Coronavirus JHM: Cell-Free Synthesis of Structural Protein p60

Sac(-) cells infected with murine coronavirus strain JHM shut off host cell protein synthesis and synthesized polypeptides with molecular weights of 150,000, 60,000, and 23,000. The 60,000- and 23,000-molecular-weight polypeptides comigrated with virion structural proteins p60 and p23, and the 60,000-molecular-weight protein was identified as p60 by tryptic peptide fingerprinting. Polyadenylate-containing RNA [poly(A) RNA] extracted from the cytoplasm of infected cells directed the synthesis of both 60,000- and 23,000-molecular-weight polypeptides in messenger-dependent cell-free systems derived from mouse L-cells and rabbit reticulocytes. The reticulocyte system also synthesized a 120,000-molecular-weight polypeptide that was specifically immunoprecipitated by antiserum raised against JHM virions. The identity of the 60,000- and 23,000-molecular-weight in vitro products was established by comigration with virion proteins, immunoprecipitation, and in the case of p60, tryptic peptide fingerprinting. The cytoplasmic poly(A) RNAs which encoded p60 and p23 sedimented in sucroseformamide gradients at 17S and 19S, respectively, and were clearly separable. These RNAs were among the major poly(A) RNA species synthesized in the cytoplasm of actinomycin D-treated cells late in infection, and the in vitro translation of size-fractionated RNA released from polysomes confirmed that they represent physiological mRNA's. These results suggest that the expression of the coronavirus JHM genome involves more than one subgenomic mRNA.     

The genome of respiratory syncytial virus is a negative-stranded RNA that codes for at least seven mRNA species.

The RNA from purified respiratory syncytial (RS) virions and the RNAs from RS virus-infected cells were isolated and characterized. The RNA from RS virions was found to be a unique species of single-stranded RNA of approximately 5 x 10(6) daltons. Specific annealing experiments demonstrated that at least 93% of the virion RNA was of negative (nonmessage) polarity. Eight major and three minor species of virus-specific RNA were detected in the cytoplasm of RS virus-infected HEp-2 cells. The largest intracellular RNA species comigrated with RNA from purified virions, was not polyadenylated, and was synthesized only in the presence of concomitant protein synthesis. The seven major smaller species of RNA were synthesized in the presence of an inhibitor of protein synthesis. These RNAs were all polyadenylated and were shown to be RS virus specific by their ability to anneal specifically to purified virion RNA. The sum of the sizes of the major RS virus-specific polyadenylated RNAs was sufficient to account for the coding capacity of the RS virus genome (within the limits of reliability of the methods we have used to determine size).     

Respiratory syncytial virus mRNA coding assignments.

The polypeptide coding assignments for six of the respiratory syncytial virus-specific mRNAs were determined by translation of the individual mRNAs in vitro. The coding assignments of the RNAs are as follows. RNA band 1 is complex and can be separated into at least two components on the basis of electrophoretic mobility (molecular weights [MWs] approximately equal to 0.21 X 10(6) and 0.31 X 10(6), respectively) that code for three polypeptides of 9.5, 11, and 14 kilodaltons (K). RNA 2 (MW, 0.39 X 10(6)) codes for a 34K polypeptide; RNA 3 (MW, 0.40 X 10(6)) codes for a 26K polypeptide; RNA 4 (MW, 0.47 X 10(6)) codes for a 42K polypeptide; and RNA 5 (MW, 0.74 X 10(6)) codes for a 59K polypeptide. By limited-digest peptide mapping, the 34, 26, and 42K polypeptides synthesized in vitro appeared to be unique. Additionally, peptide mapping showed that the 34, 26, and 42K polypeptides synthesized in vitro were indistinguishable from their counterparts synthesized in infected cells. Thus, the 34, 26, and 42K polypeptides coded for by mRNAs 2, 3, and 4, respectively, were identified as the respiratory syncytial virus phosphoprotein (34K), matrix protein (26K), and nucleocapsid protein (42K), respectively. RNA 5 was shown to code for a 59K polypeptide. The 59K polypeptide synthesized in vitro did not comigrate with any polypeptide specific to infected cells, suggesting that it is a candidate for co- or post-translational modification.     

