bluuebunny/crossref_metadata_2025 huggingface dataset:
Structures of two spliced herpes simplex virus type 1 immediate-early mRNA's which map at the junctions of the unique and reiterated regions of the virus DNA S component
Authors: Watson R J , Sullivan M , Vande Woude G F
Published: January 2020

We have examined the structures of two herpes simplex virus type 1 immediate-early (IE) RNAs (IE mRNA-4 and IE mRNA-5) which map at the junctions of the unique (Us) and reiterated regions (TRs/IRs) of the virus DNA short component. Hybrids between IE cytoplasmic RNA and herpes simplex virus type 1 DNA restriction fragments were digested with single-strand-specific nucleases S1 and exonuclease VII, and the products were analyzed by agarose gel electrophoresis. Data obtained with the nuclease digestion technique were confirmed by electron microscopy of R-loop structures formed with polyadenylated IE RNA and virus DNA fragments. It was found that both IE mRNA-4 and IE mRNA-5 contained a 260-base 5'-terminal cotranscript which mapped at equivalent loci within TRs/IRs. These 5'-terminal sequences were shown to be spliced to 3'-terminal cotranscripts of 1,450 bases (for IE mRNA-4) and 1,540 bases (for IE mRNA-5). The 3'-terminal cotranscripts contained sequences encoded by both TRs/IRs and opposite ends of Us, indicating that the introns contained by the IE mRNA-4 and IE mRNA-5 genes, found to be approximately 150 base pairs in size, mapped entirely within the reiterated sequences. The data suggest that these genes may contain common and unique components, and the implications of this model are discussed.
Evidence that the genome of hepatitis A virus consists of single-stranded RNA
Authors: Coulepis A G , Tannock G A , Locarnini S A , Gust I D
Published: January 2020

Nucleic acid was extracted from purified hepatitis A virus, radiolabeled with 125I, and shown to consist of single-stranded RNA which sediments at 35S and contains sequences of polyadenylic acid. These findings are consistent with hepatitis A virus being a member of the genus Enterovirus within the family Picornaviridae.
Inhibition of host DNA synthesis by adenovirus infection is reversed at elevated temperatures
Authors: Foster D A , Spangler R , Zubay G
Published: January 2020

Infection of KB cells by adenovirus normally results in a drastic reduction of host DNA synthesis at 33 to 37 degrees C. This inhibition was largely reversed by raising the temperature to 41 degrees C. Inhibition was reinstated if the temperature is lowered.
Complex of simian virus 40 large-T antigen and host 53,000-molecular-weight protein in monkey cells
Authors: Harlow E , Pim D C , Crawford L V
Published: January 2020

Mouse cells transformed by simian virus 40 (SV40) have been shown to contain a complex of the virus-coded large-T antigen with a host 53,000-molecular-weight (53K) protein. Initial attempts to detect a similar complex in lytically infected cells were unsuccessful, and it therefore seemed that the complex might be peculiar to transformed or abortively transformed nonpermissive cells. Immunoprecipitation of [32P]phosphate-labeled extracts of SV40-infected CV-1 African green monkey kidney cells with antibodies specific for large-T or the 53K protein revealed that the large-T-53K protein complex was formed during lytic infections. Only a minor fraction of the large-T present was associated with 53K protein, and large-T and the 53K host protein cosedimented during centrifugation through sucrose gradients. We used monospecific sera and monoclonal antibodies to study the rate of synthesis and phosphorylation of the 53K protein during lytic infections. Infection of CV-1 cells with SV40 increased the rate of synthesis of the 53K protein fivefold over that in mock-infected cells. At the same time, the rate of phosphorylation of the 53K protein increased more than 30-fold compared with control cultures. Monkey cells transformed by UV-irradiated SV40 (Gluzman et al., J. Virol. 22:256-266, 1977) also contained the large-T-53K protein complex. The formation of the complex is therefore not a peculiarity of SV40-transformed rodent cells but is a common feature of SV40 infections.
DNA sequence analysis of simian virus 40 mutants with deletions mapping in the leader region of the late viral mRNA's: mutants with deletions similar in size and position exhibit varied phenotypes
Authors: Barkan A , Mertz J E
Published: January 2020

The nucleotide sequences of 10 viable yet partially defective deletion mutants of simian virus 40 were determined. The deletions mapped within, and, in many cases, 5' to, the predominant leader sequence of the late viral mRNA's. They ranged from 74 to 187 nucleotide pairs in length. Six of the mutants had lost the sequence that corresponds to the "cap" site (5' terminus) of the most abundant class of 16S mRNA's. One of these mutants had a deletion that extended 103 nucleotide pairs into the region preceding this primary cap site and, therefore, was missing many secondary cap sites as well. A seventh mutant lacked the entire major 16S leader sequence except for the first six nucleotides at its 5' end and the last nine at its 3' end. Although these mutants differed in the size and position of their deletions, we were unable to discover any simple correlations between their growth characteristics and their DNA sequences. This finding indicates that the secondary structures of the RNA transcripts may play a more important role than the exact nucleotide sequence of the RNAs in determining how they function within the cell.
Convenient assay for interferons
Authors: Rubinstein S , Familletti P C , Pestka S
Published: January 2020

A convenient assay for interferons based on reduction of cytopathic effect was developed. The number of manipulations and the lengths of the various incubation steps were reduced to a minimum. The assay is simple to perform and can be completed within 16 h. Moreover, it can be used with various types of cells and a variety of viruses.
Dissociation of interferon effects on murine leukemia virus and encephalomyocarditis virus replication in mouse cells
Authors: Czarniecki C W , Sreevalsan T , Friedman R M , Panet A
Published: January 2020

