Translation and identification of the viral mRNA species isolated from subcellular fractions of vesicular stomatitis virus-infected cells.

The cytoplasm of vesicular stomatitis virus (VSV)-infected BHK cells has been separated into a fraction containing the membrane-bound polysomes and the remaining supernatant fraction. Total poly(A)-containing RNA was isolated from each fraction and purified. A 17S class of VSV mRNA was found associated almost exclusively with the membrane-bound polysomes, whereas 14,5S and 12S RNAs were found mostly in the postmembrane cytoplasmic supernatant. Poly(A)-containing VSV RNA synthesized in vitro by purified virus was resolved into the same size classes. The individual RNA fractions isolated from VSV-infected cells or synthesized in vitro were translated in cell-free extracts of wheat germ, and their polypeptide products were compared by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The corresponding in vivo and in vitro RNA fractions qualitatively direct the synthesis of the same viral polypeptides and therefore appear to contain the same mRNA species. By tryptic peptide analysis of their translation products, the in vivo VSV mRNA species have been identified. The 17S RNA, which is compartmentalized on membrane-bound polysomes, codes for a protein of molecular weight 63,000 (P-63) which is most probably a nonglycosylated form of the viral glycoprotein, G. Of the viral RNA species present in the remaining cytoplasmic supernatant, the 14.5S RNA codes almost exclusively for the N protein, whereas the 12S RNA codes predominantly for both the NS and M proteins of the virion.     

In Vitro Protein-Synthesizing Activity of Vesicular Stomatitis Virus-Infected Cell Extracts

Crude cytoplasmic extracts from vesicular stomatitis virus (VSV)-infected HeLa cells incorporate radioactive amino acids into hot trichloroacetic acid-precipitable material linearly for 10 to 20 min. The material synthesized in vitro corresponds in molecular weight to four of the five VSV structural proteins. However, synthesis of the viral glycoprotein (G) is significantly reduced, whereas the relative amounts of viral structural proteins L and NS synthesized are increased compared with the ratio of the proteins found in the virion. Fractionation of a VSV-infected crude cytoplasmic extract into a cytoplasmic pellet (20,000 x g for 30 min) and a cytoplasmic supernatant results in a significant reduction in protein synthesizing activity of both fractions, although both contain polysomes. The products synthesized by a cytoplasmic supernatant-directed system included all the VSV structural proteins except the glycoprotein, whereas in an in vitro system directed by the cytoplasmic pellet there is a marked reduction in synthesis of the nucleoprotein (N) and also a small relative increase in synthesis of the glycoprotein. Addition of uninfected, preincubated HeLa or L-cell S10 or a HeLa ribosomal fraction to the VSV-infected cytoplasmic pellet results in a 30- to 60-fold stimulation of (35)S-methionine incorporation. However, these uninfected extracts do not stimulate (35)S-methionine incorporation by the infected crude cytoplasmic extract or the cytoplasmic supernatant. The products synthesized by the stimulated cytoplasmic pellet now include sizeable amounts of the glycoprotein in addition to the other VSV structural proteins.     

Polyoma virus complementary RNA directs the in vitro synthesis of capsid proteins VP1 and VP2.

Polyoma virus complementary RNA, synthesized in vitro by using highly purified Escherichia coli RNA polymerase and nondefective form I polyoma DNA, was translated in a wheat germ cell-free system. Polypeptides were synthesized that comigrated on sodium dodecyl sulfate-polyacrylamide gels with the polyoma capsid proteins VP1 and VP2, although most of the cell-free products were of smaller molecular weights. The VP1-size protein specifically immunoprecipitated with anti-polyoma virus serum, and upon digestion by trypsin yielded [35S]methionine-labeled tryptic peptides that co-chromatographed with the [3H]methionine-labeled tryptic peptides of virion-derived VP1 on both cation-exchange and anion-exchange resins. The VP2-size in vitro product contained all the virion VP2 methionine-labeled tryptic peptides, as shown by cation- and anion-exchange chromatography and two-dimensional fingerprinting on cellulose. We conclude that full-length polyoma VP1 and VP2 are synthesized in response to complementary RNA and consequently that the viral capsid proteins VP1, VP2, and VP3 are entirely virus coded.     

Sub-cellular localization of vesicular stomatitis virus messenger RNAs.

Vesicular stomatitis virus (VSV) messenger RNAs (mRNAs) appear to be compartmentalized within the infected HeLa cells. Analysis by polyacrylamide gel electrophoresis in formamide of the RNA associated with the membrane bound polyribosomes from VSV-infected cytoplasmic extracts shows predominantly one size class of VSV mRNA, which is absent from the remaining cytoplasm. These results are consistent with the mRNA for the viral glycoprotein being exclusively associated with membrane bound polysomes since the latter have been shown to synthesize mainly the virion glycoprotein in an $\text{in vitro}$ translation system.     

