In vitro translation of immediate early, early, and late classes of RNA from vaccinia virus-infected cells.

Cytoplasmic RNA, isolated at various times after vaccinia virus infection, was translated in a message-dependent cell-free system prepared from rabbit reticulocytes. Supplementation of the system with calf liver tRNA specifically increased translation of viral RNA. Virtually all of the [³⁵S]methionine-labeled viral proteins from infected cells that were detected by sodium dodecyl sulfate -polyacrylamide gel electrophoresis appeared to be synthesized in the cell-free system. When programmed with RNA extracted at 2 hr after infection, early viral proteins were made and formation of cellular proteins was diminished. Primarily late proteins were synthesized using RNA extracted at 4 or more hr after infection, suggesting that the switch in protein synthesis is regulated principally by changes in RNA concentration rather than by modification of the translation apparatus of the cell. However, the vaccinia virus-mediated inhibition of host protein synthesis that occurred in the presence of actinomycin D was not associated with a decrease in translatable cellular mRNA. Immediate early RNA and early RNA were obtained by infecting cells in the presence of inhibitors of protein and of DNA synthesis, respectively. Analysis of the in vitro translation products did not reveal a class of early genes that require protein synthesis for expression. On the contrary, seven polypeptides, of which a 28,000-dalton species was most prominent, were synthesized in relatively greater amounts with immediate early RNA than with early RNA. All early and late mRNA species appear to be polyadenylylated, and a correlation between RNA sedimentation and molecular weight of translation product was obtained.     

Biosynthesis of reovirus-specified polypeptides: System-dependent effect of reovirus mRNA methylation on translation in vitro catalyzed by ascites tumor and wheat embryo cell-free extracts

The effect of methylation on the translation of reovirus messenger RNA catalyzed by the homologous mouse ascites tumor and the heterologous wheat embryo cell-free protein synthesizing systems was examined. Unmethylated and methylated reovirus mRNAs were translated with comparable efficiency in the ascites system; by contrast, in the wheat system, unmethylated reovirus mRNA was translated about 10% as efficiently as methylated viral mRNA. S-Adenosyl-l-homocysteine (SAdoHcy) inhibited S-adenosyl-l-[Me-³H]methionine-mediated methylation in both the ascites and wheat cell-free extracts; however, translation of unmethylated reovirus mRNA was blocked by SAdoHcy in the wheat, but not in the ascites, cell-free protein synthesizing system.     

Characterization of an influenza a host range mutant

A mixed infection of primary chick kidney cells at 38° with A/Ann Arbor/6/60 cold adapted virus and A/Alaska/6/77 wt virus yielded a cold-reassortant virus, CR43-clone 3, which had a host range different from that of either parent. It does not produce detectable virus when grown in Madin-Darby canine kidney cells, while growing normally in primary chick kidney cells at 33°. Both parents, however, grow well in either cell type at 33°. Genotypic analysis of viral RNA electrophoresed in polyacrylamide gels has shown that CR43-clone 3 virus has an aberrant NS gene different from the NS gene of either parent virus. Reassortant viruses made between CR43-clone 3 virus and A/California/ 10/78 (H1N1) virus in primary chick kidney cells at 33° showed the same host range restriction only if the NS gene was derived from the CR43-clone 3 virus. A mixed infection with these same parents, but in Madin-Darby canine kidney cells at 33°, produced reassortants that always contained the A/California/10/78 NS gene instead of the CR43clone 3 NS gene. Ferrets inoculated intranasally with the CR43-clone 3 reassortant do not become sick or infected, based on the lack of symptoms: no rhinitis, coryza, or fever; and no detectable virus recovered from nasopharyngeal swabs, turbinate, or lung tissues at 48 hr after infection. Thus, CR43-clone 3 virus contains an aberrant NS gene and manifests a restricted host range phenotype in Madin-Darby canine kidney cells and ferrets.     

In vitro translation of measles virus-induced RNA.

RNA was extracted from Vero cells infected with measles virus and tested for its ability to direct protein synthesis in the wheat germ cell-free translation system. When in vitro products were subjected to immunoprecipitation with immunoglobulins directed against measles virus four major bands were found to correspond to the viral proteins.     

Genome Structures of reiteration mutants of simian virus 40.

