bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Sindbis Virus Production by Media of Low Ionic Strength: Intracellular Events and Requirements for Reversal
Authors: Waite Marilynn R. F. , Pfefferkorn E. R.
Published: January 2020

Incubation of Sindbis virus-infected cultures in medium with an ionic strength of 0.105 reduced the virus yield more than 99%. This inhibition was rapidly reversed by exposing the cultures to normal medium: within 20 min the previously inhibited cultures had released as much infectious virus as normal controls had produced during hours of incubation. The following intracellular processes were essentially normal in inhibited, infected monolayers: protein and phospholipid synthesis, the synthesis of infectious viral ribonucleic acid and its incorporation into nucleocapsids, and viral modification of the cell membrane. Accelerated virus production was detected within 20 sec after exposure of inhibited cultures to normal medium. It required an ionic strength greater than 0.145, a
p
H above 6.7, and a temperature above 21 C. It was not dependent on osmotic pressure, de novo protein synthesis, or a functional energy metabolism. Virus release also occurred in sonic-treated materials of inhibited cells under the same conditions as in living cells. Potential applications of the inhibition to concentration of virus stocks or to obtaining virus in nonphysiological solutions are noted. Preliminary studies with Semiliki Forest virus, Newcastle disease virus, and vesicular stomatitis virus suggest that this phenomenon may be limited to arboviruses.
Bacteriophage Infection: Which End of the SP82G Genome Goes in First?
Authors: McAllister William T.
Published: January 2020

The transfer of deoxyribonucleic acid (DNA) from bacteriophage SP82G to its host may be halted by chilling but is affected little by chloramphenicol, actinomycin D, or cyanide. The order of entry of markers on the phage genome was determined by halting the transfer of DNA at intervals, removing the untransferred DNA by blending, and assaying for the presence of markers in the blended complexes. Markers on the phage genome are transferred in a linear, polar fashion consistent with the previously determined genetic and physical maps. Those markers concerned with early functions enter first, and the rate of transfer is temperature dependent.
Uncoating of Poliovirus by Isolated Plasma Membranes
Authors: Chan V. F. , Black F. L.
Published: January 2020

Poliovirus type I LSc strain labeled with
14
C-uridine was adsorbed onto isolated plasma membranes and incubated with them. When membranes from Hep-2 or Vero cells were used, 22% of the label was converted to a trichloroacetic acid-soluble form, when trypsin or ribonuclease was added, the fraction rendered soluble was increased, and when the two enzymes were added in sequence, 85% or more of the label became trichloroacetic acid-soluble. This labilization of poliovirus could be reproduced when butanol-solubilized proteins from membranes were substituted for the whole plasma membranes, but it did not occur with membranes from polio-virus-resistant calf kidney or BHK-21 cells.
Virus-Specific Proteins Synthesized in Cells Infected with
RNA
+
Temperature-Sensitive Mutants of Sindbis Virus
Authors: Scheele Christina M. , Pfefferkorn E. R.
Published: January 2020

All Sindbis virus temperature-sensitive mutants defective in “late” functions were systematically surveyed by acrylamide-gel electrophoresis for similarities and differences in the intracellular pattern of virus-specific proteins synthesized at the permissive and nonpermissive temperatures. Only cells infected with mutants of complementation group C showed an altered pattern. At the nonpermissive temperature, these mutants failed to induce the synthesis of a polypeptide corresponding to the nucleocapsid protein and instead overproduced a protein of higher molecular weight than either viral structural protein. This defect was shown to be irreversible by the finding that
3
H-leucine incorporated at 41.5 C specifically failed to appear in the nucleocapsid of virions subsequently released at 29 C. Attempts to demonstrate a precursor protein in wild-type infections were inconclusive.
Synthesis and Cleavage of Enterovirus Polypeptides in Mammalian Cells
Authors: Kiehn E. D. , Holland John J.
Published: January 2020

Evidence is presented that the entire enterovirus ribonucleic acid genome is translated into a single giant polypeptide of well over 200,000 daltons molecular weight. This giant protein was detected repeatedly in coxsackievirus B1-infected cells, even in the absence of amino acid analogues. The enzymes which cleave this giant protein into smaller structural and enzymatic proteins accumulate with increasing time after infection. It appears that they are either virus-coded proteolytic enzymes or else host enzymes which are activated or released from an intracellular site as a result of virus infection. These cleavage enzymes do not cause grossly apparent cleavage of host-cell proteins.
Equilibrium Density Gradient Studies on Simian Virus 40-Yielding Variants of the Adenovirus Type 2-Simian Virus 40 Hybrid Population
Authors: Wiese William H. , Lewis Andrew M. , Rowe Wallace P.
Published: January 2020

The simian virus 40 (SV40)-yielding variants of the adenovirus type 2 (Ad.2)-SV40 hybrid (Ad.2
++
) population were studied by means of fixed-angle equilibrium density gradient centrifugation in cesium chloride. The hybrid virions of the Ad.2
++
high-efficiency yielder population banded at densities of 0.004 g/cm
3
lighter than the nonhybrid Ad.2 virions. The degree of separation of the hybrid particles was sufficient to permit greater than 100-fold relative purification by two cycles of centrifugation. Hybrid particles that produce adenovirus plaques in African green monkey kidney cells by two-hit kinetics (one-hit kinetics when assayed on lawns of nonhybrid adenovirus) were not separable from the particles that yield SV40 virus. The hybrid particle in the Ad.2
++
low-efficiency yielder population was not separable from the nonhybrid Ad.2 virions.
Proteins of Vesicular Stomatitis Virus: Kinetics and Cellular Sites of Synthesis
Authors: Wagner Robert R. , Snyder Ruth M. , Yamazaki Shudo
Published: January 2020