Two subclones of Swiss mouse cells infected with Moloney murine leukemia virus (M-MuLV) were tested for their response to interferon (IFN). Whereas M-MuLV production in the two subclones was inhibited to the same extent, one of the subclones was significantly more sensitive to IFN when the antiviral effect was measured by replication of encephalomyocarditis (EMC) virus. The same subclone was also more sensitive to the anticellular activities of IFN. Additionally, NIH 3T3 cells infected with M-MuLV were completely resistant to IFN actions when EMC virus replication or the anticellular activities were tested. However, under the same conditions, M-MuLV production was completely inhibited by IFN. These results indicate that IFN may affect cell growth functions and EMC replication through mechanisms different from those by which MuLV production is inhibited.
Maturation of bacteriophage SPPI DNA: limited precision in the sizing of mature bacteriophage genomes
Authors: Humphreys G O , Trautner T A
Published: January 2020

Maturation of bacteriophage SPPI is imprecise. Although terminally redundant and circularly permuted molecules were always formed, individual molecules varied by more than 200 base pairs from each other.
Generation of novel, biologically active Harvey sarcoma viruses via apparent illegitimate recombination
Authors: Goldfarb M P , Weinberg R A
Published: January 2020

NIH 3T3 cells transfected with Harvey sarcoma virus (HSV) DNA may acquire deleted proviruses (Goldfarb and Weinberg, J. Virol. 38:125-135, 1981). Such proviruses lack the right end of the wild-type HSV DNA genome corresponding to the 3'-proximal portion of the viral RNA. As expected, the RNA transcripts of these deleted HSV (delHSV) proviruses lacked sequences normally found at the 3' end of wild-type HSV RNA. Since frequently these delHSV RNA transcripts were longer than wild-type HSV RNA, we suggest that transcription proceeded through the deleted provirus and continued into flanking nonviral sequences. When delHSV-transformed cells were infected with Moloney murine leukemia virus (M-MLV), delHSV RNA was pseudotyped into new virus particles, demonstrating that the 3'-proximal sequences of wild-type HSV RNA are not essential for virion RNA encapsidation. Cells which carried a delHSV genome and were infected with M-MLV helper released very low titers of highly transmissible sarcoma virus. The inability to rescue high titers of sarcoma virus from these cells reflected the necessary presence of the deleted 3'-terminal sequences for normal efficient transmission of the sarcoma virus genome (Goldfarb and Weinberg, J. Virol. 38:125-135, 1981). The small amount of highly transmissible sarcoma virus rescuable from delHSV-transformed cells originated via genetic recombination between del HSV and the M-MLV helper used for the sarcoma virus rescue. The recombinant sarcoma virus genomes reacquired a competent 3' genomic end from the parental M-MLV genome, which restored efficient transmissibility. The locations of sites for recombination between the delHSV and M-MLV genomes appeared to be nonrandom. These sites were in genomic regions where the parental genomes bore no detectable sequence homology. Structural mapping of these recombinant sarcoma virus genomes indicated that the HSV transformation gene lies within 2.0 kilobases of the RNA 5' end. Based upon our genetic recombination studies, we suggest a model to explain how leukemia viruses can recombine with cellular sequences to generate novel defective viruses.
Polyadenylation sites for influenza virus mRNA
Authors: Robertson J S , Schubert M , Lazzarini R A
Published: January 2020

Polyadenylated transcripts of influenza virus RNA are incomplete copies of the individual genome segments, lacking sequences complementary to the 5'-terminal nucleotides of the virion RNA. By using a procedure which depends on the polyadenylic acid tail of the mRNA being encoded in part by the genome, we have determined that the common tract of uridine residues, approximately 17 to 22 nucleotides from the 5' end of each segment, is the site of polyadenylation of influenza virus mRNA.
Adenovirus-induced mutations at the hypoxanthine phosphoribosyltransferase locus of Chinese hamster cells
Authors: Marengo C , Mbikay M , Weber J , Thirion J P
Published: January 2020

Hpt-13 is a Chinese hamster cell line deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and sensitive to a medium containing 10(-4) M hypoxanthine, 5.5 X 10(-6) M aminopterin, and 10(-4) M thymidine. In this cell line there is a high incidence of cells resistant to this selective medium after an incubation with either ethyl methane sulfonate or adenovirus type 2 complete virions or their incomplete particles. The rate of reversion in the presence of these agents was 34-fold higher with ethyl methane sulfonate and 2.5- to 5.6-fold higher with adenovirus particles than the spontaneous rate of reversion. The revertant phenotypes were stable for many generations without selective pressure. All of the revertants tested recovered the hypoxanthine phosphoribosyltransferase activity. Most of them, however, carried an enzyme of lower activity and faster electrophoretic mobility than that of the wild type. The preferential reversion to this type of enzyme was observed among spontaneous revertants as well as among those induced by mutagenesis with ethyl methane sulfonate or exposure to viral particles. Our results suggest that adenovirus particles and ethyl methane sulfonate induce mutations at the hpt locus of Hpt-13 cells through similar mechanisms.
Replication of double-stranded RNA of the virus-like particles in Saccharomyces cerevisiae
Authors: Newman A M , Elliott S G , McLaughlin C S , Sutherland P A , Warner R C
Published: January 2020