Translation of Sindbis virus 26 S RNA and 49 S RNA in lysates of rabbit reticulocytes

Sindbis virus-specific polypeptides were synthesized in lysates of rabbit reticulocytes in response to added 26 S or 49 S RNA. Sindbis 26 S RNA was translated into as many as three polypeptides which co-migrate in acrylamide gels with proteins found in infected cells.Wild type 26 S RNA was translated primarily into two polypeptides, which appear to be the Sindbis nucleocapsid protein (mol. wt 30,000) and the precursor of the two glycoproteins of the virion (mol. wt 100,000). A larger polypeptide (mol. wt 130,000) was synthesized in response to ts2 26 S RNA, a species of RNA which was isolated from cells infected with the ts2 mutant of Sindbis virus. This large polypeptide is apparently the protein which accumulates in cells infected with the mutant virus and which is thought to be a precursor of all three viral structural proteins.These results support the hypothesis that 26 S RNA is the messenger for the three structural proteins of the virion and that the RNA codes for one large polypeptide precursor. The precursor may then be cleaved at a specific site to yield the nucleocapsid protein and a second polypeptide which, in infected cells, is cleaved in a series of steps to yield the two glycoproteins of the virion.Sindbis 49 S RNA was translated into eight or nine polypeptides ranging from 60,000 to 180,000 molecular weights. The viral structural proteins, as such, were not synthesized in response to the added 49 S RNA.     

Two small virus-specific polypeptides are produced during infection with Sindbis virus.

We have identified and characterized two small virus-specific polypeptides which are produced during infection of cells with Sindbis virus, but which are not incorporated into the mature virion. The larger of these is a glycoprotein with an approximate molecular weight of 9,800 and is found predominantly in the medium of infected cells. Three independent lines of evidence demonstrate conclusively that this 9,800-dalton glycoprotein is produced during the proteolytic conversion of the precursor polypeptide, PE2, to the virion glycoprotein E2. This small glycoprotein is therefore analogous to the virion glycoprotein E3 of the very closely related alphavirus, Semliki Forest virus. The 9,800-dalton glycoprotein of Sindbis virus, unlike the E3 glycoprotein of Semliki Forest virus, is not, however, present in the viral particle. The other virus-specific polypeptide is 4,200 daltons in size, does not appear to be a glycoprotein, and is neither incorporated into the mature virus nor released into the culture medium. The gene for this small polypeptide is present in the viral 26S mRNA (the mRNA which encodes all the viral structural polypeptides) and appears to be located in the portion of the mRNA which encodes the two viral glycoproteins. The possibility that this 4,200-dalton polypeptide functions as a signal peptide during the synthesis of the viral membrane glycoproteins is discussed.     

Characterization of polypeptides immunoprecipitable from Pichinde virus-infected BHK-21 cells

Using hamster anti-Pichinde virus serum, we immunoprecipitated polypeptides from BHK-21 cells infected with Pichinde virus. Seven immunoprecipitable polypeptides exhibited a time- and multiplicity of infection-dependent appearance when the cultures were pulse-labeled with L-[35S]methionine for 1 h. The predominant polypeptide was a nucleoprotein (NP) of 64,000 daltons. Components of 48,000, 38,000, and 28,000 daltons, when analyzed by two-dimensional tryptic peptide mapping, were found to be derived from NP. After a 3-h chase period, polypeptides of 17,000, 16,500, and 14,000 daltons were evident, and peptide mapping revealed that these three polypeptides were also related to NP. During a series of pulse-chase experiments, a 79,000-dalton glycoprotein (GPC) was cleaved to glycoproteins of 52,000 and 36,000 daltons. Radiolabel in a polypeptide of approximately 200,000 daltons (L) did not chase into smaller cleavage products. L, GPC, and NP were found to be unique by two-dimensional tryptic peptide mapping. Comparison of polypeptides immunoprecipitated from infected cells with structural components of purified virus revealed that L protein was evident in both. This is the first report of a high-molecular-weight polypeptide in Pichinde virus particles and infected cells.     