Five viral peptides synthesized in L cells infected with vesicular stomatitis (VS) virus were identified by polyacrylamide gel electrophoresis and designated as follows: nucleoprotein N, a membrane glycoprotein G, a membrane surface protein S, and two nonstructural proteins NS1 and NS2. A slowly migrating minor structural protein L also present in infected cells is probably an aggregate. Incorporation of
3
H-amino acids into each viral protein could be detected by the 2nd hr after infection and even earlier for protein N which is synthesized in the greatest amount. There was no evidence of regulation of viral protein synthesis at the transcriptive level; nonstructural and structural proteins were synthesized throughout the cycle of infection. Short pulses of
3
H-amino acids revealed no uncleaved precursor peptides that could be chased into structural peptides. Proteins N and S were chased into released virions but protein G was apparently incorporated into virions as it was being synthesized. VS viral proteins of infected cells were released by mechanically disrupting cytoplasmic membrane by nitrogen decompression and fractionated by high-speed centrifugation. Protein NS1 was present in the nonsedimentable cytoplasmic fraction throughout the cycle of infection. The nucleoprotein N was recovered primarily from the nonsedimentable fraction early in infection but aggregated into a sedimentable component, presumably the nucleocapsid, later in infection. Proteins G and S were always present in the sedimentable fraction of mechanically disrupted infected cells, presumably in association with plasma membrane. Exposure of infected cells to the membrane-dissolving agent, digitonin, resulted in solubilization of most of protein G and all of protein S but not of protein N. These experiments are compatible with the hypothesis that VS viral proteins G and S are synthesized at and inserted into plasma membrane which envelopes a nucleocapsid core to form the VS virion.
Arenoviruses: Proposed Name for a Newly Defined Virus Group
Authors: Rowe Wallace P. , Murphy Frederick A. , Bergold Gernot H. , Casals Jordi , Hotchin John , Johnson Karl M. , Lehmann-Grube Fritz , Mims Cedric A. , Traub Eric , Webb Patricia A.
Published: January 2020

The name “arenoviruses” is proposed for the newly defined taxonomic group containing lymphocytic choriomeningitis, Lassa, and the Tacaribe complex viruses.
Inhibition of Translation of Preformed
lac
Messenger Ribonucleic Acid by T4 Bacteriophage
Authors: Duckworth Donna Hardy
Published: January 2020

Cells infected with bacteriophage T4 are unable to translate β-galactosidase messenger ribonucleic acid which exists in the cell before infection, unless translation is underway at the time of infection.
Effect of the “Ribonucleic Acid Control” Locus in
Escherichia coli
on T4 Bacteriophage-Specific Ribonucleic Acid Synthesis
Authors: Sköld Ola
Published: January 2020

Amino acid control of ribonucleic acid (RNA) synthesis in bacteria is known to be governed genetically by the
rel
locus. We investigated whether the
rel
gene of the host would also exert its effect on the regulation of phage-specific RNA synthesis in T4 phage-infected
Escherichia coli
cells. Since T-even phage infection completely shuts off host macromolecular synthesis, phage RNA synthesis could be followed specifically by the cumulative incorporation of radioactivity from labeled precursors into RNA of infected cells. Labeled uracil was shown to accumulate in phage-specific RNA for 30 to 35 min after infection, a phenomenon which probably reflects an expansion of the labile phage-RNA pool. Amino acid starvation was effected by the use of auxotrophic bacterial strains or thienylalanine. The latter substance is an amino acid analogue which induces a chemical auxotrophy by inhibiting the biosynthesis of phenylalanine, tyrosine, and tryptophan. Phage RNA synthesis was strictly dependent on the presence of amino acids, whereas phage deoxyribonucleic acid synthesis was not. By the use of several pairs of bacterial strains which were isogenic except for the
rel
gene, it was demonstrated that amino acid dependence was related to the allelic state of this gene. If the
rel
gene was mutated, amino acid starvation did not restrict phage RNA synthesis.
DNA-binding activity of simian virus 40 large T antigen correlates with a distinct phosphorylation state
Authors: Scheidtmann K H , Hardung M , Echle B , Walter G
Published: January 2020

