Biosynthesis of reovirus-specified polypeptides: the reovirus s1 mRNA encodes two primary translation products

Reovirus serotypes 1 (Lang strain) and 3 (Dearing strain) code for a hitherto unrecognized low-molecular-weight polypeptide of Mr approximately 12,000. This polypeptide (p12) was synthesized in vitro in L-cell-free protein synthesizing systems programmed with either reovirus serotype 1 mRNA, reovirus serotype 3 mRNA, or with denatured reovirus genome double-stranded RNA, and in vivo in L-cell cultures infected with either reovirus serotype. The synthesis of p12 in vivo was insensitive to actinomycin D, and occurred at similar times after infection as the previously identified reovirus encoded lambda, mu, and sigma polypeptides. Pulse-chase experiments in vivo, and the relative kinetics of synthesis of p12 in vitro, indicate that it is a primary translation product. Fractionation of reovirus mRNAs by velocity sedimentation and translation of separated mRNAs in vitro suggests that p12 is coded for by the s1 mRNA, which also codes for the previously recognized sigma 1 polypeptide. Synthesis of both p12 and sigma 1 in vitro in L-cell-free protein synthesizing systems programmed with denatured reovirus genome double-stranded RNA also suggests that these two polypeptides can be coded by the same mRNA species. The Mr approximately 12,000 polypeptide was not a detectable structural component of purified virions, and antiserum prepared against purified reovirions did not immunoprecipitate p12. It is proposed that the Mr approximately 12,000 polypeptide encoded by the S1 genome segment be designated sigma 1bNS, and that the polypeptide previously designated sigma 1 be renamed sigma 1a.     

A genetic map of reovirus. II. Assignment of the double-stranded RNA-negative mutant groups C, D, and E to genome segments

The double-stranded RNA (dsRNA) of recombinants derived from crosses of the dsRNA-negative, temperature-sensitve (ts) mutants of reovirus type 3 and reovirus serotypes 1 or 2 were examined by polyacrylamide gel electrophoresis. Analysis of deletions and replacements in the recombinants allowed identification of genome segments containing the ts lesions. In this way the location of the mutation of the group C prototype mutant tsC(447) os genome segment S2, that of the group D prototype mutant tsD(357) is genome segment L1, and that of the group E prototype mutant tsE(320) is genome segment S3. In addition the location of the temperature-sensitive lesion of serotype 2 is genome segment S1.     

The sequences of the S2 genome segments of reovirus serotype 3 and of the dsRNA-negative mutant ts447

The most temperature-sensitive dsRNA-negative mutant of reovirus serotype 3 is ts447; the amount of dsRNA formed in cells infected with it at 39 degrees is less than 0.1% of that formed in cells with wt virus at 37 degrees. The genome segment in which this mutation is located is S2. We compare here the sequence of the S2 genome segment of wt reovirus serotype 3 with that of mutant ts447. The two sequences differ in three locations, at two of which there are C to U transitions, while at the third there is an A to G transition. All cause amino acid changes (Ala to Val, Ala to Val, and Asn to Asp, respectively). One mutation (at nucleotide position 581, which causes an Ala to Val change) causes the length of an alpha-helix to be significantly reduced and may be that which is responsible for the ts phenotype.     

Temperature-sensitive mutants of influenza virus: A mutation in the hemagglutinin gene

A mutant (ts-61S) belonging to a single recombination-complementation group (Group VI) was obtained by segregation of an influenza virus WSN (HON1) temperature-sensitive double mutant (ts-61) that possessed mutational lesions characteristic of Groups V and VI. The segregant retained the thermolabile hemagglutinating activity of the parental mutant, ts-61, but lost the defectiveness in virion RNA synthesis manifested by the parent at the nonpermissive temperature. No hemagglutinating activity developed in cells infected with ts-61S at the nonpermissive temperature. In rescue experiments all HO-serotype progeny from the cross between ts-61S (HO-serotype) and temperature-resistant H3-serotype virus were temperature-sensitive, localizing the ts defect in the hemagglutinin gene. No glycosylated hemagglutinin polypeptide was detected in the polyacrylamide gel electropherogram of cells infected with ts-61S at the nonpermissive temperature, whereas the synthesis of neuraminidase (the other virion glycoprotein) proceeded normally at both permissive and nonpermissive temperatures. The results indicate that the Group VI mutation is in the gene coding for the viral hemagglutinin.     