We have performed structural studies of four reiteration mutant genomes of SV40 (simian virus 40) using electron microscopic heteroduplex analysis, DNA-DNA reassociation tests, and analysis of restriction endonuclease cleavage patterns. Two reiteration mutants which appeared early during serial undiluted passages of two independent SV40 stocks were found to contain similar regions of the SV40 genome in their respective monomer fragments and no monkey substitution sequences. This observation is consistent with the presence of preferred sites for intramolecular recombination in the wildtype SV40 genome. Another reiteration mutant was shown to have a 530 base pair segment of the SV40 genome encompassing the origin for DNA replication which was linked to a 350 base pair monkey DNA sequence. Reassociation kinetic studies revealed that this monkey sequence was reiterated 10 to 20 times per diploid genome. All of the reiteration mutants studied preserved and amplified the origin for SV40 DNA replication.     

In vitro translation products of turnip crinkle virus RNA

Turnip leaves infected with turnip crinkle virus accumulate a 35-kd polypeptide which comigrates with the major protein from isolated virions. RNA from TCV virions directs the synthesis of a number of polypeptides in vitro including a 35-kd protein which is immunoprecipitable with antiserum to virus particles. Translation of viral RNA size-fractionated on sucrose gradients or methyl mercurichydroxide-containing gels shows that the TCV coat protein is synthesized primarily from RNA fragments which are smaller than the genomic RNA. A satellite RNA species found in virions does not direct the synthesis in vitro of any identifiable protein.     

Temperature-sensitive mutants of influenza A/Udorn/72 (H3N2) virus. III. Genetic analysis of temperature-dependent host range mutants

One hundred thirty-three ts mutants of influenza A/Udorn/72 virus were arranged into eight complementation groups, A-H, on Madin-Darby canine kidney (MDCK) monolayer cultures at the restrictive temperature of 40 degrees. The eight complementation groups, A-H, on MDCK cells corresponded to the eight recombination groups, A-H, on rhesus monkey kidney (RMK) cells, respectively, and this suggested that each MDCK complementation group represented one of the eight influenza A RNA gene segments. These ts viruses were used to identify the locus of the ts mutation in temperature-dependent host range (td-hr) mutants of the A/Udorn/72 virus. Sixteen of the 133 ts mutants exhibited distinct host (MDCK)-dependent restriction of plaque formation at 40 degrees but not at 34 degrees and were referred to as td-hr mutants. These 16 td-hr mutants were ts+ (not ts) on RMK cells but ts on MDCK cells. The td-hr mutants did not share a common lesion and the ts lesions were distributed among the eight complementation groups, A-H, when tested on MDCK cells. An analysis of one of the td-hr mutants indicated that an extrageneic RMK-dependent suppressor mutation did not account for the td-hr phenotype. These data suggested that a host-dependent ts mutation was responsible for the td-hr restriction of this mutant. Representation of td-hr mutations in each of the eight complementation groups indicates that the influenza A virus genome can undergo mutation leading to an altered host range in any of its eight RNA segments.     

In vitro translation of tobacco etch virus RNA

The wheat-embryo-derived cell-free system was optimized for translation of tobacco etch virus (TEV) RNA. When examined by polyacrylamide gel electrophoresis, the product of the TEV RNA stimulated system proved to be a single protein with a molecular weight of 40,000. This finding is different from results obtained when TEV RNA is used as a template in the rabbit reticulocyte lysate in vitro protein-synthesizing system.     

Inhibition of vaccinia virus late protein synthesis by isatin-beta-thiosemicarbazone: characterization and in vitro translation of viral mRNA.

Thespecific effect of istin-βthiosemicarbozone (IBT) was manifested after vaccinia virus late protein synthesis had commenced. At 6 hr after infection, viral protein synthesis was inhibited by about 9596. We confirmed that λ portion of the virus-specific RNA appears to be degraded (B. Woodson and W. K. Joklik, 1965, Proc. Nat. Acad. Sci. USA 54,946–953). Nevertheless, the amount of viral RNA that was capped, properly methylated, and polyadenylylated, was reduced by only about 50%. Moreover, RNA from IBT-treated cells stimulated cell-free protein synthesis to one-half the level obtained with RNA from control cells. Polyacrylamide gel electrophoretic analysis further demonstrated that RNA from IBT-treated cells was translated into late viral proteins in vitro. Thus, it seems possible that the inhibition of protein synthesis in IBT-treated cells does not result entirely or directly from either an inhibition of mRNA synthesis or from λ depletion of mRNA caused by accelerated degradation. An alternative possibility, that accelerated degradation is secondary to λ more immediate effect of the drug on protein synthesis, was considered.     