Isolation and characterization of canine distemper virus-specific RNA

Ten species of virus-specific RNA were detected in Vero cells infected with the FXNO strain of canine distemper virus (CDV). The largest RNA was the genome-sized RNA and the nine smaller species were polyadenylated RNAs. Similar results were obtained for nine other strains of CDV. The molecular weights of these ten RNAs were determined to be 4.61 X 10(6), 2.46 X 10(6), 1.52 X 10(6), 1.32 X 10(6), 1.19 X 10(6), 1.07 X 10(6), 0.77 X 10(6), 0.65 X 10(6), 0.58 X 10(6), and 0.48 X 10(6). By in vitro translation of the polyadenylated RNAs in a rabbit reticulocyte lysate system, three different proteins which probably correspond to H, NP, and M were synthesized from the fraction containing RNAs 7, 8, 9, and 10.     

In Vitro Translation of Uukuniemi Virus-Specific RNAs: Identification of a Nonstructural Protein and a Precursor to the Membrane Glycoproteins

We isolated the virus-specific RNA species from Uukuniemi virus-infected chicken embryo cells and fractionated them by sucrose gradient centrifugation. In addition to three RNA species cosedimenting with the three viral RNA segments L (29S), M (23S), and S (17S), a fourth major RNA species, sedimenting at about 12S (S2), was found early in the infection. Annealing experiments indicated that the cytoplasmic L and M RNA species consisted of both plus and minus strands, with the plus strands in slight excess. Most of the S1 RNA was of negative polarity, whereas S2 was of positive polarity. The S2 RNA specifically annealed to the virion S RNA segment, indicating that it is transcribed from this segment. In vitro translation of the individual RNA species in micrococcal nuclease-treated cell-free reticulocyte extracts showed that an mRNA cosedimenting with the virion M RNA directed the synthesis of a virus-specific 110,000-dalton polypeptide (p110). This polypeptide could be immunoprecipitated with antiserum prepared against purified virions. When translation was carried out in the presence of dog pancreas microsomes, p110 was absent. Instead, an immunoprecipitable polypeptide band, with a molecular weight of about 70,000 and migrating between the virion surface glycoproteins G1 and G2, was observed. It is thus likely that the glycoproteins are synthesized as a precursor (p110), which during translation is cleaved roughly in the middle to yield G1 and G2. The 12S RNA species directed the synthesis of the nucleocapsid protein and a novel polypeptide with an apparent molecular weight of about 30,000. The latter was not precipitated with antivirion serum and was absent from lysates programmed with the corresponding RNA fraction from a mock-infected extract. Since, in addition, it was not found in purified virions and was present in the cytoplasm of infected cells but not in uninfected cells, it probably represents a nonstructural polypeptide.     

Synthesis of Capsid and Noncapsid Viral Proteins in Response to Encephalomyocarditis Virus Ribonucleic Acid in Animal Cell-Free Systems

The polypeptide products formed in two cell-free protein-synthetic systems programmed with encephalomyocarditis (EMC) virus ribonucleic acid (RNA) have been compared with the virus-specific proteins found in EMC-infected cells and with the capsid proteins of the purified virion. Tryptic peptides of (35)S-methioninelabeled proteins from these three sources were compared by co-chromatography and electrophoresis and by isoelectric focussing. Fifty-two methionine-containing peptides were resolved in digests of material from infected cells, of which about one-third were also clearly present in digests of the virion capsid proteins. The product formed in response to EMC RNA in cell-free systems from Krebs mouse ascites tumor cells yielded 26 to 29 such peptides. Most of these peptides were shown to behave identically with virus-specific peptides from infected cells, whereas just under half of them appeared to be identical with peptides from the virion capsid proteins. The product formed in response to EMC RNA in the L-cell cell-free system was similar, whereas six additional EMC-specific peptides were detected in mixed Krebs L-cell systems. The results indicate that the EMC RNA genome is partially translated in the mouse cell-free systems used to yield products containing both virion capsid and virus-specific noncapsid polypeptides.     