Synthesis of exported proteins by membrane-bound polysomes from Escherichia coli.

A membrane-bound fraction of polysomes of Escherichia coli has been isolated after lysis of cells without the use of lysozyme. Protein-synthesis studies in vitro show that membrane-bound and free polysomes are different in the following respects. 1. Membrane-bound polysomes synthesize proteins which are exported from the cell. The products include proteins of the outer membrane and a secreted periplasmic protein, the maltose-binding protein. 2. The major product synthesized by free polysomes is elongation factor Tu, a soluble cytoplasmic protein. 3. The activity of membrane-bound polysomes in vitro is more resistant to puromycin than is the activity of free polysomes. In addition, the mRNA associated with membrane-bound polysomes is more stable than the bulk of cellular mRNA as revealed by studies with rifampicin.     

Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein g mRNA into membrane-bound polyribosome complexes

Glycoprotein mRNA (G mRNA) of vesicular stomatitis virus is synthesized in the cytosol fraction of infected HeLa cells. Shortly after synthesis, this mRNA associates with 40S ribosomal subunits and subsequently forms 80S monosomes in the cytosol fraction. The bulk of labeled G mRNA is then found in polysomes associated with the membrane, without first appearing in the subunit or monomer pool of the membrane-bound fraction. Inhibition of the initiation of protein synthesis by pactamycin or muconomycin A blocks entry of newly synthesized G m RNA into membrane-bound polysomes. Under these circumstances, labeled G mRNA accumulates into the cytosol. Inhibition of the elongation of protein synthesis by cucloheximide, however, allows entry of 60 percent of newly synthesized G mRNA into membrane-bound polysomes. Furthermore, prelabeled G mRNA associated with membrane-bound polysomes is released from the membrane fraction in vivo by pactamycin or mucomycon A and in vitro by 1mM puromycin - 0.5 M KCI. This release is not due to nonspecific effects of the drugs. These results demonstrate that association of G mRNA with membrane-bound polysomes is dependent upon polysome formation and initiation of protein synthesis. Therefore, direct association of the 3' end of G mRNA with the membrane does not appear to be the initial event in the formation of membrane-bound polysomes.     

Tubulin Synthesis in Rat Forebrain: Studies with Free and Membrane-Bound Polysomes

Abstract: Free and membrane-bound polysomes were prepared from rat forebrain and added to a cell-free system containing rabbit reticulocyte factors and L-[³⁵S]methionine. The translation products were analyzed by two-dimensional gel electrophoresis followed by autoradiography. The free polysomes synthesized actin and at least four major tubulin subunits (α¹, α², β¹, and α²) that are found in rat forebrain cytoplasm. The membrane-bound polysomes synthesized predominantly one protein (MB) in the tubulin region of the two-dimensional gel. MB has a molecular weight and isoelectric point similar to α-tubulin. Only trace amounts of α- and β-tubulin and actin were synthesized by the membrane-bound polysomes. MB co-purified with cytoplasmic tubulin after two cycles of aggregation and disaggregation. MB synthesized in vitro (from membrane-bound polysomes) and α- and β-tubulin and actin subunits (synthesized from free polysomes) were digested with Staphylococcus aureus V8 protease, and the resulting peptides were separated by slab gel electrophoresis followed by autoradiography. The peptide pattern of MB was similar but not identical to the peptide patterns of α- and β-tubulin; MB yielded peptides not found in tubulin. We conclude that membrane-bound polysomes from rat forebrain do not synthesize significant amounts of the predominant tubulin subunits synthesized by free polysomes. A major protein (MB) is synthesized by membrane-bound polysomes and is similar, but not identical, to α-tubulin synthesized by free polysomes on the basis of molecular weight, isoelectric point, and peptide analysis.     

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.     

How a single sindbis virus mRNA directs the synthesis of one soluble protein and two integral membrane glycoproteins

Previous work has shown that the 26S RNA found in Sindbis-infected chicken embryo fibroblasts encodes the three viral structural proteins, one internal protein, core, and two membrane glycoproteins, E₁ and E₂. This mRNA has one initiation site; core, E₁, and E₂ are derived by proteolytic cleavage. Here we show that during infection, the 26S RNA is found mainly in membrane-bound polysomes which synthesize all three virion structural proteins. These polysomes are released from the membrane upon treatment with puromycin and high salt. Newly synthesized core protein is localized on the cytoplasmic side of endoplasmic reticulum membranes, while newly synthesized envelope proteins are sequestered by the lipid bilayer. These results suggest that the nascent glycoproteins, presumably their amino termini, are of major importance in directing the binding of polysomes containing 26S mRNA to endoplasmic reticulum membranes and the subsequent transfer of glycoproteins into the bilayer.     