The mode of replication of the L double-stranded RNA (dsRNA) present in virus-like particles in Saccharomyces cerevisiae was examined by density transfer experiments. After transfer to light medium, significant amounts of fully heavy dsRNA persisted over a number of cell doublings. In addition, very little material of hybrid density was ever formed, and the accumulation of fully light material began as early as 0.5 doubling after transfer to light medium. Our results are compatible with a conservative mode of replication or with a semiconservative mode of replication carried out by a small portion of the total dsRNA population. In additional experiments the synthesis of dsRNA relative to the cell cycle was studied. This was done by determining the ratio of short-term to long-term radioactive label in size-separated cell fractions of a prelabeled exponential culture. The ratio of short-term to long-term label remained constant for all fractions, implying that dsRNA is synthesized throughout the cell cycle, increasing through the cell cycle at an exponential rate.
Characterization of an immunosuppressive parvovirus related to the minute virus of mice
Authors: McMaster G K , Beard P , Engers H D , Hirt B
Published: January 2020

We have characterized an immunosuppressive parvovirus related to the minute virus of mice (MVM). The parvovirus, MVM(i), grew efficiently on the murine lymphoma cell line EL-4 and not on the A-9 strain of L-cells which is a host for the prototype MVM. MVM(i) was immunosuppressive for allogeneic mixed leukocyte cultures, inhibiting the generation of cytolytic T lymphocytes. MVM had no effect on mixed leukocyte cultures. MVM and MVM(i) particles were similar in buoyant density, sedimentation rate, appearance in the electron microscope, and polypeptide composition. We present restriction enzyme maps of the DNAs of MVM and MVM(i) which show that they are closely related. Out of 109 restriction endonuclease cleavage sites (representing together about 10% of the nucleotide sequence), 86 sites were shared by MVM and MVM(i), whereas 22 sites were absent from one of the two viruses. MVM(i) DNA had an apparent deletion of about 60 nucleotides relative to MVM, located near the 5' terminus of viral DNA.
Comparison of canine parvovirus with mink enteritis virus by restriction site mapping
Authors: McMaster G K , Tratschin J D , Siegl G
Published: January 2020

The genomes of canine parvovirus and mink enteritis virus were compared by restriction enzyme analysis of their replicative-form DNAs. Of 79 mapped sites, 68, or 86%, were found to be common for both types of DNA, indicating that canine parvovirus and mink enteritis virus are closely related viruses. Whether they evolved from a common precursor or whether canine parvovirus is derived from mink enteritis virus, however, cannot be deduced from our present data.
Effect of polymerase mutations on packaging of primer tRNAPro during murine leukemia virus assembly
Authors: Levin J G , Seidman J G
Published: January 2020

The role of reverse transcriptase in selective encapsidation of the murine leukemia virus (MuLV) tRNA primer, tRNAPro, was investigated by examining the tRNA composition of several nonconditional pol mutants. One mutant, clone 23, which contains an altered polymerase about 40% smaller than the wild-type enzyme (B. I. Gerwin et al., J. Virol. 31:741-751, 1979) had a typical viral tRNA pattern, including normal levels of tRNAPro in free and 70S-associated 4S RNA. Another class of mutants, produced by Moloney murine leukemia virus-infected cell clone M13 and subclone M13/1, does not contain any detectable polymerase protein (A. Shields et al., Cell 14:601-609, 1978) and was found to have reduced amounts of tRNAPro in free 4S RNA. However, the level of tRNAPro associated with the genome was normal in the mutant virions. These results suggest that the reverse transcriptase protein is involved in the initial selection of tRNA primer during virus assembly, but not in the subsequent association of this tRNA with genomic RNA.
Rapid and sensitive quantitative immunoassay for the large simian virus 40 T antigen
Authors: Kilton L J , Bradley M , Mehta C , Livingston D M
Published: January 2020

A quantitative, enzyme-linked immunoadsorbent assay has been developed for the simian virus 40 large T antigen. When hamster anti-simian virus 40 tumor serum was used, this method permitted specific identification of large T antigen and its analog, the D2 hybrid protein, a molecule with the same C-terminal approximately 600 amino acids as large T antigen. The sensitivity limit of this test was 0.63 ng of protein. The slopes of the regression lines of the enzyme-linked immunosorbent assay titrations performed with highly purified D2 or simian virus 40 large T antigen and with crude extracts of simian virus 40-infected monkey and transformed human cells were identical. Thus, the curve generated with a purified protein, such as D2, can serve as a quantitative standard for the measurement of large T antigen in a wide variety of extracts. Furthermore, solutions containing high salt concentrations and buffers containing up to 0.1% Nonidet P-40 did not interfere with the assay, making it applicable to the measurement of large T antigen in a variety of chromatographic fractions. The enzyme-linked immunosorbent assay was three times more sensitive, was significantly faster to perform, and was quantitatively valid over a much broader large-T-antigen concentration range than the complement fixation test. As such, it should be useful in future studies of the structure and function of this protein.
Integrated Simian Virus 40 DNA: Nucleotide Sequences at Cell-Virus Recombinant Junctions
Authors: Stringer James R.
Published: January 2020

DNA fragments containing the integrated viral DNA present in the simian virus 40 (SV40)-transformed rat cell lines SVRE9 and SVRE17 were cloned in procaryotic vectors, and the DNA sequences linking SV40 and cell DNA were determined. Comparison of the DNA sequences at the SV40-cell junctions in SVRE9 and SVRE17 cells with those of a previously characterized viral insertion from SV14B cells shows that no specific viral or cellular sequences occur at SV40-cell junctions and that the cellular DNA sequences adjacent to integrated SV40 DNA do not display the direct repeat structure characteristic of transposons and retrovirus proviruses.
Analysis of JC virus DNA purified directly from human progressive multifocal leukoencephalopathy brains
Authors: Rentier-Delrue F , Lubiniecki A , Howley P M
Published: January 2020

