Expression in Escherichia coli of the large genomic segment of infectious pancreatic necrosis virus

A complete cDNA clone of the larger A segment of the genome of infectious pancreatic necrosis virus (IPNV) was expressed in Escherichia coli in an effort to develop a vaccine for IPNV in fish. When the cDNA insert was positioned in the correct orientation to the pUC19 lacZ promoter, the viral proteins VP2, NS, and VP3 were synthesized and processed as observed in infected cells. When the insert was placed in the opposite orientation, VP3 and a 38-kDa virus-specific polypeptide were also synthesized. In addition, specific deletions made from the 3' end into the NS gene of the cloned A segment led to inactivation of the NS proteolytic activity and subsequently, the synthesis of an unprocessed VP2-NS polyprotein precursor. Antiserum to this polyprotein distinguished NS (28.5 kDa) from VP3 (31 kDa) and led to the identification of a previously undescribed 38-kDa virus-specific polypeptide in infected cells. Thus, both internal translational initiation and proteolytic cleavage could lead to the synthesis of VP2, NS, and VP3 from a single mRNA with a single open reading frame. A trpE expression vector, pATH2, was used to synthesize large quantities of the A-segment-encoded proteins in bacteria. The resulting bacterial lysate was very effective in inducing protective immunity in rainbow trout fry.     

Polypeptides specified by the influenza virus genome 2. Assignment of protein coding functions to individual genome segments by in vitro translation

Cytoplasmic RNA extracted from chick embryo fibroblasts infected with influenza A (fowl plague) virus (FPV) was translated in a wheat germ cell-free protein-synthesizing system. Polypeptides which comigrated during SDS-polyacrylamide gel electrophoresis with marker virus-specific polypeptides P₁, P₂, P3, NP, M, and NS were synthesized in vitro. The NP, M, and NS polypeptides were positively identified by tryptic peptide mapping. The polypeptide component of the virus glycoprotein HA was also synthesized in vitro, and was identified by tryptic peptide mapping. RNA extracted from purified FPV (vRNA) did not direct the synthesis of any recognizable virus-specific polypeptides in vitro, either as a total preparation, or as individual RNA genome segments. The protein coding functions of the vRNA segments were identified by hybridization of individual segments to a preparation of infected cell cytoplasmic RNA. On subsequent translation of the RNA in vitro, synthesis of the virus-specific polypeptide corresponding to the hybridized vRNA segment was specifically reduced. We conclude that, for FPV, virion RNA segments 1–3 code for the three P polypeptides and segments 4, 5, 6, 7, and 8 code for polypeptides HA, NP, NA, M, and NS, respectively.     

Biosynthesis of reovirus-specified polypeptides. Multiplication rate but not yield of reovirus serotypes 1 and 3 correlates with the level of virus-mediated inhibition of cellular protein synthesis

Kinetic analysis of mouse L fibroblast cells infected with reovirus revealed that serotypes 1 (Lang strain) and 3 (Dearing strain) differ significantly from each other in terms of their rates of multiplication and their effects on cellular protein synthesis. Serotype 1 did not significantly affect the synthesis of cellular polypeptides in monolayer cultures of L cells at late times after infection when virus-specific protein synthesis was at a maximum. By contrast, under identical culture conditions, serotype 3 essentially completely inhibited the synthesis of cellular polypeptides at late times when viral protein synthesis was at a maximum. The kinetics of virus-specific polypeptide synthesis and the production of infectious progeny were considerably slower for the serotype 1 Lang strain as compared to the serotype 3 Dearing strain, both at 30 and 37 degrees. However, the relative pattern of viral polypeptide synthesis and the final yield of infectious progeny did not differ significantly between serotypes 1 and 3. For both serotypes, the maximum yield of infectious progeny was obtained shortly after the maximum rate of viral polypeptide synthesis was reached. These results suggest that the rate of multiplication, but not the final yield, of reovirus serotypes 1 and 3 is related to the extent of virus-mediated inhibition of cellular protein synthesis.     

Comparison of capsid polypeptides of group B coxsackieviruses and polypeptide synthesis in infected cells

Summary Capsid polypeptides of all six types (B1–6) of group B coxsackieviruses were compared by high-resolution gel electrophoresis, and synthesis of protein and RNA in B4- or B5-infected HeLa cells was analyzed. Four polypeptides, VP1–4, were detected in each type. Another polypeptide, VP0, slightly larger than VP1, was also detected in trace amounts in some types. VP1–3 showed different but characteristic molecular weights (VP1, 34,500 to 37,000; VP2, 31,000 to 36,000; VP3, 26,000 to 32,500), and presented well-defined and reproducible differences in electrophoretic mobility. The molecular weight of VP4 ranged from 5,000 to 5,500. VP1 was largest in B2 and B4, smallest in B1, and of intermediate size in the other types. VP2 was largest in B4 and smallest in B2; VP3 was largest in B5 and B6 and smallest in B4. In B4- or B5-infected HeLa cells, host protein synthesis began to decline after 2 hours postinfection and was less than 20 percent of the control by 6 hours postinfection. Actinomycin D-resistant viral RNA synthesis started at about 2 hours postinfection, peaked by 5 hours, and then declined rapidly. Virus-specific protein synthesis began while host protein synthesis was declining, increased during the ensuing period, and declined in late infection. A number of virus-specific proteins with molecular weights from 23,500 to >92,500 were detected in the host cytoplasm. At least three of these proteins were also present in the nucleus. The kinetics of processing of virus-specific proteins were examined by pulse-chase experiments in B5-infected cells. The relative intensities of [³⁵S]-methionine-labeled polypeptides suggest that a number of smaller, stable chains (MW 23,500 to 38,000) are generated by cleavage of a precursor polypeptide (MW 92,500 to 100,000).With 7 Figures     