Characterization of Measles Virus-Specific Proteins Synthesized In Vivo and In Vitro from Acutely and Persistently Infected Cells

Measles virus protein synthesis has been analyzed in acutely and persistently infected cells. To assess the role of measles in subacute sclerosing panencephalitis (SSPE), measles viral proteins synthesized in vivo or in vitro were tested for reactivity with serum from a guinea pig(s) immunized with measles virus and sera from patients with SSPE. Guinea pig antimeasles virus serum immunoprecipitates the viral polypeptides of 78,000 molecular weight (glycosylated [G]), 70,000 molecular weight (phosphorylated [P]), 60,000 molecular weight (nucleocapsid [N]), and 35,000 molecular weight (matrix [M]) from cells acutely infected with measles virus as well as from chronically infected cells, but in the latter case, immunoprecipitated M protein has a reduced electrophoretic migration. Sera of SSPE patients immunoprecipitated all but the G protein in acutely infected cells and only the P and N proteins from chronically infected cells. In immunoprecipitates of viral polypeptides synthesized in a reticulocyte cell-free translation system, in response to mRNA from acutely or persistently infected cells, the 78,000-molecular-weight form of the G protein was not detected among the cell-free products of either mRNA. Guinea pig antimeasles virus serum immunoprecipitated P, N, and M polypeptides from the products of either form of mRNA, whereas SSPE serum immunoprecipitated the P and N polypeptides but not the M polypeptide. The differences in immunoreactivity of the antimeasles virus antiserum and the SSPE serum are discussed in terms of possible modifications of measles virus proteins in SSPE.     

The varicella-zoster virus immediate-early protein IE62 is a major component of virus particles.

Varicella-zoster virus (VZV) open reading frame (ORF) 62 potentially encodes a protein with considerable amino acid homology to the herpes simplex virus (HSV) immediate-early regulatory polypeptide ICP4 (or IE3). To identify and characterize its protein product(s) (IE62), we used a rabbit antiserum prepared against a synthetic peptide corresponding to the C-terminal 13 amino acids of the predicted protein. This antiserum reacted with phosphorylated polypeptides of 175 to 180 kDa that were made in VZV-infected cells and in cells infected with a vaccinia virus recombinant expressing IE62, but not in control-infected cells, confirming its specificity and reactivity to the IE62 protein. The antiserum recognized a 175-kDa polypeptide in purified virions that comigrated with a major structural protein. Comparison of this reactivity with that of an antipeptide antiserum directed against the VZV ORF 10 product (homologous to the HSV major structural protein VP16) indicates similar levels of ORF 62 and ORF 10 polypeptides in VZV virions. In contrast, antipeptide antiserum directed against the VZV ORF 29 product, the homolog of the HSV major DNA-binding protein, failed to recognize any protein in our virion preparations. Treatment of virions with detergents that disrupt the virion envelope did not dissociate IE62 from the nucleocapsid-tegument structure of the virion. Differential sensitivity of VZV virion IE62 to trypsin digestion in the presence or absence of Triton X-100 indicates that IE62 is protected from trypsin degradation by the virus envelope; since it is not a nucleocapsid protein, we conclude that it is part of the tegument. Finally, we show that the virion 175-kDa protein either can autophosphorylate or is a major substrate in vitro for virion-associated protein kinase activity.     

Characterization of adenovirus-associated virus-induced polypeptides in KB cells.