Human polyomavirus JC DNA was purified directly from the diseased brain tissue of two patients with progressive multifocal leukoencephalopathy (PML) by a method employing differential salt precipitation (B. Hirt, J. Mol. Biol. 26:365-369, 1967). Each of the viral genomes (JC-NIH-1 and JC-NIH-2) was molecularly cloned intact in Escherichia coli, using pBR322, at their unique EcoRI (0.00 map unit) and BamHI (0.51 map unit) sites. The JC-NIH-1 genome was approximately 50 base pairs larger and the JC-NIH-2 genome was approximately 50 base pairs smaller than the prototype human polyomavirus JC (Mad-1) DNA. Analysis of the restriction endonuclease cleavage fragments of these two DNAs and the human polyomavirus JC (Mad-1) DNA revealed only slight differences which mapped in a region of the genome extending from 0.67 to 0.74 map unit. From previous homology studies, this region of variance corresponds to the noncoding region to the late side of the origin of DNA replication.
Bunyamwera Virus Replication in Cultured
Aedes albopictus
(Mosquito) Cells: Establishment of a Persistent Viral Infection
Authors: Newton Susan E. , Short Nicholas J. , Dalgarno Lynn
Published: January 2020

Bunyamwera virus replication was examined in
Aedes albopictus
(mosquito) cell cultures in which a persistent infection is established and in cytopathically infected BHK cells. During primary infection of
A. albopictus
cells, Bunyamwera virus reached relatively high titers (≃10
7
PFU/ml), and autointerference was not observed. Three virus-specific RNAs (L, M, and S) and two virion proteins (N and G1) were detected in infected cells. Maximum rates of viral RNA synthesis and viral protein synthesis were extremely low, corresponding to <2% of the synthetic capacities of uninfected control cells. Viral protein synthesis was maximal at 12 h postinfection and was shut down to barely detectable levels at 24 h postinfection. Virus-specific RNA and nucleocapsid syntheses showed similar patterns of change, but later in infection. The proportions of cells able to release a single PFU at 3, 6, and 54 days postinfection were 100, 50, and 1.5%, respectively. Titers fell to 10
3
to 10
5
PFU/ml in carrier cultures. Persistently infected cultures were resistant to superinfection with homologous virus but not with heterologous virus. No changes in host cell protein synthesis or other cytopathic effects were observed at any stage of infection. Small-plaque variants of Bunyamwera virus appeared at approximately 7 days postinfection and increased gradually until they were 75 to 95% of the total infectious virus at 66 days postinfection. Temperature-sensitive mutants appeared between 23 and 49 days postinfection. No antiviral activity similar to that reported in
A. albopictus
cell cultures persistently infected with Sindbis virus (R. Riedel and D. T. Brown, J. Virol.
29:
51-60, 1979) was detected in culture fluids by 3 months after infection. Bunyamwera virus replicated more rapidly in BHK cells than in mosquito cells but reached lower titers. Autointerference occurred at multiplicities of infection of ≃10. Virus-specific RNA and protein syntheses were at least 20% of the levels in uninfected control cells. Host cell protein synthesis was completely shut down, and nucleocapsid protein accumulated until it was 4% of the total cell protein. We discuss these results in relation to possible mechanisms involved in determining the outcome of arbovirus infection of vertebrate and mosquito cells.
Salmonella
Bacteriophage Glycanases: Endorhamnosidase Activity of Bacteriophages P27, 9NA, and KB1
Authors: Wollin Ralfh , Eriksson Ulla , Lindberg Alf A.
Published: January 2020

Four bacteriophages, P22, P27, 9NA, and KB1, active on smooth
Salmonella
strains belonging to serogroups A, B, and D1 were investigated for endoglycosidase activity and specificity in enzyme hydrolysis assays. Purified phage was incubated with phenol-water-extracted lipopolysaccharide preparations which had been partially delipidated. Dialyzable oligosaccharides, released by phage glycosidase activity, were analyzed by sugar and methylation analyses. Phages P27, 9NA, and KB1, as well as P22 assayed earlier (U. Eriksson et al., J. Gen. Virol.
43:
503-511, 1979; S. Iwashita and S. Kanegasaki, Biochem. Biophys. Res. Commun.
55:
403-409, 1973), were all found to have phage-associated endorhamnosidase activity hydrolyzing the O-polysaccharide chain common to bacteria of serogroups A, B, and D1 [Formula: see text] between the
l
-rhamnose and
d
-galactose residues. The nature of the R monosaccharide, abequose, tyvelose, or paratose, had no effect on the activity or specificity of the endorhamnosidase, whereas a change of the
d
-galactose →
d
-mannose linkage from α1,2 to α1,6 made the O-polysaccharide chain resistant to the endorhamnosidases. Modification of the O chain by glucosylation of the
d
-galactose residue at
O
-4 or
O
-6 revealed two glycosidase specificities: the phage P22 and P27 enzymes hydrolyzed O chains glucosylated at
O
-4 but not
O
-6, whereas the phage 9NA and KB1 enzymes hydrolyzed chains glucosylated at
O
-6 but not
O
-4. Phage KB1, like P22 and P27, had a short, noncontractile tail containing a base plate with tail spikes (morphologically Bradley group C), whereas 9NA had a long, flexible tail ending with a base plate-like appendage (Bradley group B), which suggests that the endorhamnosidase activity can be associated with different tail structures.
Two forms of VPg on poliovirus RNAs
Authors: Richards O C , Hey T D , Ehrenfeld E
Published: January 2020