The state of phosphorylation and the relationship of various subclasses of simian virus 40 large T antigen (large T) differing in DNA-binding activity, degree of oligomerization, age, and subcellular distribution were investigated. Young large T (continuously labeled for 4 h late in infection) comprised about 20% of the total cellular large T. It was phosphorylated to a low degree and existed primarily in a monomeric form, sedimenting at 5S. More than 50% of this fraction bound to simian virus 40 DNA, preferentially to origin-containing sequences. Old large T (continuously labeled for 17 h, followed by a 4-h chase) represented the majority of the population. It was highly phosphorylated and predominantly in an oligomeric form, sedimenting at 15S to 23S. Only 10 to 20% of this fraction bound to simian virus 40 DNA. Another subclass of large T which was extracted from nuclei with 0.5 M salt resembled newly synthesized molecules in all properties tested; it was phosphorylated to a low degree, sedimented at 5S, and bound to viral DNA with high efficiency (greater than 70%). Two-dimensional phosphopeptide analysis of the individual subclasses revealed two distinct phosphorylation patterns, one characteristic for young, monomeric, and DNA-binding large T, the other for old, oligomeric, and non-DNA-binding large T. All sites previously identified in unfractionated large T (K.H. Scheidtmann et al., J. Virol. 44:116-133, 1982) were also phosphorylated in the various subclasses, but to different degrees. Peptide maps of the DNA-binding fraction, the 5S form, and the nuclear high-salt fraction showed two prominent phosphopeptides not previously characterized. Both peptides were derived from the amino-terminal region of large T, presumably involved in origin binding, and probably represent partially phosphorylated intermediates of known phosphopeptides. Our data show that the DNA-binding activity, age, and oligomerization of large T correlate with distinct states of phosphorylation. We propose that differential phosphorylation might play a role in the interaction of large T with DNA.
Nucleotide sequence of v-fps in the PRCII strain of avian sarcoma virus
Authors: Huang C C , Hammond C , Bishop J M
Published: January 2020

PRCII is an avian retrovirus whose oncogene (v-fps) induces fibrosarcomas in birds. The viral gene v-fps arose by transduction of an undetermined portion of a cellular gene known as c-fps. PRCII is weakly oncogenic when compared with Fujinami sarcoma virus, another transforming virus containing v-fps. As a first step in the elucidation of the molecular basis for the decreased virulence of PRCII, we have determined the entire nucleotide sequence of v-fps in the PRCII genome. The v-fps domain in PRCII encodes a polypeptide with a molecular weight of ca. 60,500 fused to a portion of the polyprotein encoded by the viral structural gene gag. The hybrid gag-fps polyprotein of PRCII would have a molecular weight of ca. 98,100, in accord with results of previous studies of the protein encoded by the PRCII genome. The leftward junctions between fps and gag in Fujinami sarcoma virus and PRCII are located at the same position in fps, but at different positions in gag. A sequence of 1,020 nucleotides, bounded by direct repeats of 6 nucleotides, is present in v-fps of Fujinami sarcoma virus but absent from PRCII. Our data should permit further explorations of the relationship between structure and function in the transforming protein encoded by v-fps.
Evidence That the 32,000-Dalton Protein Encoded by Bottom-Component RNA of Cowpea Mosaic Virus is a Proteolytic Processing Enzyme
Authors: Franssen Henk , Moerman Marja , Rezelman Geertje , Goldbach Rob
Published: January 2020

Translation of middle-component RNA of cowpea mosaic virus in vitro produced two polypeptides of 95 and 105 kilodaltons (95K and 105K, respectively) with overlapping amino acid sequences, which were specifically cleaved by a protease encoded by the bottom-component RNA. The proteolytic cleavage was studied by the addition of antibodies raised against various bottom-component RNA-encoded proteins to extracts prepared from bottom-component RNA-inoculated cowpea protoplasts. Since antiserum to the 32K polypeptide efficiently inhibited the proteolytic activity of such extracts, although antiserum to VPg or to the 170K polypeptide did not, evidence was obtained which indicates that the 32K polypeptide represents the protease involved. Fractionation of proteolytically active extract by glycerol gradient centrifugation demonstrated that 32K polypeptides do not exist as free proteins but are aggregated to the bottom-component RNA-encoded 170K, 84K, 60K, or 58K polypeptides. Maximal proteolytic activity was observed for 32K polypeptides associated with 170K polypeptides, suggesting that the activity was unstable and confined to newly synthesized molecules.
Influenza virus-induced immune complexes suppress alveolar macrophage phagocytosis
Authors: Astry C L , Jakab G J
Published: January 2020

Immune complexes in the lungs are capable of inducing adverse responses. Herein we have detailed the formation of immune complexes in the lungs of influenza virus-infected mice and examined their effect on alveolar macrophage defenses. On days 3, 7, 10, 15, and 30 after aerosol infection with influenza A/PR8/34 virus, the acellular pulmonary lavage fluid was tested for viral antigen, specific viral antibody, and immune complexes by immunoassays. Whereas peak viral antigen (day 3) diminished to undetectable levels by day 10, specific viral antibody remained at a low concentration until day 10, after which it rapidly increased. Immune complex concentrations increased through day 7, peaked at day 10, and gradually returned to the control level by day 30. These data demonstrate that immune complexes of detectable size are induced by influenza virus infection during the interface between antigen excess and antibody excess conditions. Since alveolar macrophages are the pivotal phagocytic defense cells in the lung, the ability of normal alveolar macrophages to ingest opsonized erythrocytes was quantitated in the presence of immune complexes from lavage fluid. Immune complexes from day 10 virus-infected lungs caused a dose-dependent suppression of antibody-mediated phagocytosis to 30% of control values. In contrast, although these immune complexes also markedly decreased the phagocytosis of antibody-coated yeast cells, they did not significantly impair the antibody-independent ingestion of unopsonized yeast cells by macrophages. the suppressive effects of immune complexes on alveolar macrophages may, in part, explain the phagocytic dysfunction that occurs 7 to 10 days after influenza virus pneumonia.
Role of recombinant ecotropic and polytropic viruses in the development of spontaneous thymic lymphomas in HRS/J mice
Authors: Thomas C Y , Khiroya R , Schwartz R S , Coffin J M
Published: January 2020

