Packaging of the Virion Host Shutoff (Vhs) Protein of Herpes Simplex Virus: Two Forms of the Vhs Polypeptide Are Associated with Intranuclear B and C Capsids, but Only One Is Associated with Enveloped Virions

ABSTRACT The virion host shutoff (Vhs) protein (UL41) is a minor component of herpes simplex virus virions which, following penetration, accelerates turnover of host and viral mRNAs. Infected cells contain 58-kDa and 59.5-kDa forms of Vhs, which differ in the extent of phosphorylation, yet only a 58-kDa polypeptide is incorporated into virions. In pulse-chase experiments, the primary Vhs translation product comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the 58-kDa virion polypeptide, and could be chased to 59.5 kDa. While both 59.5-kDa and 58-kDa forms were found in nuclear and cytoplasmic fractions, the 59.5-kDa form was significantly enriched in the nucleus. Both forms were associated with intranuclear B and C capsids, yet only the 58-kDa polypeptide was found in enveloped cytoplasmic virions. A 58-kDa form, but not the 59.5-kDa form, was found in L particles, noninfectious particles that contain an envelope and tegument but no capsid. The data suggest that virions contain two populations of Vhs that are packaged by different pathways. In the first pathway, the primary translation product is processed to 59.5 kDa, is transported to the nucleus, binds intranuclear capsids, and is converted to 58 kDa at some stage prior to final envelopment. The second pathway does not involve the 59.5-kDa form or interactions between Vhs and capsids. Instead, the primary translation product is phosphorylated to the 58-kDa virion form and packaged through interactions with other tegument proteins in the cytoplasm or viral envelope proteins at the site of final envelopment.

Proteolytic Processing of a Serotype 8 Human Astrovirus ORF2 Polyprotein

ABSTRACT Astroviruses require the proteolytic cleavage of the capsid protein to infect the host cell. Here we describe the processing pathway of the primary translation product of the structural polyprotein (ORF2) encoded by a human astrovirus serotype 8 (strain Yuc8). The primary translation product of ORF2 is of approximately 90 kDa, which is subsequently cleaved to yield a 70-kDa protein (VP70) which is assembled into the viral particles. Limited trypsin treatment of purified particles containing VP70 results in the generation of polypeptides VP41 and VP28, which are then further processed to proteins of 38.5, 35, and 34 kDa and 27, 26, and 25 kDa, respectively. VP34, VP27 and VP25 are the predominant proteins in fully cleaved virions, which correlate with the highest level of infectivity. Processing of the VP41 protein to yield VP38.5 to VP34 polypeptides occurred at its carboxy terminus, as suggested by immunoblot analysis using hyperimmune sera to different regions of the ORF2, while processing of VP28 to generate VP27 and VP25 occurred at its carboxy and amino terminus, respectively, as determined by immunoblot, as well as by N-terminal sequencing of those products. Based on these data, the processing pathway for the 90-kDa primary product of astrovirus Yuc8 ORF2 is presented.

Furin Is Involved in Baculovirus Envelope Fusion Protein Activation

ABSTRACT The Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) Se8 gene was recently shown to encode the viral envelope fusion (F) protein. A 60-kDa C-terminal subunit (F1) of the 76-kDa primary translation product of this gene was found to be the major envelope protein of SeMNPV budded virus (BV) (W. F. J. IJkel, M. Westenberg, R. W. Goldbach, G. W. Blissard, J. M. Vlak, and D. Zuidema, Virology 275:30–41, 2000). A specific inhibitor was used to show that furin is involved in cleavage of the precursor envelope fusion (F0) protein. BV produced in the presence of the inhibitor possesses the uncleaved F0 protein, while an F protein with a mutation in the furin cleavage site was translocated to the plasma membrane but lost its fusogenic activity. These results indicate that cleavage of F0 is required to activate the SeMNPV F protein and is necessary for BV infectivity. Specific antibodies against F1 and against the putative N terminus (F2) of the primary translation product were used to show that the F protein is BV specific and that BVs contain both the 60- (F1) and 21-kDa (F2) cleavage products. In nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis both subunits migrate as a single 80-kDa protein, indicating that the subunits remain associated by a disulfide linkage. In addition, the presence of the F protein predominately as a monomer suggests that disulfide links are not involved in oligomerization. Thus, the envelope fusion protein from group II nucleopolyhedroviruses of baculoviruses has properties similar to those of proteins from a number of vertebrate viruses.

A processed form of the Spatzle protein defines dorsal-ventral polarity in the Drosophila embryo

Stein et al. (1991) identified a soluble, extracellular factor that induces ventral structures at the site where it is injected in the extracellular space of the early Drosophila embryo. This factor, called polarizing activity, has the properties predicted for a ligand for the transmembrane receptor encoded by the Toll gene. Using a bioassay to follow activity, we purified a 24 × 10(3) M(r) protein that has polarizing activity. The purified protein is recognized by antibodies to the C-terminal half of the Spatzle protein, indicating that this polarizing activity is a product of the spatzle gene. The purified protein is smaller than the primary translation product of spatzle, suggesting that proteolytic processing of Spatzle on the ventral side of the embryo is required to generate the localized, active form of the protein.