Interferon action against reovirus: activation of interferon-induced protein kinase in mouse L929 cells upon reovirus infection

Experiments are presented which indicate that reovirus infection results in an activation of the interferon (IFN)-induced protein kinase in mouse L929 cells. This is indicated by (i) the phosphorylation in vivo of a 67,000 M_r (67K) polypeptide, which is characteristic of the IFN-induced protein kinase, within a few hours after reovirus infection of IFN-treated mouse L929 cells, and (ii) the phosphorylation in vitro of the 67K polypeptide as well as the α-subunit of exogenously added initiation factor eIF-2 in extracts of IFN-treated reovirus-infected cells without the addition of double-stranded RNA which is required in similar extracts from uninfected cells. In NIH 3T3 cells, which are deficient in (2′,5′)oligoadenylate-activated endonuclease, the replication of reovirus is inhibited by IFN treatment, though EMC virus replication is insensitive. It is suggested that this kinase activation observed upon reovirus infection may play a role in the antiviral action of IFN against reovirus.     

Genetic analysis of adenovirus type 2 IV. Coordinate regulation of polypeptides 80K,Illa, and V

An adenovirus type 2 temperature-sensitive mutant, ts3, deficient in virion assembly was studied by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electron microscopy. A total of 28 virus-induced polypeptides could be detected, four of which were precursor proteins. At the nonpermissive temperature, ts3 failed to synthesize a newly identified nonstructural polypeptide 80K. Polypeptide V was absent, but it may have been replaced by an arginine-rich polypeptide which was oversynthesized and migrated as a 50K band regardless of temperature. Similarly, polypeptide 55K (= IVa₂) was also oversynthesized and migrated slower than WT-55K (= IVa₂). In addition, several polypeptides were synthesized at increased or decreased levels, compared with WT. Polypeptide IIIa was sometimes resolved into three bands (66K, 67K, 68K) in WT, while ts3-IIIa lacked the 66K and 68K components. Polypeptide 36K was found to be rich in arginine. Consistent with the notion that the processing of PVII into VII requires virion assembly, no processing of ts3-PVII could be observed. By contrast, the cleavage products, VI and VIII, appeared normally, thus disputing the virion assembly requirement previously postulated for these polypeptides. Electron microscopy of ts3-infected cells revealed two hitherto undescribed intranuclear structures, possible tubular forms in addition to normal virions at the permissive temperature, and roughly spherical, core-like structures of 80–100 nm at the restrictive temperature. These results suggest that the pleiotropic effects of the ts3 mutation arrest virus assembly at a corelike structure.     

Studies on temperature-sensitive events in synthesis of poliovirus ts mutants

Summary Studies on temperature-sensitive events of four poliovirus ts mutants indicated that the temperature-sensitive stage of ts2 and ts5 mutants occurs late in the reproduction cycle, but for ts 11 and tsM/23 it occurs both at early and at late stages of the cycle. RNA production by ts2, ts11 and tsM/23 mutants (RNA^–) under nonpermissive conditions (40° C) is 13–14 per cent as compared with RNA production at 36° C. All the mutants under study form both 35S and 20S RNA under nonpermissive conditions, but the relative amount of 35S RNA synthesized at 40° C is considerably lower than at 36° C. A degradation of viral RNA under nonpermissive conditions is detected. Obtained data suggest that ts5 mutant (RNA⁺) under nonpermissive conditions is incapable of assembly of virus particles from synthesized components, but the multiplication of ts2, ts11 and tsM/23 mutants does not occur because of strong inhibition of RNA synthesis. Protein subvirion structures induced by ts5 mutant under nonpermissive conditions after subsequent transfer of the infected cells to permissive conditions, are incorporated into mature virus particles. Subvirion structures induced by ts M/23 mutant do not participate in further stages of morphogenesis after the infected cells are shifted to permissive conditions.     