Electrophoretic analysis of KB cells coinfected with adenovirus-associated virus (AAV) type 2, a defective parvovirus, and adenovirus type 5 (as helper) have revealed the synthesis in vivo of at least five AAV-specific polypeptides. The three largest polypeptides, with molecular weights of 90,700, 71,600, and 60,000 comigrated in polyacrylamide gels with the three AAV structural polypeptides. The remaining two polypeptides had molecular weights of 24,900 and 15,800. The concentrations of the AAV-induced polypeptides relative to one another remained approximately constant during the infectious cycle, and the structural components were present in proportions similar to those found in purified virions. As determined by pulse-chase experiments, all polypeptides were generated at the level of protein synthesis and not by posttranslational proteolytic processing. Although inhibitors of proteolytic enzymes failed to influence the pattern of AAV-induced polypeptides, and amino acid analog, L-canavanine, blocked the appearances of both the major structural polypeptide (60,000 daltons) and the larger nonstructural polypeptide (24,900 daltons). Taken in conjunction with pulse-chase data, this result supports a model whereby the major virion polypeptide is produced by proteolytic cleavage of the nascent polypeptide chain.     

Rabies virus protein synthesis in infected BHK-21 cells.

Rabies virus specific polypeptide synthesis was examined under hypertonic conditions, which selectively inhibit cellular protein synthesis. The rabies virus proteins (L, G, N, M1, M2) were synthesized throughout the course of infection, with little change in their relative rates of synthesis. The rates of synthesis of the G and M1 polypeptides were more sensitive to increasing osmolarity than those of the L, N, and M2 polypeptides. Extrapolation to isotonicity of the results obtained under hypertonic conditions indicated that the molar ratios of the polypeptides synthesized under normal conditions were 0.4 (L), 64 (G), 100 (N), 75 (M1) and 35 (M2). A high-molecular-weight polypeptide (190,000), designated polypeptide L, was repeatedly detected both in infected cells and in extracellular virus. The estimated number of L polypeptide molecules per virion was 33. The synthesis of a viral glycoprotein precursor, designated gp78, , preceded the appearance of the mature viral glycoprotein in infected cells labeled with [3H]glucosamine under isotonic conditions. In cells labeled under hypertonic conditions, little or no mature viral glycoprotein was detected, but a virus-specific glycoprotein with an electrophoretic mobility similar to that of gp78 was observed. This glycoprotein could be chased into mature viral glycoprotein when the hypertonic conditions were made isotonic. These results suggest that a reversible block of viral glycoprotein synthesis occurs under hypertonic conditions.     

Identification of virus-coded nonstructural polypeptides in bunyavirus-infected cells.

Analyses of bunyavirus-infected cell extracts identified at least two virus-induced nonstructural polypeptides. With snowshoe hare (SSH), La Crosse (LAC), and six SSH-LAC reassortant viruses, it was shown that one of these nonstructural polypeptides (NSs, approximate molecular weight, 7.4 X 10(3)) is coded by the SSH small (S)-size viral RNA species. This nonstructural polypeptide was not detected (at least in the same relative abundancies) in LAC virus-infected cells or in cells infected with reassortants having LAC S RNA. For SSH virus, tryptic peptide analyses of either [3H]leucine- or [3H]arginine-labeled NSs indicated that it contains unique sequences not present in the SSH nucleocapsid (N) polypeptide (also coded by the S RNA; J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978). Analyses of SSH virus-infected cell extracts and extracts of cells infected with SSH-LAC reassortants having SSH medium (M)-size RNA species indicated that a nonstructural polypeptide (NSM; approximate molecular weight, 12 X 10(3)) is coded by the SSH M RNA species. In extracts of LAC virus-infected cells (or cells infected with SSH-LAC reassortants having LAC M RNA), a polypeptide with an electrophoretic mobility slightly faster than that of the SSH NSM polypeptide was observed (approximate molecular weight, 11 X 10(3)); it has been designated LAC NSM. The relationships of the NSM polypeptides to the other M RNA-coded polypeptides (G1 and G2; J. R. Gentsch and D. H. L. Bishop, J. Virol. 30;767-770, 1979) have not been determined. Two additional polypeptides present in both LAC- and SSH-infected cell extracts also appear to be virus induced (one with an approximate molecular weight of 10 X 10(3), p10; the other with an approximate molecular weight of 18 X 10(3), p18). Whether these polypeptides are virus coded has not been determined.     