Elevated in vitro translation of a 25-kDa protein in renal cell carcinoma

As a first step in understanding the changes in protein synthesis that occur in renal cell carcinoma, we have prepared poly(A)+ RNA from surgically removed tumors and from their normal tissue counterpart. These RNAs were then translated in vitro in the rabbit reticulocyte lysate system and the synthesized labeled polypeptides were separated by one- and two-dimensional gel electrophoresis. A major 25-kDa primary translation product was observed with all renal cell carcinomas. The synthesis of this protein was barely detectable with the RNA from normal tissue adjacent to the tumor. To determine if this protein could be further processed (removal of signal peptide and (or) core glycosylation), canine pancreatic microsomal membranes were added to the system. This addition resulted in the formation of a vertical row of three additional spots, with the same isoelectric point as the primary translation product and with molecular masses ranging from 27 to 31 kDa. The 31-kDa protein was retained on Concanavalin A. After digestion with endoglycosidase H, it was no longer visible on sodium dodecyl sulfate gels and a new 27-kDa band was generated suggesting that the mature protein was indeed a glycoprotein. Future experiments will be aimed at identifying this protein and examining its potential value as a marker of renal cell carcinoma.Key words: renal cancer, post-translational modifications, glycosylation, tumor markers.

Proviral insertional activation of c-erbB: differential processing of the protein products arising from two alternate transcripts

Proviral insertional activation of c-erbB results in the expression of two alternate transcripts (ENV+ and ENV-). We used cDNA clones representing the two alternate transcripts to generate stably transformed quail fibroblast cell lines which express the products of these transcripts independently. Analysis of the co- and posttranslational processing of the insertionally activated c-erbB products expressed in these cell lines revealed that the protein products of the ENV+ and ENV- transcripts were processed differently. The ENV+ transcript produced a primary translation product which was rapidly cotranslationally cleaved near the amino terminus to form a 79,000-Mr product. This protein product was efficiently converted to a higher-molecular-weight form, of between 82,000 and 88,000 (gp82-88), which was terminally glycosylated and expressed on the cell surface. A small portion of the ENV+ primary translation product underwent a second proteolytic cleavage to generate an unglycosylated 53,000-Mr species. In contrast, the primary translation product of the ENV- transcript, p80, was not proteolytically processed; this precursor form was rapidly converted to two discrete glycosylation intermediates, gp82 and go84. Only a small portion (less than 10%) of the total ENV- insertionally activated c-erbB product was slowly converted to the terminally glycosylated cell surface form, gp85-88. The processing differences that distinguished the ENV+ and ENV- products were similar to processing differences that we observed in parallel studies on the viral erbB products of the avian erythroblastosis viruses AEV-H and AEV-R, respectively. Since all four erbB protein products shared the same number, position, and sequence context of potential N-linked glycosylation sites, yet differed in the extent of their carbohydrate maturation, these data suggest that the mechanisms used by these truncated receptor molecules to associate with cellular membranes may be distinct.

Proviral insertional activation of c-erbB: differential processing of the protein products arising from two alternate transcripts.

Proviral insertional activation of c-erbB results in the expression of two alternate transcripts (ENV+ and ENV-). We used cDNA clones representing the two alternate transcripts to generate stably transformed quail fibroblast cell lines which express the products of these transcripts independently. Analysis of the co- and posttranslational processing of the insertionally activated c-erbB products expressed in these cell lines revealed that the protein products of the ENV+ and ENV- transcripts were processed differently. The ENV+ transcript produced a primary translation product which was rapidly cotranslationally cleaved near the amino terminus to form a 79,000-Mr product. This protein product was efficiently converted to a higher-molecular-weight form, of between 82,000 and 88,000 (gp82-88), which was terminally glycosylated and expressed on the cell surface. A small portion of the ENV+ primary translation product underwent a second proteolytic cleavage to generate an unglycosylated 53,000-Mr species. In contrast, the primary translation product of the ENV- transcript, p80, was not proteolytically processed; this precursor form was rapidly converted to two discrete glycosylation intermediates, gp82 and go84. Only a small portion (less than 10%) of the total ENV- insertionally activated c-erbB product was slowly converted to the terminally glycosylated cell surface form, gp85-88. The processing differences that distinguished the ENV+ and ENV- products were similar to processing differences that we observed in parallel studies on the viral erbB products of the avian erythroblastosis viruses AEV-H and AEV-R, respectively. Since all four erbB protein products shared the same number, position, and sequence context of potential N-linked glycosylation sites, yet differed in the extent of their carbohydrate maturation, these data suggest that the mechanisms used by these truncated receptor molecules to associate with cellular membranes may be distinct.