In vitro synthesis of structural and nonstructural proteins of Sendai and SV5 viruses

The messenger RNAs of SV5 and two strains of Sendai virus were isolated from infected cells and translated in a wheat germ cell-free system. Comparison of the peptide maps of the polypeptides synthesizedin vivo andin vitro established that the nonglycosylated polypeptides P, NP, and M of both SV5 and Sendai virus had been synthesizedin vitro. In immunoprecipitation studies of the putative SV5 polypeptides synthesizedin vitro, antiserum against whole virions precipitated NP, P, and M, and other polypeptides which did not comigrate with mature virion polypeptides. Monospecific antisera against the HN and F glycoproteins precipitated two of the latter polypeptides with molecular weights of ～55,000 and 50,000, respectively, suggesting that the nonglycosylated forms of these polypeptides had been synthesizedin vitro. Polypeptide C, previously found in Sendai virus-infected cells and proposed to be a virus-specific nonstructural polypeptide, has been found to exhibit strain-specific differences in migration in polyacrylamide gels. Polypeptides with the appropriate strain-specific migration have been synthesizedin vitro with mRNAs from cells infected with the different Sendai virus strains, and shown by peptide mapping to be the same polypeptides as those synthesizedin vivo. The results have thus provided further evidence that C is a virus-coded nonstructural protein. Another polypeptide (C′), with migrates slightly slower than C, has been found in infected cells and synthesizedin vitro. This polypeptide also exhibits strain-specific differences in migration in polyacrylamide gel electrophoresis, and has been shown by peptide mapping to be similar to C. The explanation for the difference in migration between C and C′ is as yet unknown.     

Affective disorders and cognitive performance. A clinical report

A study of 27 manic-depressives (some with histories of up to 65 years' duration and 100 attacks) showed that it is a disorder from which the patient can make a complete recovery without any cognitive deterioration. Many of these patients have had courses of the traditional drug and physical treatments (including ECT) used in mental hospitals over the last half century. This indicates that such treatments themselves need not produce deterioration.     

Isolation and preliminary characterization of temperature-sensitive mutants of lumbo virus

Temperature-sensitive (ts) mutants were isolated from Lumbo virus (Bunyaviridae) after chemical mutagenesis. Genetic tests showed that these mutants are distributed in two complementation-recombination groups.     

The synthesis and processing of the nepovirus grapevine fanleaf virus proteins in rabbit reticulocyte lysate

Translation of a mixture of the two grapevine fanleaf virus (GFLV) RNAS in reticulocyte lysates in the presence of the amino acid analogs canavanine, S-aminoethyl-cysteine, and p-fluorophenylalanine gave two products of molecular weights (Mr) 220,000 (from RNA-1) and 125,000 (from RNA-2). In the absence of these analogs a protease apparently induced by RNA-1 catalyzed cleavage of the RNA-2 product (Mr 125,000) into two proteins of Mr 68,000 and 58,000. The 58,000 Mr protein was shown to be the likely virus coat protein by peptide mapping after partial proteolysis. Cleavage of the RNA-2 product of GFLV was not catalyzed by the translation products of another nepovirus, tobacco ringspot virus.     

Host-range mutants of adenovirus type 5 defective for growth in HeLa cells

Host-range mutants of adenovirus type 5 have been selected on human embryo kidney cells transformed by sheared adeno 5 DNA (293 cells). These mutants grow well on 293 cells, but are restricted on HeLa cells. By complementation tests on HeLa cells, these mutants were classified into two groups, neither of which was found to correspond to any of the 17 temperature-sensitive complementation groups. Members of the two complementation groups of host-range mutants differ in their host range on other cell types: Members of one group grow only on 293 cells, while those of the other group on both normal and transformed human embryo kidney cells. In the second group, cell-associated adeno gene functions are not needed for mutant growth, but, with the other group, it is possible that an adeno gene product made in the transformed cell complements the growth of the mutants. Recombination tests carried out with the host-range mutants and Ad5 temperature-sensitive mutants indicate that the host-range mutations map within the left half of the genetic map and that some (hr 1 and hr 2) probably lie very close to the left end of the map. The results presented in this communication are briefly discussed in relation to recent unpublished work concerning the biochemical characteristics of the mutants and the transformed 293 cells and to the role of adeno genes in transformation.     