The protein (VPg) linked to the 5' termini of poliovirus RNAs resolved into two species when subjected to non-equilibrium electrofocusing. The differently charged forms of VPg were not due to protein phosphorylation nor to variability of the number of phosphate residues associated with the nucleotide moiety remaining after RNase digestion of the nucleoprotein. Single-stranded viral RNA isolated from mature virions contained predominantly the more basic form of VPg, whereas unpackaged single-stranded RNa remaining in cells at the end of the virus replication cycle contained predominantly the more acidic form of VPg. Replicative-form (RF) molecules also contained both species of VPg, with the more acidic form representing the major species. Both plus and minus RNA strands in RF had similar VPg compositions; however, there appeared to be a strongly selective loss of VPg from only the minus strand in RF, particularly at late times postinfection.
Characterization of AKR murine leukemia virus sequences in AKR mouse substrains and structure of integrated recombinant genomes in tumor tissues
Authors: Quint W , Quax W , van der Putten H , Berns A
Published: January 2020

A specific cDNA probe of AKR murine leukemia virus (AKR-MLV) was prepared to detect AKR-MLV sequences in normal and tumor tissues in a variety of AKR mouse substrains. AKR strains contained up to six endogenous AKR-MLV genomes. All substrains tested had one AKR-MLV locus in common, and closely related substrains had several proviruses integrated in an identical site. Virus-induced tumors in the AKR/FuRdA and AKR/JS strains showed a reintegration pattern of AKR-MLV sequences unique for the individual animal, suggesting a monoclonal origin for the outgrown tumors. An analysis of tumor DNAs from the AKR/FuRdA and AKR/JS substrains with restriction enzymes cleaving within the proviral genome revealed a new EcoRI restriction site and BamHI restriction site not present in normal tissues. The positions of these sites corresponded both with cleavage sites of EcoRI and BamHI in integrated Moloney recombinants and with the structure of isolated AKR mink cell focus-forming viruses. All tumors analyzed to data contain nearly identical integrated recombinant genomes, suggesting a causal relationship between the formation of recombinants and the leukemogenic process.
Discontinuous synthesis of both strands at the growing fork during polyoma DNA replication in vitro
Authors: Närkhammar-Meuth M , Eliasson R , Magnusson G
Published: January 2020

In discontinuous polyoma DNA replication, the synthesis of Okazaki fragments is primed by RNA. During viral DNA synthesis in nuclei isolated from infected cells, 40% of the nascent short DNA fragments had the polarity of the leading strand which, in theory, could have been synthesized by a continuous mechanism. To rule out that the leading strand fragments were generated by degradation of nascent DNA, they were further characterized. DNA fragments from a segment of the genome which replication forks pass in only one direction were strand separated. The sizes of the fragments from both strands were similar, suggesting that one strand was not specifically degraded. Most important, however, the majority of the Okazaki fragments of both strands were linked to RNA at their 5' ends. For identification, the RNA was labeled at the 5' ends by [beta-32P]GTP, internally by [3H]CTP, [3H]GTP, and [3H]UTP, or at the 3' ends by 32P transfer from adjacent [32P]dTMP residues. All three kinds of labeling indicated that an equal proportion of DNA fragments from the two strands was linked to RNA primers.
Fine-structure mapping of herpes simplex virus type 1 temperature-sensitive mutations within the short repeat region of the genome
Authors: Preston V G
Published: January 2020

Cloned herpes simplex virus type 1 (HSV-1) DNA fragments were used to fine-structure map the temperature-sensitive (ts) lesions from four mutants, ts T, D, c75, and K, by marker rescue. These mutants all overproduced immediate-early viral polypeptides at the nonpermissive temperature. Although one of these viruses, ts K, gave a more restricted infected-cell polypeptide profile under these conditions than the other three, no complementation was detected between pairwise crosses of these mutants in the yield test. Recombination, however, was obtained between all mutant pairs except ts T and D. In physical mapping experiments, ts+ virus was recovered from cells coinfected with DNA of ts T, D, or c75 and BamHI fragment k from wild-type strain 17 HSV-1 DNA cloned in pAT153, whereas ts K was rescued by cloned HSV-1 BamHI-y. Both of these cloned DNA fragments contained sequences from the short repeat region of the HSV-1 genome. The ts mutations were more precisely mapped by marker rescue, using restriction enzyme fragments within BamHI-k and -y from cloned DNA. The smallest fragment able to rescue a mutant was 320 base pairs long. The order of the four mutations derived from these studies was consistent with the assignment by genetic recombination. All four lesions mapped within the coding sequences of the immediate-early polypeptide Vmw IE 175 (ICP4) which lie outside the "a" sequence. The results showed that mutations in different regions of the gene encoding Vmw IE 175 could produce similar phenotype effects at the nonpermissive temperature.
Integrated polyoma genomes in inducible permissive transformed cells
Authors: Chartrand P , Gusew-Chartrand N , Bourgaux P
Published: January 2020