Characterization of rotavirus replication intermediates: a model for the assembly of single-shelled particles

The segmented double-stranded (ds)RNA genome of the rotaviruses is replicated asymmetrically with viral mRNA serving as the template for minus-strand RNA synthesis. To identify intermediate structures in rotavirus replication, subviral particles (SVPs) purified from the cytoplasm of simian rotavirus SA11-infected cells were assayed for RNA polymerase activity in a cell-free system that supports viral RNA replication. Intact SVPs containing newly made RNA were resolved by electrophoresis under nondenaturing conditions on 0.6% agarose gels (50 mM Tris-glycine, pH 8.8). This gel system was found to separate without disrupting SA11 single- and double-shelled virions and virion-derived core particles. SVPs from the cell-free system that contained newly made dsRNA migrated in the agarose gels at positions between virion-derived cores and intermediate of single- and double-shelled virions. SVPs containing newly made dsRNA were eluted from the gel and analyzed for protein content by electrophoresis on polyacrylamide gels. The results showed that three distinct types of replication intermediates (RIs) were present in SA11-infected cells. The smallest intermediate (precore RI, 45 nm, 220 S) contained the structural proteins VP1, VP3, and VP9 and the nonstructural proteins NS53, NS35, and NS34. A second intermediate (core RI, 60 nm, 310 S) contained the core proteins VP1, VP2, and VP3 and the proteins VP9, NS35 and NS34. The largest RI (single-shelled RI, 75 nm, 420 S) contained the inner shell proteins VP1, VP2, VP3, and VP6 and the proteins VP9, NS35 and NS34. Analysis of the formation and turnover of RIs in infected cells pulse-labeled with 35S-amino acids supports a hypothesis that rotavirus single-shelled particles are assembled in vivo by the sequential addition of VP2 and VP6 to precore RIs consisting of VP1, VP3, VP9, NS35, and NS34.     

Control of vesicular stomatitis virus protein synthesis.

The five structural polypeptides of vesicular stomatitis virus are synthesized in infected cells at nonequimolar rates which correspond to their relative amounts in the virus particle. These experiments are concerned with the mechanism(s) responsible for the relative rates of viral polypeptide synthesis.Treatment of infected cells with amino acid analogs in order to prevent formation of functional viral proteins resulted in inhibition of viral genome replication and virion assembly. This treatment, however, did not alter the relative rates of viral polypeptide synthesis. This suggested that the mechanisms determining these rates do not require the function of newly-synthesized viral proteins and do not involve direct coupling between protein synthesis and virion assembly.Relative translation frequencies of viral messenger RNA molecules coding for each polypeptide were compared by treating cells with low levels of cycloheximide and anisomycin, inhibitors of protein synthesis that interfere preferentially with polypeptide chain elongation. Under these conditions the rate of synthesis of each viral polypeptide should be proportional only to the amount of its messenger RNA available for translation. The relative rates of viral polypeptide synthesis were not altered by this treatment, suggesting that viral messenger RNA molecules coding for each polypeptide are translated with similar average frequencies.Rates of peptide chain growth were compared by following the incorporation of ³H-amino acids into completed viral polypeptides following a pulse-label. The time necessary to achieve maximum incorporation of isotope into each completed viral polypeptide was taken as its translation time, the time necessary to synthesize and release a completed molecule. We were able to determine translation times for the M and G polypeptides by this method and found that they correlated directly with their molecular weights, suggesting that nascent chains of these two polypeptides are propagated at similar rates.     

Bovine viral diarrhea virus induced apoptosis correlates with increased intracellular viral RNA accumulation

Non-cytopathic (NCP) and cytopathic (CP) parent-daughter pairs are often isolated from cattle with bovine viral diarrhea virus (BVDV) induced mucosal disease. Alignment of these pair genomes revealed that genetic changes in CP BVDV involve the NS2-3 coding region and correlate with expression of NS3. However, additional mutations are present elsewhere in the genomes of these natural pairs, precluding unambiguous mapping of this function to the NS2-3 region. To evaluate this phenomenon in identical genetic backgrounds, we have constructed an NCP isogenic pair of the NADL by deletion of the cIns from NS2 region. The levels of viral protein synthesis in infected cells revealed no marked difference between the CP and the isogenic NCP BVDV mutant. In contrast, RNA accumulation in cells infected with CP virus was up to 25 times higher than that in cells infected with NCP BVDV. No significant difference in growth kinetics and viral yields were observed between the CP BVDV and the isogenic NCP pair. Analyses of additional NCP/CP parent-daughter field BVDV isolates revealed a similar pattern of macromolecular synthesis, suggesting the generality of this phenomenon. These results implicate increased levels of RNA accumulation in CP BVDV infected cells, along with the production of NS3 as potential contributors to viral cytopathogenicity.