The biological and genetic characteristics of murine leukemia viruses (MuLV) derived from leukemic and normal HRS/J mice were studied. T1-oligonucleotide fingerprinting and mapping of viral RNAs from unpassaged isolates revealed the presence of complex mixtures of viral genomes. MuLV that were purified by endpoint dilution were genetically heterogeneous. Thus, endogenous retroviral sequences expressed in the tissues of HRS/J mice readily recombined with one another. Furthermore, the regular recovery of recombinant ecotropic MuLV suggested reciprocal in vivo complementation of a genetic defect(s) in each of the endogenous ecotropic proviruses Emv-1 and Emv-3. Some recombinant ecotropic viruses contained sequences in the p15E-U3 region that were not derived from Emv-1 and Emv-3 but were found in recombinant polytropic HRS/J viruses. Finally, comparison of the genetic structures of leukemogenic and nonleukemogenic MuLV of this strain implied that the oncogenic phenotype of these MuLV is encoded within env or the U3 region of the genome or both. Our results are consistent with a stepwise convergent evolution of recombinant MuLV in vivo in individual HRS/J mice. Ultimately, this process of selection results in formation of leukemogenic polytropic viruses.
Independent mutations in Ad2ts111 cause degradation of cellular DNA and defective viral DNA replication
Authors: Stillman B W , White E , Grodzicker T
Published: January 2020

An adenovirus mutant, Ad2ts111, has previously been shown to be temperature sensitive for viral DNA replication in vivo and also to induce degradation of cellular DNA. Soluble nuclear extracts prepared from Ad2ts111-infected HeLa cells grown at either the permissive (32 degrees C) or the nonpermissive (39.5 degrees C) temperature are thermolabile for elongation but not for initiation of DNA replication in vitro. Adenovirus single-stranded-DNA-binding protein purified from wild-type-infected cells can complement these extracts at the restrictive temperature in vitro. The DNA-binding protein synthesized in Ad2ts111-infected cells is stable at the nonpermissive temperature and is phosphorylated, as is the wild-type protein. In contrast, the mutant DNA-binding protein synthesized in Ad5ts125-infected cells is unstable. Ad2ts111 and Ad5ts125 do not complement each other for virus growth in vivo. These results suggest that Ad2ts111 contains a mutation in the DNA-binding protein that affects viral DNA synthesis. Finally, we demonstrated that, unlike viral DNA synthesis, the induction of cellular DNA degradation in Ad2ts111-infected cells is not temperature sensitive and that this phenotype is a result of a mutation in early region 1 on the virus genome. Thus, the two phenotypes displayed in Ad2ts111-infected cells, namely, the temperature-sensitive replication of viral DNA and the degradation of cell DNA, are the result of two separate mutations.
Physical maps of varicella-zoster virus DNA derived with 11 restriction enzymes
Authors: Mishra L , Dohner D E , Wellinghoff W J , Gelb L D
Published: January 2020

Varicella-zoster virus DNA was digested with 11 restriction endonucleases, and the resulting fragments were separated on agarose gels. Terminal fragments were identified by lambda exonuclease digestion. Physical maps were then constructed using a combination of double restriction enzyme digestion and hybridization to cloned BamHI fragments to place the remaining fragments in order.
Analysis of DNA sequences which regulate the transcription of a herpes simplex virus immediate early gene
Authors: Preston C M , Cordingley M G , Stow N D
Published: January 2020

The locations and functions of DNA sequences involved in transcription of the gene encoding herpes simplex virus type 1 immediate early (IE) mRNAs 4 and 5 were analyzed by use of a transient-expression assay. The region upstream of the genes encoding IE mRNAs 4 and 5 was fused to the thymidine kinase gene coding sequences, and production of enzyme or RNA was measured after transfection of plasmids into BHK cells. The effect of deletions in the upstream region was determined in the absence or presence of a virus structural component which stimulates herpes simplex virus IE transcription. Two distinct units were identified. One of these was a promoter which required not more than 69 base pairs of DNA specific for the genes encoding IE mRNAs 4 and 5 upstream from the mRNA 5' terminus. The other was a far-upstream region which mediated the response to the virion component and had an upstream boundary between nucleotides -347 and -335. An origin of DNA replication was interposed between these two units. The element TAATGAGATAC , which represents a consensus sequence present in the upstream regions of all herpes simplex type 1 IE genes, appeared to be essential for stimulation by the virion component. The activity of this element was modulated by the sequences which flank it, especially by regions having extremely high contents of guanine plus cytosine and which contain a conserved unit CCCGCCC or its complement GGGCGGG .
Nucleotide sequence and host La protein interactions of rabies virus leader RNA
Authors: Kurilla M G , Cabradilla C D , Holloway B P , Keene J D
Published: January 2020