Using the approach described by Botchan, Topp, and Sambrook (Cell 9:269-287, 1976), we analyzed the organization of the integrated viral sequences in five clonal isolates from the same permissive, inducible cell line (Cyp line) transformed by the tsP155 mutant of polyoma virus. In all five clones, viral sequences were found that could be assigned to a common integration site, as they were joined to the cellular DNA in the same fashion in every instance. However, the sequences comprised between these points differed markedly from clone to clone, as if cell propagation had been accompanied by amplification or recombination or both within the viral insertion. When the clones were compared, no correlation could be found between the abundance, or the organization, of the integrated viral sequences and the amount, or the nature, of the free viral DNA molecules produced during induction. Altogether, our findings suggest that specific events, occurring during either the excision or the subsequent replication of the integrated viral sequences, are responsible for the predominant production of nondefective viral DNA molecules by permissive transformed cells, such as Cyp cells.
Monoclonal antibodies to the influenza A virus nucleoprotein affecting RNA transcription
Authors: van Wyke K L , Bean W J , Webster R G
Published: January 2020

Monoclonal antibodies were used to map the antigenic domains of the A/WSN/33 (HoN1) influenza virus nucleoprotein. Three nonoverlapping antigenic regions of the nucleoprotein were identified by using competitive-binding enzyme-linked immunosorbent assays. Monoclonal antibodies to two nucleoprotein domains inhibited in vitro transcription of viral RNA, suggesting that these specific regions of the nucleoprotein are topographically or functionally involved in RNA transcription.
Characterization of the Coat Protein mRNA of Southern Bean Mosaic Virus and Its Relationship to the Genomic RNA
Authors: Ghosh Amit , Rutgers Tineke , Ke-Qiang Mang , Kaesberg Paul
Published: January 2020

RNA isolated from southern bean mosaic virions contains, in small amount, a subgenomic RNA (molecular weight, 0.38 × 10
6
) that serves in vitro as an mRNA for southern bean mosaic virus coat protein. The RNA has a 5′-linked protein indistinguishable from the protein linked to the 5′ end of full-length genomic RNA. Its base sequence, determined to 91 bases from the 3′ end, is identical to the 3′-terminal sequence of the genomic RNA. The results suggest that the coat protein messenger sequence exists as a “silent” cistron near the 3′ end of the genomic RNA.
Specific Sindbis virus-coded function for minus-strand RNA synthesis
Authors: Sawicki D L , Sawicki S G , Keränen S , Kääriäinen L
Published: January 2020

The synthesis of minus-strand RNA was studied in cell cultures infected with the heat-resistant strain of Sindbis virus and with temperature-sensitive (ts) belonging to complementation groups A, B, F, and G, all of which exhibited an RNA-negative (RNA-) phenotype when infection was initiated and maintained at 39 degrees C, the nonpermissive temperature. When infected cultures were shifted from 28 degrees C (the permissive temperature) to 39 degrees C at 3 h postinfection, the synthesis of viral minus-strand RNA ceased in cultures infected with ts mutants of complementation groups B and F, but continued in cultures infected with the parental virus and mutans of complementation groups A and G. In cultures infected with ts11 of complementation group B, the synthesis of viral minus-strand RNA ceased, whereas the synthesis of 42S and 26S plus-strand RNAs continued for at least 5 h after the shift to 39 degrees C. However, when ts11-infected cultures were returned to 28 degrees C 1 h after the shift to 39 degrees C, the synthesis of viral minus-strand RNA resumed, and the rate of viral RNA synthesis increased. The recovery of minus-strand synthesis translation of new proteins. We conclude that at least one viral function is required for alphavirus minus-strand synthesis that is not required for plus-strand synthesis. In cultures infected with ts6 of complementation group F, the syntheses of both viral plus-strand and minus-strand RNAs were drastically reduced after the shift to 39 degrees C. Since ts6 failed to synthesize both plus-strand and minus-strand RNAs after the shift to 39 degrees C, at least one common viral component appears to be required for the synthesis of both minus-strand and plus-strand RNAs.
Nucleotide sequences of the mRNA's encoding the vesicular stomatitis virus G and M proteins determined from cDNA clones containing the complete coding regions
Authors: Rose J K , Gallione C J
Published: January 2020

The complete nucleotide sequences of the vesicular stomatitis virus mRNA's encoding the glycoprotein (G) and the matrix protein (M) have been determined from cDNA clones that contain the complete coding sequences from each mRNA. The G protein mRNA is 1,665 nucleotides long, excluding polyadenylic acid, and encodes a protein of 511 amino acids including a signal peptide of 16 amino acids. G protein contains two large hydrophobic domains, one in the signal peptide and the other in the transmembrane segment near the COOH terminus. Two sites of glycosylation are predicted at amino acid residues 178 and 335. The close correspondence of the positions of these sites with the reported timing of the addition of the two oligosaccharides during synthesis of G suggests that glycosylation occurs as soon as the appropriate asparagine residues traverse the membrane of the rough endoplasmic reticulum. The mRNA encoding the vesicular stomatitis virus M protein is 831 nucleotides long, excluding polyadenylic acid, and encodes a protein of 229 amino acids. The predicted M protein sequence does not contain any long hydrophobic or nonpolar domains that might promote membrane association. The protein is rich in basic amino acids and contains a highly basic amino terminal domain. Details of construction of the nearly full-length cDNA clones are presented.
Morphological and immunological comparison of caprine arthritis encephalitis and ovine progressive pneumonia viruses
Authors: Dahlberg J E , Gaskin J M , Perk K
Published: January 2020