The stability of satellite viral RNAs in vivo and in vitro

Like the satellite RNA (Sat-RNA) of cucumber mosaic virus (CMV), the RNA of satellite tobacco necrosis virus (STNV-RNA) was shown to be capable of surviving in vivo without replication for at least 10 days in the absence of its helper tobacco necrosis virus (TNV). However, under similar conditions, the genomic RNA 3 of CMV failed to survive for 48 hr. It has been demonstrated that both STNV-RNA and Sat-RNA are significantly more resistant to inactivation in vitro than the RNAs of their helper viruses. The thermal denaturation kinetics of STNV-RNA and Sat-RNA, unlike those of TNV-RNA and CMV-RNA, are more like those of transfer RNA (tRNA) indicating that a high proportion of their nucleotides are involved in base pairing. STNV-RNA, Sat-RNA, and tRNA also show similar degrees of resistance to degradation by the single strand-specific S(1) nuclease. It is suggested that both STNV-RNA and Sat-RNA may owe their in vitro stability to features of their molecular structure which may also account for their ability to survive in vivo for prolonged periods without replication. Similarities and differences between satellites and viroids are discussed and it is concluded that these two classes of RNAs are unlikely to be related. The possible evolutionary origins of the satellites are also considered.     

Formation of P22 generalized transducing particles in vitro.

Biologically active Salmonella phage P22 generalized transducing particles (i.e., P22 capsids filled with bacterial DNA) are formed in vitro in extracts in which phage DNA is encapsulated to produce viable phage.     

An avian oncovirus mutant deficient in genomic RNA: characterization of the packaged RNA as cellular messenger RNA.

SE 21Qlb is a recombinant avian oncovirus which is continuously produced by a line of transformed quail cells. These cells produce no detectable infectious viral progeny. Virions of SE 21Q1b contain 1–2% of the normal amounts of RSV-specific RNA found in PR-RSV-C virions and substantial quantities of heterogeneously sedimenting nonviral RNA (M. Linial, E. Medeiros, and W. S. Hayward, 1978b, Cell, 15:1371–1381). RNA extracted from purified virions of SE 21Q1b is as efficient a template for amino acid incorporation in a mRNA-dependent reticulocyte lysate as oligo(dT)-cellulose purified cellular mRNA. Virion RNA from SE 21Q1b serves as a template in vitro for a small amount of the precursor to the internal structural proteins (gag proteins). But unlike RNA from other avian oncoviruses, SE 21Q1b RNA makes, in addition, a number of proteins whose sizes on sodium dodecyl sulfate (SDS)-polyacrylamide gels resemble those of a number of uninfected quail fibroblast proteins. The proteins range in size from 15,000–220,000 daltons. Immunoprecipitation of proteins synthesized in vitro from SE 21Qlb virion RNA with antisera against the gag, pol, and env gene products shows that this RNA serves as a template for only one viral gene product, the recombinant gag gene precursor of 72,000 daltons (ΔPr76) (R. Shaikh, M. Linial, J. Coffin, and R. Eisenman, 1978, Virology87, 326–338). Addition of lysed virus to proteins made in vitro from PR-C RNA or RNA from PR-E-95c, a recombinant whose gag gene structure is similar to that of PR-E-21Qlb, results in the cleavage of gag protein precursors (K. von der Helm, 1977, Proc. Nat. Acad. Sci. USA74, 911–915) into several of the internal structural proteins. On the other hand, products of translation of PR-E-21Q1b RNA are not detectably cleaved into the internal structural proteins, suggesting that a very low amount of PR-E-21Q1b translation products are gag related. A protein of 37,000 daltons constitutes about 15% of the total protein synthesized from SE 21Qlb RNA. Data from serological and peptide mapping studies indicate that this protein is unrelated to the virion gag, pol, or env proteins. However, the major tryptic peptide of this protein appears to be identical to the major peptide of a prominent quail cell protein having the same apparent molecular weight as the in vitro translation product. Thus, several lines of evidence suggest that SE 21Q1b virions contain substantial amounts of functional cellular messenger RNAs.