Synthesis of virus-specific polypaptides by temperature-sensitive mutants of herpes simplex virus type 1.

The polypeptide phenotypes of 22 temperature-sensitive (ts) mutants of herpes simplex virus type 1 were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis of mutant-infected cells at permissive and nonpermissive temperatures. Following analysis of isotopically labeled polypeptides synthesized from 4–24 hr postinfection, the mutants were divided into four major phenotypic groups which include: (1) DNA^?ts mutants which share several common polypeptide defects, (2) DNA^±ts mutants which exhibit polypeptide profiles resembling the DNA^?ts mutants, (3) DNA⁺ts mutants which exhibit polypeptide phenotypes differing only slightly from that observed in wild-type virus-infected cells grown at 39°, and (4) DNA⁺ts mutants which exhibit no detectable alterations in their polypeptide profiles when compared with that of the wildtype virus. When the polypeptide phenotypes of the mutants were compared with previously determined mutant characteristics, including synthesis of viral DNA, thymidine kinase, DNA polymerase, and physical virus particles, a correlation was consistently observed between mutant polypeptide and viral DNA phenotypes.     

Changes in membrane polypeptides that occur when chick embryo fibroblasts and NRK cells are transformed with avian sarcoma viruses

Studies were designed to detect changes in the plasma membrane polypeptide composition of chick embryo fibroblasts (CEF) and Normal rat kidney (NRK) cells following infection with avian RNA tumor viruses. A method capable of isolating large fragments of plasma cell membranes, based on fractionation in a two-phase polymer system, was developed for this purpose.Infection with the Prague (subgroup C) or SR (subgroup A) strains of RSV, or with ts 68 of SR-RSV-A or ts 339 of B77 at the permissive temperature, caused CEF to transform morphologically; simultaneously a limited number of changes occurred in their plasma cell membrane composition. They were: (a) a large increase in the rate of labeling with ¹⁴C-valine and in the total amount of a polypeptide with an apparent MW of 73,000; a similar small increase occurred with respect to a polypeptide with an MW of 95,000; neither could be labeled with ¹⁴C-glucosamine; (b) a decrease in the rate of labeling and in the total amount of high molecular weight glycopoly-peptides with MWs of about 250,000; (c) a decrease in the rate of labeling and in the total amount of a polypeptide with an MW of 39,000. Infection with ts 68 and ts 339 at the nonpermissive temperature, or with a nontransforming mutant of SR-RSV-A, or with RAV-7, did not cause alterations (a) and (b), but did cause alteration (c).Identical membrane polypeptide changes were observed in NRK cells infected with the Prague strain of RSV or with ts 339 of B77 at the permissive but not at the nonpermissive temperature. The significance of the fact that these changes, in particular the increases in the amounts of the 73,000 and 95,000 dalton polypeptides, occurred to the same extent in the membranes of both avian and mammalian cells is discussed.     

The nature of the polypeptide encoded by each of the 10 double-stranded RNA segments of reovirus type 3

Under suitable conditions of denaturation, the double-stranded (ds) RNA segments of reovirus can be translated in cell-free protein synthesizing systems. Since all 10 segments of reovirus ds RNA can be isolated in virtually pure form, this provides a means for determining the nature of the polypeptide encoded by each individual segment. The complete coding assignment set was determined for the Dearing strain of reovirus serotype 3. Polypeptide identification was made not only on the basis of electrophoretic migration rates in both the phosphate- and Tri-glycine (Laemmli)-based polyacrylamide gel systems, but also on the basis of comparing peptide profiles of in vitro translation products and authentic reovirus polypeptides after digestion with staphylococcal V8 protease. The latter method provides absolute identification. The assignment set is (using the commonly accepted designation for the ds RNA segments, but a newly proposed nomenclature for the polypeptides): Segment L1 codes for the minor virion component λ3, and segments L2 and L3 code for the two major virion core components λ2 and λ1, respectively; segment M1 codes for a minor virion component μ2, segment M2 codes for the polypeptide that is present in virions both in the form of the minor component μ1 and as the major component μ1C which is derived from it by cleavage, and segment M3 codes for the nonstructural polypeptide μNS; and segment S1 codes for the minor outer capsid shell component σ1, segment S2 codes for the core component σ2, segment S3 codes for the nonstructural polypeptide σNS, and segment S4 codes for the major outer capsid shell component σ3.     