Caprine arthritis encephalitis virus (CAEV) causes a variety of pathological conditions ranging from mild to very severe and from acute to chronic, depending upon the age of initial infection and other variables. Although the virus has been reported to have properties of characteristic of retroviruses and to be related to maedi-visna virus (also called progressive pneumonia virus [PPV]), relatively little information about its morphological and immunological characteristics has been reported. We describe the morphological features of CAEV replicating in cultured caprine cells. Although the virus replicates slowly and very little virus is released from productively infected cells, it is apparent that the morphogenesis of CAEV is strikingly similar to that of maedi-visna. After the transmission of CAEV to a more permissive permanent cell line derived from Himalayan tahr ovary, it was possible to grow and purify enough virus to initiate biochemical characterization. The structural proteins of CAEV are generally very similar to those of PPV, suggesting that the two viruses are closely related but not identical. This was substantiated by showing that serum from a CAEV-infected goat immunoprecipitated both CAEV and PPV virion structural antigens from extracts of radiolabeled virus and also precipitated putative nonstructural viral antigens from extracts of both CAEV- and PPV-infected cells.
Ribonucleic Acid Synthesis in Cells Infected with Herpes Simplex Virus
Authors: Wagner Edward K. , Roizman Bernard
Published: January 2020

HEp-2 cells were pulse-labeled at different times after infection with herpes simplex virus, and nuclear ribonucleic acid (RNA) and cytoplasmic RNA were examined. The data showed the following: (i) Analysis by acrylamide gel electrophoresis of cytoplasmic RNA of cells infected at high multiplicities [80 to 200 plaque-forming units (PFU)/cell] revealed that ribosomal RNA (rRNA) synthesis falls to less than 10% of control (uninfected cell) values by 5 hr after infection. The synthesis of 4
S
RNA also declined but not as rapidly, and at its lowest level it was still 20% of control values. At lower multiplicities (20 PFU), the rate of inhibition was slower than at high multiplicities. However, at all multiplicities the rates of inhibition of 18
S
and 28
S
rRNA remained identical and higher than that of 4
S
RNA. (ii) Analysis of nuclear RNA of cells infected at high multiplicities by sucrose density gradient centrifugation showed that the synthesis and methylation of 45
S
rRNA precursor continued at a reduced but significant rate (ca. 30% of control values) at times after infection when no radioactive uridine was incorporated or could be chased into 28
S
and 18
S
rRNA. This indicates that the inhibition of rRNA synthesis after herpesvirus infection is a result of two processes: a decrease in the rate of synthesis of 45
S
RNA and a decrease in the rate of processing of that 45
S
RNA that is synthesized. (iii) Hybridization of nuclear and cytoplasmic RNA of infected cells with herpesvirus DNA revealed that a significant proportion of the total viral RNA in the nucleus has a sedimentation coefficient of 50
S
or greater. The sedimentation coefficient of virus-specific RNA associated with cytoplasmic polyribosomes is smaller with a maximum at 16
S
to 20
S
, but there is some rapidly sedimenting RNA (> 28
S
) here too. (iv) Finally, there was leakage of low-molecular weight (4
S
) RNA from infected cells, the leakage being approximately three-fold that of uninfected cells by approximately 5 hr after infection.
Inhibition of Interjacent Ribonucleic Acid (26
S
) Synthesis in Cells Infected by Sindbis Virus
Authors: Scheele Christina M. , Pfefferkorn E. R.
Published: January 2020

The interrelationship of viral ribonucleic acid (RNA) and protein synthesis in cells infected by Sindbis virus was investigated. When cultures were treated with puromycin early in the course of infection, the synthesis of interjacent RNA (26
S
) was preferentially inhibited. A similar result was obtained by shifting cells infected by one temperature-sensitive mutant defective in RNA synthesis from the permissive (29 C) to the nonpermissive (41.5 C) temperature. Under both conditions, the viral RNA produced appeared to be fully active biologically. Once underway, the synthesis of viral RNA in wild-type Sindbis infections did not require concomitant protein synthesis.
Ribonucleic Acid Synthesis of Vesicular Stomatitis Virus
Authors: Stampfer Martha , Baltimore David , Huang Alice S.
Published: January 2020

Plaque-forming B particles of vesicular stomatitis virus (VSV) induce the synthesis of virus-specific ribonucleic acid (RNA) in Chinese hamster ovary cells, whereas defective T particles do not. Infection with low input multiplicities of B results in the formation of four species of RNA. During infection with high multiplicities, RNA synthesis begins with mainly these four species of RNA but gradually shifts to a new pattern of RNA synthesis involving five other species of RNA. The change can also be induced by superinfection with T at 2.5 hr after infection with a low multiplicity of B. T added at the same time as B prevents virtually all RNA synthesis. Synthesis of the first group of RNA species correlates with the formation of B particles, whereas synthesis of the second group correlates with the formation of T particles. The various species of RNA formed after infection with VSV particles include single-stranded RNA, a completely double-stranded RNA, and RNA with partially double-stranded regions. These observations begin to establish a molecular basis for understanding the ability of T particles to interfere with the growth of B particles.
Some Properties of Influenza Virus Nucleocapsids
Authors: Kingsbury D. W. , Webster R. G.
Published: January 2020

Nucleocapsids released from influenza virions by sodium deoxycholate sedimented heterogeneously in sucrose gradients. Highly infectious virus (complete) preparations yielded nucleocapsids with peak distributions at 64 and 56
S
; von Magnus type virus (incomplete) lacked 64
S
nucleocapsids. Treatment of influenza virus nucleocapsids with pancreatic ribonuclease rendered the associated viral ribonucleic acid (RNA) molecules acid-soluble, indicating that capsid proteins do not completely surround the viral RNA's. However, the capsid proteins remained associated after enzymatic hydrolysis of the RNA, as judged by persistently high sedimentation rates. Sedimentation rates of viral nucleocapsids reflected the sedimentation rates of the associated RNA's: 64
S
nucleocapsids contained 18
S
RNA, whereas 56
S
nucleocapsids contained 15
S
RNA, although in both cases RNA's sedimenting at 4 to 13
S
were also recovered. Furthermore, just as incomplete virions lacked 64
S
nucleocapsids, they also lacked 18
S
RNA. These findings support the hypothesis that the influenza virus genome is divided among several distinct pieces of RNA.
Characterization of
Xanthomonas phaseoli
Bacteriophages
Authors: Vidaver Anne K. , Schuster Max L.
Published: January 2020

