatitis C virus H strain (HCV-H) polyprotein is cleaved to produce at least 10 distinct products, in the order of NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NSSA-NS5B-COOH. An HCV-encoded serine proteinase activity in NS3 is required for cleavage at four sites in the nonstructural region (3/4A, 4A/4B, 4B/5A, and 5A/SB). In this report, the HCV-H serine proteinase domain (the N-terminal 181 residues of NS3) was tested for its ability to mediate trans-processing at these four sites. By using an NS3-SB substrate with an inactivated serine proteinase domain, trans-cleavage was observed at all sites except for the 3/4A site. Deletion of the inactive proteinase domain led to efficient trans-processing at the 3/4A site. Smaller NS4A-4B and NS5A-5B substrates were processed efficiently in trans; however, cleavage of an NS4B-5A substrate occurred only when the serine proteinase domain was coexpressed with NS4A. Only the N-terminal 35 amino acids of NS4A were required for this activity. Thus, while NS4A appears to be absolutely required for trans-cleavage at the 4B1/5A site, it is not an essential cofactor for serine proteinase activity. To begin to examine the conservation (or divergence) of serine proteinase-substrate interactions during HCV evolution, we demonstrated that similar trans-processing occurred when the proteinase domains and substrates were derived from two different HCV subtypes. These results are encouraging for the development of broadly effective HCV serine proteinase inhibitors as antiviral agents. Finally, the kinetics of processing in the nonstructural region was examined by pulse-chase analysis. NS3-containing precursors were absent, indicating that the 2/3 and 3/4A cleavages occur rapidly. In contrast, processing of the NS4A-5B region appeared to involve multiple pathways, and significant quantities of various polyprotein intermediates were observed. NS5B, the putative RNA polymerase, was found to be significantly less stable than the other mature cleavage products. This instability appeared to be an inherent property of NS5B and did not depend on expression of other viral polypeptides, including the

Hepatitis C virus (HCV) nonstructural protein 4A (NS4A) is only 54 amino acids (aa) in length, yet it is a key regulator of the essential serine protease and RNA helicase activities of the NS3-4A complex, as well as a determinant of NS5A phosphorylation. Here we examine the structure and function of the C-terminal acidic region of NS4A through site-directed mutagenesis of a Con1 subgenomic replicon and through biophysical characterization of a synthetic peptide corresponding to this region. Our genetic studies revealed that in 8 of the 15 C-terminal residues of NS4A, individual Ala substitutions or charge reversal substitutions led to severe replication phenotypes, as well as decreased NS5A hyperphosphorylation. By selecting for replication-competent mutants, several second-site changes in NS3 were identified and shown to suppress these defects in replication and NS5A hyperphosphorylation. Circular-dichroism spectroscopy and nuclear magnetic resonance spectroscopy on a peptide corresponding to the C-terminal 19 aa of NS4A revealed that this region can adopt an alpha-helical conformation, but that this folding requires neutralization of a cluster of acidic residues. Taken together, these data suggest that the C terminus of NS4A acts as a dynamic regulator of NS3-4A interaction, NS5A hyperphosphorylation, and HCV replicase activity.