Genetics of reovirus: Identification of the ds RNA segments encoding the polypeptides of the μ and σ size classes

Recombinants derived from crosses between reovirus serotypes 1, 2, and 3 were utilized to identify the ds RNA segments encoding polypeptides of the μ and σ size classes. The results indicate the following assignments: M1 ds RNA encodes polypeptide μ2, M2 encodes μ1, M3 encodes μNS, S1 encodes σ1, S2 encodes σ2, S3 encodes σNS, and S4 encodes σ3. In addition polypeptide species μ2 has been conclusively identified as a structural component of the reovirus core. A new nomenclature for the viral polypeptides is discussed.     

Studies on the role of M protein in virus assembly using a ts mutant of HVJ (Sendai virus).

A temperature-sensitive mutant of HVJ, HVJ cl.151, was isolated from BHK cells persistently infected with HVJ and characterized. HVJ c1.151 virion had an M polypeptide different in apparent molecular weight from that of HVJ wild-type, that is 36,000 and 34,000 daltons, respectively. HVJ c1.151 was blocked in a late function required for virus maturation. M protein antigen of HVJ c1.151 was detected in infected cells by immunofluorescent microscopy only at permissive temperature but not at nonpermissive temperature, although GP and NP antigens were detected at both temperatures. Further, analysis of the infected cells by SDS-polyacrylamide gel electrophoresis showed that viral structural polypeptides P, F₀, NP, and F and nonstructural polypeptide C were synthesized in infected cells at nonpermissive temperature and these structural polypeptides were incorporated into virions upon temperature shiftdown, whereas polypeptides HN and M, which may be synthesized at nonpermissive temperature, were not able to be incorporated into virions upon temperature shift down. Thus, the temperature-sensitive lesion of HVJ c1.151 is considered to be in HN and M proteins. Membrane fluorescense, immunoferritin electron microscopy, and SDS-polyacrylamide gel electrophoresis of plasma membranes showed that migration of F₀, and F to the cell surface occurred normally even at nonpermissive temperature. Immunoferritin electron microscopy also demonstrated that the viral glycoproteins which arrived at the plasma membrane were dispersed on the entire surface of the membrane at the nonpermissive temperature and that viral components synthesized at this temperature could not assemble at the plasma membrane. In addition, it was found that antibody-induced redistribution of viral glycoproteins on the surface of cells infected with HVJ c1.151 and incubated at nonpermissive temperature occurred very rapidly; in contrast, such redistribution of viral glycoproteins occurred more slowly and less completely in cells incubated at permissive temperature, suggesting that viral glycoproteins on the plasma membrane of cells at nonpermissive temperature have a high degree of mobility in the plane of the membrane as compared with those on the plasma membrane of cells at permissive temperature. These results suggest strongly that a function which fixes the viral glycoproteins at restricted areas of plasma membrane to form a viral envelope is blocked in HVJ c1.151-infected cells at nonpermissive temperature. Analysis of plasma membrane by SDS-polyacrylamide gel electrophoresis showed that viral glycopolypeptides but no NP were present on membranes isolated from HVJ cl.151-infected cells at nonpermissive temperature in spite of the presence of a large amount of NP in the whole cells, whereas plasma membranes isolated from cells at permissive temperature contained all viral structural polypeptides. The possible roles of M protein of HVJ in formation of the viral envelope and association of nucleocapsid with the envelope during assembly are discussed based on these results.     