Characterization of the Polypeptides Formed in Response to Encephalomyocarditis Virus Ribonucleic Acid in a Cell-Free System from Mouse Ascites Tumor Cells

The polypeptide products synthesized at different times in a cell-free system from Krebs mouse ascites tumor cells in response to the addition of encephalomyocarditis (EMC) virus ribonucleic acid (RNA) were characterized by electrophoresis on polyacrylamide gels and fingerprint analysis of their tryptic peptides. Translation of the EMC RNA genome with time occurred in a nonrandom fashion in these systems, to yield products containing sequences characteristic of both virion capsid polypeptides and EMC-specific polypeptides present only in the infected cell. The molecular weights of the products fell in a series from 20,000 to 140,000 daltons, although occasionally traces of larger polypeptides were also observed. All of the major polypeptides appeared to arise from partial or complete translation of about 60% of the EMC RNA genome. They were not formed by cleavage of a large precursor molecule. It is suggested that they are artifacts generated by premature "termination" of nascent polypeptide chains at preferred sites.     

Three structural polypeptides coded for by minite virus of mice, a parvovirus.

Purified full and empty virions of minute virus of mice were separated on CsCl gradients, and their polypeptides were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The empty particle contains two polypeptides, A (83,300 daltons) and B (64,300 daltons), which are 15 to 18% and 82 to 85%, respectively, of the virion mass. The full particle contains the single-stranded DNA genome, proteins A and B, and a third polypeptide, C (61,400 daltons). Again A is 15 to 18% of the protein mass, but the amounts of B and C vary inversely in different preparations of full particles. These polypeptides comprise greater than 99.6% of the protein in either virion, and their molecular weights and molar ratios are independent of the species of host cell on which the virus is propagated, They are not found in uninfected cells, and no protein component of uninfected cells copurifies with either virion under our conditions. Pulse-chase experiments show that the three proteins are synthesized only after virus infection and are therefore probably virus coded. Sequential harvesting from the nuclei of cells infected under one cycle growth conditions shows an increase in the proportion of C in full particles as infection progresses, suggesting that C is derived from B in a late maturation step.     

Cell-free synthesis of mouse mammary tumor virus Pr77 from virion and intracellular mRNA.

Mouse mammary tumor virus (MuMTV) was purified from two cell lines (GR and Mm5MT/c1), and the genomic RNA was isolated and translated in vitro in cell-free systems derived from mouse L cells and rabbit reticulocytes. The major translation product in both systems was a protein with the molecular weight 77,000. Several other products were also detected, among them a 110,000-dalton and in minor amounts a 160,000-dalton protein. All three polypeptides were specifically immunoprecipitated by antiserum raised against the major core protein of MuMTV (p27), but they were not precipitated by antiserum against the virion glycoprotein gp52. Analysis of the in vitro products by tryptic peptide mapping established their relationship to the virion non-glycosylated structural proteins. The 77,000-dalton polypeptide was found to be similar, if not identical, to an analogous precursor isolated from MuMTV-producing cells. Peptide mapping of the 110,000-dalton protein shows that it contains all of the methionine-labeled peptides found in the 77,000-dalton protein plus some additional peptides. We conclude that the products synthesized in vitro from the genomic MuMTV RNA are related to the non-glycosylated virion structural proteins. Polyadenylic acid-containing RNA from MuMTV-producing cells also directed the synthesis of the 77,000-dalton polypeptide in the L-cell system. If this RNA preparation was first fractionated by sucrose gradient centrifugation the 77,000-dalton protein appeared to be synthesized from mRNA with a sedimentation coefficient between 25 and 35S.