Rabies virus leader RNA was detected in infected BHK-21 cell extracts by hybridization to end-labeled genomic RNA. Similar to the leader RNA of vesicular stomatitis virus, the leader RNA of rabies virus was also found to be associated with the La protein by specific immunoprecipitation with antisera from lupus patients. The 3' end of the genomic RNA of rabies virus was sequenced, and the size and termination site of leader RNA were determined. In addition, extension of the sequence into the nucleocapsid gene of rabies virus showed an open reading frame for at least 37 amino acid residues. Sequence relationships between rabies virus and vesicular stomatitis virus leader genes and the possible involvement of the La protein in rhabdovirus biology are discussed.
Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus
Authors: Laprevotte I , Hampe A , Sherr C J , Galibert F
Published: January 2020

The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing.
Location of DNA-binding proteins and disulfide-linked proteins in vaccinia virus structural elements
Authors: Ichihashi Y , Oie M , Tsuruhara T
Published: January 2020

Treatment with sodium dodecyl sulfate (SDS) converted the vaccinia virus strain IHD-J into particles of two types: (i) ghosts which possessed a thin-membrane vesicle derived from basement part of the virus membrane with attached lateral bodies and a membranous structure derived from the core wall and (ii) aggregates of a DNA-nucleoprotein eluted from the core. These particles lacked lipids, and all the viral phospholipids were detected in the SDS-soluble fraction. The viral membrane was composed of an SDS-soluble coat layer and the basement membrane, and the basement membrane was maintained by a mechanism other than the lipid bilayer. By comparisons of protein species in morphologically distinct subviral particles prepared by several solubilizing methods, protein compositions of viral structural elements were suggested as follows: 25,000-molecular-weight viral protein-17,000-molecular-weight viral protein ( VP25K - VP17K ), viral basement membrane; VP13 . 8K , major component of the lateral body; VP70K , VP69K , VP66K , and VP64K , minor components of the lateral body; VP61K , outer layer of core wall; VP57K - VP22K , inner layer of core wall; and VP27K - VP13K , nucleoprotein. These structural elements found in the SDS-insoluble particles dissolved in the same SDS solution under reducing conditions, indicating that the disulfide linkages seem to have a principal role in maintaining their morphological integrity. VP57K , VP27K , VP13 . 8K , and VP13K were revealed to possess affinity for DNA. Denatured calf thymus DNA and viral DNA in double- or single-stranded form associated equally well with these proteins, but RNA did not bind. Therefore, it was strongly suggested that disulfide-linked VP27K - VP13K represented the nucleoproteins of vaccinia virus. A structural model of vaccinia virus is proposed and discussed.
Influenza viruses are T cell-independent B cell mitogens
Authors: Anders E M , Scalzo A A , White D O
Published: January 2020

UV-inactivated influenza virus A strains of subtypes H1, H2, H3, and H6 were shown to be mitogenic for unprimed splenic lymphocytes from BALB/c mice. Representative viruses of these four subtypes all behaved as T cell-independent B cell mitogens. The magnitude of the proliferative response was determined by the subtype of the hemagglutinin molecule: H2 and H6 viruses were the most potent mitogens, and H3 viruses were moderately mitogenic, whereas H1 viruses induced only low, but significant, levels of proliferation. Mitogenesis was inhibited by antiviral sera and by monoclonal antibodies directed against hemagglutinin.
Uukuniemi virus maturation: immunofluorescence microscopy with monoclonal glycoprotein-specific antibodies
Authors: Kuismanen E , Bång B , Hurme M , Pettersson R F
Published: January 2020

Monoclonal antibodies directed against Uukuniemi virus glycoproteins G1 and G2 in combination with polyclonal antibodies against the nucleoprotein (N) were used to study the maturation of the virus in Golgi complexes of infected chicken embryo fibroblasts and BHK cells. Of 25 monoclonal antibodies obtained, 10 were shown to be G1 specific and 15 were shown to be G2 specific by immunoblotting and immunoprecipitation. In double-staining experiments, some of the monoclonal antibodies gave similar distributions of fluorescence as compared with the staining obtained from polyclonal rabbit anti-G1-G2 antibodies. Others, however, preferentially stained either the glycoproteins in the Golgi complex or those at the cell surface. This may indicate that the glycoproteins underwent conformational changes during their transport. Uukuniemi virus infection resulted in the vacuolization of the membranes of Golgi complexes where the maturation of the virus was taking place. Double-staining experiments with monoclonal antibodies which preferentially stained the Golgi-associated viral glycoproteins and with anti-N polyclonal rabbit antiserum showed a correlation between the progressive vacuolization of the Golgi complex and the accumulation of viral nucleoprotein in the Golgi region, suggesting that a morphological alteration of the Golgi complex may be a prerequisite for intracellular maturation of the virus. Treatment of Uukuniemi virus-infected cells with tunicamycin, a drug which inhibits N-linked glycosylation, resulted in the accumulation of both glycoproteins at an intracellular location, apparently representing the endoplasmic reticulum. Double-staining experiments showed a parallel accumulation of nucleoprotein at these sites, indicating that local accumulation of glycoproteins is required for nucleoprotein binding to intracellular membranes.
Physical mapping of the herpes simplex virus type 2 nuc- lesion affecting alkaline exonuclease activity by using herpes simplex virus type 1 deletion clones
Authors: Wathen M W , Hay J
Published: January 2020