Reovirus inhibition of cellular RNA and protein synthesis: role of the S4 gene

Type 2 reovirus inhibits L cell RNA and protein synthesis more rapidly and efficiently than type 3. By using recombinant reoviruses containing various combinations of double-stranded RNA segments derived from reovirus type 2 and 3 we have found that the S4 double-stranded RNA segment (the segment that encodes the σ3 polypeptide, the major outer capsid polypeptide) is responsible for the capacity of type 2 to inhibit L cell RNA and protein synthesis.     

Phenotypic characterization and physical mapping of a temperature-sensitive mutant of Autographa californica nuclear polyhedrosis virus defective in DNA synthesis

A ts mutant of Autographa californica nuclear polyhedrosis virus (AcMNPV), ts8, was shown to be defective in viral DNA synthesis at the nonpermissive temperature. Ts8-infected cells synthesized only early viral polypeptides at the nonpermissive temperature, and in contrast to wild-type (WT)-infected cells, showed no inhibition of host cell protein synthesis. The effect of the mutation on viral DNA synthesis was not immediately reversed after shifting infected cells down from the nonpermissive temperature to the permissive temperature; rather, a delay of several hours occurred before viral DNA synthesis was detected. The rate of accumulation of viral DNA in ts8-infected cells failed to increase after a shift from the permissive temperature to the nonpermissive temperature. This indicated that the ts8 mutation was involved in the synthesis of proteins required for viral DNA synthesis. The mutation was mapped by marker rescue to the region lying between 60.1 and 62.0% of the AcMNPV physical map.     

Control of protein synthesis in herpesvirus-infected cells: analysis of the polypeptides induced by wild type and sixteen temperature-sensitive mutants of HSV strain 17.

The polypeptides induced in cells infected with a Glasgow isolate of HSV-I (17 syn+) have been characterized by SDS polyacrylamide gel electrophoresis. Study of the kinetics of synthesis in three cell lines has detected a total of 52 polypeptides, 33 of which can be identified in polypeptide profiles of purified virions. These include six low mol. wt. polypeptides that have not been previously reported. Several polypeptides were labelled with glucosamine in infected BHK cells. The different polypeptide patterns obtained at permissive (31 degrees C) and nonpermissive (38 degrees C) temperature in cells infected with 16 temperature-sensitive (ts) mutants are reported. The effect of multiplicity of infection (m.o.i.) on the polypeptide profile has been examined for two of the DNA -ve mutants: below ten, the profile varied with the m.o.i. whereas above ten it was constant. All mutants were therefore examined at an m.o.i. of approx. 20. Mutants from the same complementation group showed very similar profiles. A number of general conclusions concerning control of protein synthesis in HSV infected cells can be made: (I) As most of the 16 ts mutants affected the synthesis of several or many polypeptides it follows that a large proportion of genome specifies controlling functions. (2) The high frequency with which some polypeptides were affected suggests they are at or near the terminus of biosynthetic pathways which are under multiple control. (3) Conversely, some polypeptides were affected with a low frequency suggesting that their synthesis is not dependent on the expression of many virus functions. (4) Several individual ts mutations lead to the synthesis of increased amounts of different large polypeptides. (5) Analysis of every band detectably affected by at least one ts mutation has disclosed nine classes of dependence relationship between polypeptide synthesis and the DNA phenotype of the mutants, illustrating that this relationship is complex and different for different polypeptides. (6) The inhibition of host protein synthesis by the virus may not be a simple single step process.     