Tryptic peptide analysis on nonstructural and structural precursor proteins from Semliki Forest virus mutant-infected cells.

Analysis of [35S]methionine-labeled tryptic peptides of the large proteins induced by temperature-sensitive mutants of Semliki Forest virus was carried out. The 130,000-molecular-weight protein induced by ts-2 and ts-3 mutants contained the peptides of capsid protein and of both major envelope proteins E1 and E2. The ts-3-induced protein with molecular weight of 97,000 contained peptides of the capsid and envelope protein E2 but not those of E1. Two proteins with molecular weights of 78,000 and 86,000 from ts-1-infected cells did not contain the peptides of the virion structural proteins. They are evidently expressions of the nonstructural part of the 42S RNA genome of Semliki Forest virus.     

Evidence for Simian Virus 40 (SV40) Coding of SV40 T-Antigen and the SV40-Specific Proteins in HeLa Cells Infected with Nondefective Adenovirus Type 2-SV40 Hybrid Viruses

HeLa cells infected with the nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses (Ad2(+)ND1, Ad2(+)ND2, Ad2(+)ND4, and Ad2(+)ND5) synthesize SV40-specific proteins ranging in size from 28,000 to 100,000 daltons. By analysis of their methionine-containing tryptic peptides, we demonstrated that all these proteins shared common amino acid sequences. Most methionine-containing tryptic peptides derived from proteins of smaller size were contained within the proteins of larger size. Seventeen of the 21 methionine-containing tryptic peptides of the largest SV40-specific protein (100,000 daltons) from Ad2(+)ND4-infected cells were identical to methionine-containing peptides of SV40 T-antigen immunoprecipitated from extracts of SV40-infected cells. All of the methionine-containing tryptic peptides of the Ad2(+)ND4 100,000-dalton protein were found in SV40 T-antigen immunoprecipitated from SV40-transformed cells. All SV40-specific proteins observed in vivo could be synthesized in vitro using the wheat germ cell-free system and SV40-specific RNA from hybrid virus-infected cells that was purified by hybridization to SV40 DNA. As proof of identity, the in vitro products were shown to have methionine-containing tryptic peptides identical to those of their in vivo counterparts. Based on the extensive overlap in amino acid sequence between the SV40-specific proteins from hybrid virus-infected cells and SV40 T-antigen from SV40-infected and -transformed cells, we conclude that at least the major portion of the SV40-specific proteins cannot be Ad2 coded. From the in vitro synthesis experiments with SV40-selected RNA, we further conclude that the SV40-specific proteins must be SV40 coded and not host coded. Since SV40 T-antigen is related to the SV40-specific proteins, it must also be SV40 coded.     

Chinese hamster ovary cells replicate adenovirus deoxyribonucleic acid.

Chinese hamster ovary (CHO) cells infected with adenovirus type 2 (Ad2) produced amounts of viral deoxyribonucleic acid (DNA) equal to that synthesized in permissively infected HeLa cells. However, there was 6,000-fold less virion produced in CHO cells. Since the structural viral polypeptides were not detected by pulse-labeling CHO cells at various times postinfection, the block in virion formation is located between the synthesis of viral DNA and late proteins. Extracts of CHO cells could also function in a recently reported in vitro Ad2 DNA synthesis system which is dependent upon the addition of exogenous Ad2 DNA covalently linked to a 5'-terminal protein (Ikeda et al., Proc. Natl. Acad. Sci. U.S.A. 77:5827-5831, 1980). Extracts of infected CHO cytoplasm were able to complement uninfected CHO nuclear extracts to synthesize viral DNA on Ad2 templates. This in vitro replication system has the potential to probe host DNA synthesis requirements as well as viral factors.     

In Vitro Translation of Cardiovirus Ribonucleic Acid by Mammalian Cell-free Extracts

Cell-free extracts prepared from Ehrlich ascites and mouse L cells synthesize viral proteins in response to encephalomyocarditis virus, mouse Elberfeld virus, and mengovirus ribonucleic acid. Although HeLa cell extracts are inactive, their ribosomes are functional in the presence of heterologous supernatant fractions. Synthesis depends upon the addition of adenosine triphosphate, guanosine triphosphate, an energy-generating system, and 4 mm Mg(2+). Initiation is completed during the first 10 to 20 min of incubation, but chain elongation continues for 1 hr or more. The products are of higher molecular weight than virion structural proteins and resemble polypeptides formed in virus-infected cells during a short pulse. Tryptic peptides of virion proteins and in vitro products are similar for all three cardioviruses.     