The nuc- lesion affecting alkaline exonuclease activity in the herpes simplex virus type 2 (HSV-2) mutant ts1348 had previously been mapped to the EcoRI-D restriction enzyme fragment of HSV-1. Eight clones with deletions representing most of HSV-1 EcoRI fragment D were selected with lambda gtWES hybrids. These clones were tested for their ability to rescue the alkaline exonuclease activity of HSV-2 nuc- ts1348 virus. The sequences colinear with the HSV-2 nuc- lesion were found to map between 0.169 and 0.174 map units on the HSV-1 Patton genome, representing an 0.8-kilobase-pair region that is 12.9 to 13.7 kilobase pairs from the left end of HSV-1 EcoRI fragment D.
Immunogenic proteins of feline rhinotracheitis virus
Authors: Maes R K , Fritsch S L , Herr L L , Rota P A
Published: January 2020

Feline rhinotracheitis virus is an upper-respiratory-tract pathogen of cats. It may also cause generalized infections or abortions. Antigens present in [35S]methionine- or [14C]glucosamine-labeled purified virions, in Nonident P-40 (NP-40) extracts of a mixture of virions and infected cells, and in virion-free cell culture medium, along with mock-infected Crandell -Rees feline kidney cell controls, were analyzed by direct sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or by SDS-PAGE preceded by Staphylococcus aureus protein A immunoprecipitation. The direct SDS-PAGE analysis revealed at least 17 virus-specific peptides with molecular weights ranging from less than 200,000 ( 200K ) to more than 30K . Three of these peptides were glycosylated and had molecular weights of 105K , 68K , and 60K. Immunoprecipitates of purified virions and NP-40 extracts contained three major glycoproteins with the same estimated molecular weights as those found by the direct analysis. A prominent 105K glycoprotein was present in virion-free cell culture medium immunoprecipitates. In addition, a number of nonglycosylated feline rhinotracheitis virus-specific polypeptides (eight in virions, three in NP-40 extracts, and nine in virion-free cell culture medium), ranging in molecular weight from 145K to 32K, were present in the various immunoprecipitates.
Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants
Authors: Hermonat P L , Labow M A , Wright R , Berns K I , Muzyczka N
Published: January 2020

We constructed insertion and deletion mutants with mutations within the adeno-associated virus (AAV) sequences of the infectious recombinant plasmid pSM620. Studies of these mutants revealed at least three AAV phenotypes. Mutants with mutations between 11 and 42 map units were partially or completely defective for rescue and replication of the AAV sequences from the recombinant plasmids (rep mutants). The mutants could be complemented by mutants with replication-positive phenotypes. The protein(s) that is affected in rep mutants has not been identified, but the existence of the rep mutants proves that at least one AAV-coded protein is required for viral DNA replication. Also, the fact that one of the rep mutant mutations maps within the AAV intron suggests that the intron sequences code for part of a functional AAV protein. Mutants with mutations between 63 and 91 map units synthesized normal amounts of AAV duplex DNA but could not generate single-stranded virion DNA (cap mutants). The cap phenotype could be complemented by rep mutants and is probably due to a defect in the major AAV capsid protein, VP3. This suggests that a preformed capsid or precursor is required for the accumulation of single-stranded AAV progeny DNA. Mutants with mutations between 48 and 55 map units synthesized normal amounts of AAV single-stranded and duplex DNA but produced substantially lower yields of infectious virus particles than wild-type AAV (lip mutants). The lip phenotype is probably due to a defect in the minor capsid protein, VPI, and suggests the existence of an additional (as yet undiscovered) AAV mRNA. Evidence is also presented for recombination between mutant AAV genomes during lytic growth.
Antigenic relationships among proteins of bovine coronavirus, human respiratory coronavirus OC43, and mouse hepatitis coronavirus A59
Authors: Hogue B G , King B , Brian D A
Published: January 2020

Antisera prepared against each of three single and one pair of major structural proteins of the bovine coronavirus (Mebus strain) were used in immunoblotting studies to measure cross-reactivity with the structural proteins of the human coronavirus OC43 and the mouse hepatitis coronavirus A59. We conclude that the bovine coronavirus is comprised of four major structural proteins, gp190 (normally present as 120- and 100-kilodalton subunits), gp140, pp52, and gp26. The human coronavirus OC43 has an antigenically homologous counterpart of similar molecular mass to each of these proteins. The mouse hepatitis coronavirus A59 has an antigenically homologous counterpart to only three of these proteins: gp190, pp52 and gp26. There is no counterpart in the mouse virus to the 140-kilodalton glycoprotein, the apparent hemagglutinin of the bovine coronavirus.
Evidence for translational regulation of herpes simplex virus type 1 gD expression
Authors: Johnson D C , Spear P G
Published: January 2020

We compared the rates of synthesis of herpes simplex virus type 1 glycoproteins C and D and quantitated the accumulation of translatable mRNA for each glycoprotein at various times after infection. The rate of synthesis of gD increased sharply early in the infection, peaked by 4 to 6 h after infection, and declined late in the infection. In contrast, the rate of synthesis of gC increased steadily until at least 15 h after infection. The levels of mRNA for both of these glycoproteins, as detected by hybridization and by translation in vitro, continued to increase until at least 15 or 16 h after infection. Synthesis of both gC and gD and their respective mRNAs was found to be sensitive to inhibition of viral DNA replication with phosphonoacetic acid. The finding that reduced amounts of gD were synthesized late in the replicative cycle, whereas gD mRNA continued to accumulate in the cytoplasm, argues that the synthesis of gD is regulated, in part, at the level of translation.
Mutations in the hemagglutinin receptor-binding site can change the biological properties of an influenza virus
Authors: Naeve C W , Hinshaw V S , Webster R G
Published: January 2020