SV40 T-antigen-related surface antigen: correlated expression with nuclear T-antigen in cells transformed by an SV40 A-gene mutant

Rat embryo cells transformed by the SV40 A-gene mutant tsA 28.3 were analyzed at permissive and nonpermissive temperatures for the expression of SV40 nuclear T-antigen (T-Ag) and of SV40 T-Ag-related surface antigen (“surface T”) by indirect immunofluorescence microscopy. At permissive temperature both antigens were detected with rabbit antiserum prepared against purified SDS-denatured T-Ag. Upon shift of tsA 28.3 cells to the nonpermissive temperature, expression of both nuclear T-Ag and of surface T decreased simultaneously. When the cells were returned to the permissive temperature, both nuclear T-Ag and surface T reappeared. Analysis of extracts from tsA 28.3 cells grown and labeled with [³⁵S]methionine either at permissive or at nonpermissive temperature by immunoprecipitation and SDS-polyacrylamide gel electrophoresis demonstrated that tsA 28.3 cells synthesized the T-Ag polypeptide only at the permissive temperature. The T-Ag polypeptide was not detected in extracts of cells grown and labeled at nonpermissive temperature. These results demonstrate a strong correlation between the expression of nuclear T-Ag, surface T, and the synthesis of the T-Ag polypeptide, suggesting that both nuclear T-Ag and surface T are products of the SV40 A-gene.     

Characterization of the polypeptides synthesized in cells infected with a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture

Summary The intracellular synthesis of virus-specific polypeptides in cells infected with the wild-type virus of HVJ (HVJ-W) (haemagglutinating virus of Japan—the Sendai strain of parainfluenza 1 virus) and with a temperature-sensitive(ts) mutant (HVJ-pB) derived from an HVJ carrier culture has been analysed by polyacrylamide gel electrophoresis. At the permissive temperature (32° C), all of the known virus structural polypeptides were identified in cells infected with each strain of virus and in addition to the non-structural polypeptides B and C, another polypeptide at the region with a molecular weight of 26,000 to 27,000 (26 to 27K) could be detected in infected cells. At the non-permissive temperature (38° C), the synthesis of the polypeptide M was markedly restrained in cells infected with HVJ-pB, while other major virus polypeptides were present in approximately comparable amounts to cells infected with the wild-type virus. A non-structural polypeptide with a molecular weight of 105K was dominant ints mutant infected cells at higher temperatures and disappeared after temperature-shift from 38° to 32° C. The production of the non-structural polypeptides B and 27K was also temperature-sensitive. The molecular weights of the polypeptides B, M and 27K in HVJ-pB infected cells were larger than those of the corresponding polypeptides in HVJ-W infected cells. The synthesis of the M protein in HVJ-pB infected cells started just after lowering the incubation temperature and the newly made M protein was successfully incorporated into virus particles.With 4 Figures     

Genetic analysis of adenovirus type 2: VII. Cleavage-modified affinity for DNA of internal virion proteins

The core structures derived from wild-type (wt) Ad2 and ts1 virions, by disintegration with N-laury sarcosine and pyridine, were analyzed biochemically and by electron microscopy. Disintegration of wt virus with Sarkosyl yielded cores of density 1.58 g/ml (in CsCl) whereas the particles synthesized by ts1 at 39° (ts1-39°) generated subviral structures ranging in density from 1.58 to 1.72, indicating the increased lability of the mutant virions. The wt core (1.58 g cm^?3) contained predominantly polypeptide VII, while the ts1-39° cores only contained traces of this polypeptide. Significantly, Pre-VII, which is the principal form of this polypeptide in the ts1-39° virions, was completely absent from the cores. Electron microscopy of wt and ts1-Sarkosyl cores revealed identical types of structures on negative staining, although the ts1-39° cores were found to unfold more rapidly in the presence of high salt. The polypeptide analysis of the pyridine cores showed significant differences between wt and ts1. Wild-type core contained polypeptides V, VII, and 14K; ts1-33° core contained V, Pre-VII, VII, traces of IVa2, Pre-VI, VI, and possibly IX and X; while ts1-39° core contained V, Pre-VI, and Pre-VII. These findings clearly establish structural differences between wt and ts1 cores, most important the differential affinity of precursor polypeptides for DNA in ts1 virions under different conditions of disintegration. It is suggested that the loss of infectivity by the mutant is due to the altered composition and structure of the viral core.