Ten bacteriophages for
Xanthomonas phaseoli
were characterized. On the basis of adsorption rates, latent period and burst size, plaque morphology, host range, efficiency of plating, ultrastructure, sensitivity to osmotic shock, streptomycin sensitivity, temperature effects on plating efficiency, and serology, the phages were separated into at least three groups. Some of the phages were infectious for
Pseudomonas phaseolicola
(four strains) and
P. syringae
(one strain) in a narrow temperature range. The taxonomic and ecological significance of this finding is discussed.
Incorporation of Precursors into Ribonucleic Acid, Protein, Glycoprotein, and Lipoprotein of Avian Myeloblastosis Virions
Authors: Baluda M. A. , Nayak D. P.
Published: January 2020

Freshly explanted leukemic myeloblasts produce avian myeloblastosis virus (AMV) at a constant rate without any obvious cytopathic effect; therefore, subviral components are continually synthesized at a steady rate. The incorporation of various radioactive precursors into virions was monitored by determination of radioactivity in purified virus after density equilibrium sedimentation in preformed sucrose gradients. The kinetics of incorporation of
3
H-uridine have shown that there is an average time interval of 3 to 4 hr (half-life) between the time viral ribo-nucleic acid (RNA) is synthesized and the time it is released as a mature virus particle; this represents the average time interval spent by AMV-RNA in an intracellular pool. Studies with
14
C-phenylalanine have revealed that some protein synthesis takes place at or near the cell surface immediately prior to maturation and release of virus.
14
C-glucosamine also appears to be incorporated into the outer viral envelope shortly before maturation. On the other hand, there is an average lag of about 16 to 20 hr before
14
C-ethanolamine incorporated into intracellular lipoprotein appears in free virions; this probably reflects the kinetics of replacement of cellular surface membrane. Actinomycin D inhibits AMV-RNA within 30 min but permits the maturation of AMV to continue for at least 2 hr. AMV released in the presence of actinomycin D contains AMV-RNA synthesized before the addition of the drug.
Distinct Slime Polysaccharide Depolymerases of Bacteriophage-Infected
Pseudomonas aeruginosa:
Evidence of Close Association with the Structured Bacteriophage Particle
Authors: Bartell Pasquale F. , Orr Thomas E.
Published: January 2020

Five new polysaccharide depolymerases were isolated from cultures of
Pseudomonas aeruginosa
infected with phages 6, 7, 8, 9, and 10. The production of enzyme paralleled the release of phage. Depolymerase associated with phage 8 was active on slime polysaccharide A, whereas depolymerases associated with phages 6, 7, 9, and 10, like pseudomonas phage 2, hydrolyzed slime polysaccharide B. None of the depolymerases was active on slime polysaccharide C. Despite exhaustive purification, depolymerase activity was found to band with the phage particles at a density of 1.49 to 1.51 g/ml in a density gradient composed to cesium chloride. These results suggest that the depolymerases are firmly bound to the phage particles.
Role of Lysine in the Replication of Reovirus
Authors: Loh Philip C. , Oie Herbert K.
Published: January 2020

Lysine is essential for the replication of infectious reovirus. Omission of lysine from the extracellular medium not only permitted the continued synthesis of structural viral proteins and viral double-stranded ribonucleic acid (RNA), but also caused an enhanced formation of viral structures which were separable by isopycnic sedimentation of CsCl into a top band consisting of empty particles with a buoyant density of 1.29 g/cm
3
and essentially free of viral RNA, and two lower bands which were difficult to resolve and had an average buoyant density of 1.37 g/cm
3
. The lower bands contained most of the viral nucleic acid. The above effects were reversed when lysine was restored early after infection. In contrast, a single band with a buoyant density of 1.38 g/cm
3
was obtained from lysine-plus infected cells.
Comparison of phosphorylation of two polyoma virus middle T antigens in vivo and in vitro
Authors: Schaffhausen B , Benjamin T L
Published: January 2020

Two species of polyoma virus middle T antigen were detected in both lytically infected and transformed cells by in vitro kinase assay of immunoprecipitates. A minor species with an apparent molecular weight of 58,000 (58K) represented less than 10% of the total middle T protein. This species was roughly 10 times more active as a phosphate acceptor than was the predominant 56K form. Partial proteolytic mapping experiments showed that the same site was phosphorylated in both species. Mapping of the middle T antigens from a series of deletion mutants suggested that the major site of phosphorylation is tyrosine residue 315. Phosphorylation occurred on both middle T species in vivo, involving sites predominantly other than the tyrosine labeled in vitro. The 56K and 58K middle T forms differed from each other in their in vivo phosphorylation patterns. Some phosphate was incorporated into the 58K species in a region of the molecule to which at least part of the apparent molecular weight different could be mapped. hr-t mutant NG-59, which codes for a slightly altered middle T, produced only a single species (56K) which was inactive in the in vitro kinase reaction. Moreover, no 58K species appeared in vivo with this mutant. hr-t mutants are therefore defective in both aspects of phosphorylation. Phenotypically normal revertant cells of a polyoma transformed line failed to express any middle T antigens or associated kinase activity.