Avian influenza virus reassortants containing human influenza virus hemagglutinins do not replicate in ducks. Two mutations in the receptor-binding site of a human hemagglutinin at residues 226 and 228 allowed replication in ducks. The mutations resulted in a receptor-binding-site sequence identical to the known avian influenza virus sequences.
Macrophage Fc receptors control infectivity and neutralization of canine distemper virus-antibody complexes
Authors: Appel M J , Mendelson S G , Hall W W
Published: January 2020

Dogs that are persistently infected or that become moribund after exposure to canine distemper virus (CDV) have antibody that neutralized CDV when tested in dog lung macrophage cultures but failed to neutralize CDV when tested in epithelial, fibroblastic, or lymphatic cells. The antibody attached to protein A and was found in the immunoglobulin G fraction. The antibody bound complement and lysed CDV-infected target cells. The neutralizing activity in macrophages could be abolished (i) by pepsin digestion and removal of Fc portions from the antibody, (ii) by blocking the Fc receptors of macrophages with heat-treated normal dog serum, and (iii) by binding of protein A to Fc portions of the antibody. It was concluded that attachment of the CDV-antibody complex to Fc receptors of macrophages was essential for virus neutralization. If this attachment was hindered, the CDV-antibody complex became infectious for macrophages. In contrast, serum from recovering dogs neutralized CDV when tested in epithelial, fibroblastic, or lymphatic cells as well as in macrophages.
Heterogeneity of the polyribocytidylic acid tract in aphthovirus: biochemical and biological studies of viruses carrying polyribocytidylic acid tracts of different lengths
Authors: Costa Giomi M P , Bergmann I E , Scodeller E A , Augé de Mello P , Gomez I , La Torre J L
Published: January 2020

In this paper we report a study of a sample of foot-and-mouth disease virus carrying two polyribocytidylic acid [poly(C)] tracts of different lengths. By plaque purification in tissue culture, we isolated two populations of particles, one carrying the long poly(C) tract and the other carrying only the short homopolymer. The fingerprints of both viruses were indistinguishable from each other and from that of the virus present in the original sample, suggesting that the main difference between the two types of particles is limited to the poly(C) tracts of their genomic RNAs, to the flanking sequences of the poly(C) tract, or to both. In addition, some biological properties of these viruses are reported, such as stability upon serial passages in different cell lines, plaque size, and pathogenicity for cattle. The results indicate that the size of the poly(C) tract is not directly related to the virulence of these viruses. However, the size of the homopolymer could play a role in determining their efficiency of replication, and it appears that the particles with the short poly(C) tract might have some replicative advantage over those carrying the long one.
Translation efficiency of adenovirus early region 1A mRNAs deleted in the 5' untranslated region
Authors: Spindler K R , Berk A J
Published: January 2020

Adenovirus deletion mutants were studied to examine the influence of the 5' untranslated sequence on the translation of early region 1A mRNAs. Alterations of the 5' untranslated sequence, including complete deletion of the wild-type 5' untranslated sequence, did not significantly affect the rate of translation.
Role of cholesterol in fusion of Semliki Forest virus with membranes
Authors: Kielian M C , Helenius A
Published: January 2020

The low pH-triggered membrane fusion activity of Semliki Forest virus is dependent on the presence of cholesterol in the target membrane. When liposomes containing phospholipids and cholesterol analogs were used, fusion activity was observed with steroids which did not have a planar nucleus or an isooctyl side chain at C-17, but fusion activity was not observed when analogs which lacked the 3 beta-OH group were used. Binding of virus to liposomes at low pH was similarly, but not totally, dependent on the presence of a 3 beta-OH sterol.
Mapping a new gene that encodes an 11,600-molecular-weight protein in the E3 transcription unit of adenovirus 2
Authors: Wold W S , Cladaras C , Magie S C , Yacoub N
Published: January 2020

The DNA sequence of the early E3 transcription unit of adenovirus 2 (Ad2) (J. Hérissé et al., Nucleic Acids Res. 8:2173-2192, 1980), indicates that an open reading frame exists between nucleotides 1860 and 2163 that could encode a protein of Mr 11,600 (11.6K). We have determined the DNA sequence of the corresponding region in Ad5 (closely related to Ad2) and have established that this putative gene is conserved in Ad5 (a 10.5K protein). To determine whether this protein is expressed, we prepared an antiserum in rabbits against a synthetic peptide corresponding to amino acids 66 to 74 in the 11.6K protein of Ad2. The peptide antiserum immunoprecipitated a ca. 13K-14K protein doublet, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from [35S]methionine-labeled Ad2- or Ad5-early-infected KB cells. The antiserum also immunoprecipitated a 13K-14K protein doublet translated in vitro from Ad2 or Ad5 early E3-specific mRNA purified by hybridization to Ad2 EcoRI-D (nucleotides -236 to 2437). The synthetic peptide successfully competed with the 13K-14K protein doublet in immunoprecipitation experiments, thereby confirming the specificity of the antiserum. As deduced from the DNA sequence, the 11.6K protein (and the corresponding 10.5K Ad5 protein) has a conserved 22-amino-acid hydrophobic domain, suggesting that the protein may be associated with membranes. We conclude that a gene located at nucleotides 1860 to 2143 in the Ad2 E3 transcription unit (nucleotides 1924 to 2203) in the Ad5 E3 transcription unit) encodes an 11.6K protein (10.5K in Ad5).
Characterization of the guinea pig cytomegalovirus genome by molecular cloning and physical mapping
Authors: Gao M , Isom H C
Published: January 2020