Synthesis of S100 Protein on Free and Membrane-Bound Polysomes of the Rabbit Brain

Abstract: Free and membrane-bound polysomes were isolated from the cerebral hemispheres and cerebellum of the young adult rabbit. The two polysomal populations were translated in an mRNA-dependent cell-free system derived from rabbit reticulocytes. Analysis of the [³⁵S]methionine-labeled translation products on two-dimensional polyacrylamide gels indicated an efficient separation of the two classes of brain polysomes. The relative synthesis of S100 protein by free and membrane- bound polysomes was determined by direct immuno-precipitation of the cell-free translation products in the presence of detergents to reduce nonspecific trapping. Synthesis of S100 protein was found to be twofold greater on membrane-bound polysomes compared with free polysomes isolated from either the cerebral hemispheres or the cerebellum. In addition, the proportion of poly- (A+)mRNA coding for SlOO protein was also twofold greater in membrane-bound polysomes compared with free polysomes isolated from the cerebral hemispheres. These results indicate that the cytoplasmic S100 protein is synthesized predominantly on membrane-bound polysomes in the rabbit brain. We suggest that the nascent S100 polypeptide chain translation complex is attached to the rough endoplasmic reticulum by an ionic interaction involving a sequence of 13 basic amino acids in S100 protein.     

Membrane association of functional vesicular stomatitis virus matrix protein in vivo.

The matrix (M) protein of vesicular stomatitis virus (VSV) is a major structural component of the virion which is generally believed to bridge between the membrane envelope and the ribonucleocapsid (RNP) core. To investigate the interaction of M protein with cellular membranes in the absence of other VSV proteins, we examined its distribution by subcellular fractionation after expression in HeLa cells. Approximately 90% of M protein, expressed without other viral proteins, was soluble, whereas the remaining 10% was tightly associated with membranes. A similar distribution in VSV-infected cells has been observed previously. Conditions known to release peripherally associated membrane proteins did not detach M protein from isolated membranes. Membrane-associated M protein was soluble in the detergent Triton X-114, whereas soluble M protein was not, suggesting a chemical or conformational difference between the two forms. Membranes containing associated M protein were able to bind RNP cores, whereas membranes lacking M protein were not. We suggest that this membrane-bound M fraction constitutes a functional subset of M protein molecules required for the attachment of RNP cores to membranes during normal virus budding.     

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.     

Unique peptide maps of the three largest proteins specified by the flavivirus Kunjin.

Tryptic digests of four polypeptides found in Kunjin virus-infected Vero cells, NV5, NV4, V3, and NV3, were compared by peptide mapping. The polypeptides to be analyzed were labeled with radioactive methionine and separated by electrophoresis through polyacrylamide gels containing sodium dodecyl sulfate. Because infection of Vero cells by Kunjin virus does not inhibit host cell protein synthesis, radioactively labeled viral polypeptides prepared from infected cells migrate coincidentally during sodium dodecyl sulfate-gel electrophoresis with some of the labeled host proteins. Thus, the genuine viral methionine-containing peptides in tryptic digests of viral proteins have been identified by co-analyzing polypeptides from [3H]methionine-labeled uninfected cells and [35S]methionine-labeled infected cells and determining the 35S/3H ratio in the peptides resolved in two dimensions on thin-layer chromatography plates. The peptide map of NV3 demonstrated that it is host coded, whereas NV5, NV4, and V3 have unique peptide maps and, therefore, account for approximately one-half of the coding potential of Kunjin virus RNA.     

Both linked and unlinked mutations can alter the intracellular site of synthesis of exported proteins of Escherichia coli.

It previously has been demonstrated that synthesis of the periplasmic maltose-binding protein (MBP) and alkaline phosphatase (AP) of Eschericha coli predominantly occurs on membrane-bound polysomes. In this study, signal sequence alterations that adversely affect export of MBP and AP, resulting in their cytoplasmic accumulation as unprocessed precursors, were investigated to determine whether they have an effect on the intracellular site of synthesis of these proteins. Our findings indicate that export-defective MBP and AP are not synthesized or are synthesized in greatly reduced levels on membrane-bound polysomes. In some instances, a concomitant increase in the amount of these proteins synthesized on free polysomes was clearly discerned. We also determined the site of synthesis of MBP and AP in strains harboring mutations thought to alter the cellular secretion machinery. It was found that the presence of a prlA suppressor allele partially restored synthesis of export-defective MBP on membrane-bound polysomes. On the other hand, the absence of a functional SecA protein resulted in the synthesis of wild-type MBP and AP predominantly on free polysomes.