Fragments of guinea pig cytomegalovirus (GPCMV) DNA produced by HindIII or EcoRI restriction endonuclease digestion were cloned into vectors pBR322 and pACYC184, and recombinant fragments representing ca. 97% of the genome were constructed. Hybridization of 32P-labeled cloned and gel-purified HindIII, EcoRI, and XbaI fragments to Southern blots of HindIII-, EcoRI-, and XbaI-cleaved GPCMV DNA verified the viral origin of cloned fragments and allowed construction of HindIII, EcoRI, and XbaI restriction maps. On the basis of the cloning and mapping experiments, the size of GPCMV DNA was calculated to include 239 kilobase pairs, corresponding to a molecular weight of 158 X 10(6). No cross-hybridization between any internal fragments was seen. We conclude that the GPCMV genome consists of a long unique sequence with terminal repeat sequences but without internal repeat regions. In addition, GPCMV DNA molecules exist in two forms. In the predominant form, the molecules demonstrate sequence homology between the terminal fragments; in the minor population, one terminal fragment is smaller by 0.7 X 10(6) daltons and is not homologous with the fragment at the other end of the physical map. The structural organization of GPCMV DNA is unique for a herpesvirus DNA, similar in its simplicity to the structure reported for murine cytomegalovirus DNA and quite dissimilar from that of human cytomegalovirus DNA.
Aberrant polyadenylation by a vesicular stomatitis virus mutant is due to an altered L protein
Authors: Hunt D M , Smith E F , Buckley D W
Published: January 2020

TsG16(I) is a temperature-sensitive mutant of vesicular stomatitis virus, Indiana serotype. Our stocks of this mutant overproduce polyadenylic acid in an in vitro transcription system. The overproduction of polyadenylic acid occurs at all temperatures tested (27, 31, 35, and 39 degrees C) and is apparently not due to an alternation in the N protein-RNA template. To characterize the altered moiety in tsG16(I) responsible for this phenotype, virions were fractionated and the polyadenylation phenotype in homologous and heterologous reconstitution assays was determined. The aberrant polyadenylation phenotype correlated with the presence of ts L protein but not ts NS or ts M protein fractions. Results of experiments in which solubilized tsG16(I) and wild-type virion components were mixed indicated that the altered moiety behaved as if present in stoichiometric amounts relative to active L protein. The effects of raising the temperature from 31 to 39 degrees C in such mixes were as would be predicted upon the assumption that the polyadenylation phenotype was associated with a thermosensitive transcriptase component [the L protein of tsG16(I) is known to be thermosensitive]. We conclude that the data strongly support the hypothesis that L is the altered protein responsible for the aberrant polyadenylation phenotype of tsG16(I).
Structural organization and polypeptide composition of the avian adenovirus core
Authors: Li P , Bellett A J , Parish C R
Published: January 2020

CELO virus (fowl adenovirus 1) contained three core polypeptides of molecular weights 20,000, 12,000, and 9,500. The core was similar to that of human adenoviruses, with some evidence of compact subcore domains. Micrococcal nuclease digestion of CELO virus cores produced a smear of DNA fragments of gradually decreasing size, with no nucleosome subunit or repeat pattern. Moreover, when digested cores were analyzed without protease treatment, there was again no evidence of a nucleosome substructure; neither DNA fragments nor core proteins entered a 4% polyacrylamide gel. The organization of the core is thus quite unlike that of chromatin. Restriction endonuclease analysis of the DNA from digested cores showed that the right end was on the outside of the core. We suggest that adenovirus DNA is condensed into the core by cross-linking and neutralization by the core proteins, beginning with the packaging sequence at the center of the core and ending with the right end of the DNA on the outside.
Expression of murine leukemia viruses in B-cell lymphomas of CWD/Agl mice
Authors: Angel J M , Bedigian H G
Published: January 2020

The inbred mouse strain CWD/Agl has a high incidence of spontaneous B-cell lymphomas characterized by gross splenomegaly and lymph node enlargement. The endogenous ecotropic retrovirus of CWD/Agl mice is expressed in the spleen within the first 2 weeks of age and in the thymus by 1 month of age. Endogenous xenotropic virus is expressed in the spleen and bone marrow of the earliest age group examined (4 months). Restriction enzyme analysis of DNA extracted from tumorous tissues suggests that mink cell focus-forming viruses are not required for B-cell lymphomagenesis in CWD/Agl mice. CWD/Agl mice provide an important new experimental model for the study of B-cell lymphoma.