bluuebunny/crossref_metadata_2025 huggingface dataset:
Human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus Nef use distinct but overlapping target sites for downregulation of cell surface CD4
Authors: Hua J , Cullen B R
Published: January 2020

Although the Nef proteins encoded by human immunodeficiency virus type 1 (HIV-1) and simian immuno-deficiency virus (SIV) are known to induce the efficient internalization and degradation of cell surface CD4, it remains unclear whether this process involves a direct interaction between Nef and CD4. Here, we report that CD4 downregulation by HIV-1 and SIV Nef requires distinct but overlapping target sites within the CD4 intracytoplasmic domain. In particular, mutation of a glutamic acid residue located at CD4 residue 405 or of arginine and methionine residues located, respectively, at residue 406 and 407 results in a mutant CD4 protein that is efficiently downregulated by HIV-1 Nef but refractory to downregulation by SIV Nef. However, both HIV-1 and SIV Nef require an isoleucine located at residue 410 and the dileucine motif found at CD4 residues 413 and 414. CD4 downregulation induced by the Nef protein encoded by HIV-2 is shown to require a CD4 target sequence that is similar to, but distinct from, that observed with SIV Nef. These data explain the previous finding that the murine CD4 protein, which has an alanine at residue 405, is refractory to downregulation by SIV, but not HIV-1, Nef (J. L. Foster, S.J. Anderson, A. L. B. Frazier, and J. V. Garcia, Virology 201:373-379, 1994). In addition, these observations provide strong genetic support for the hypothesis that the Nef-mediated downregulation of cell surface CD4 requires a direct Nef-CD4 interaction.
Functional and structural analysis of the sialic acid-binding domain of rotaviruses
Authors: Isa P , López S , Segovia L , Arias C F
Published: January 2020

The infectivity of most animal rotaviruses is dependent on the interaction of the virus spike protein VP4 with a sialic acid (SA)-containing cell receptor, and the SA-binding domain of this protein has been mapped between amino acids 93 and 208 of its trypsin cleavage fragment VP8. To identify which residues in this region are essential for the SA-binding activity, we performed alanine mutagenesis of the rotavirus RRV VP8 expressed in bacteria as a fusion polypeptide with glutathione S-transferase. Tyrosines were primarily targeted since tyrosine has been involved in the interaction of other viral hemagglutinins with SA. Of the 15 substitutions carried out, 10 abolished the SA-dependent hemagglutination activity of the protein, as well as its ability to bind to glycophorin A in a solid-phase assay. However, only alanine substitutions for tyrosines 155 and 188 and for serine 190 did not affect the overall conformation of the protein, as judged by their interaction with a panel of conformationally sensitive neutralizing VP8 monoclonal antibodies (MAbs). These findings suggest that these three amino acids play an essential role in the SA-binding activity of the protein, presumably by interacting directly with the SA molecule. The predicted secondary structure of VP8 suggests that it is organized as 11 beta-strands separated by loops; in this model, Tyr-155 maps to loop 7 while Tyr-188 and Ser-190 map to loop 9. The close proximity of these two loops is also supported by previous results from competition experiments with neutralizing MAbs directed at RRV VP8.
Effects of nucleocapsid mutations on human immunodeficiency virus assembly and RNA encapsidation
Authors: Zhang Y , Barklis E
Published: January 2020

The human immunodeficiency virus (HIV) Pr55Gag precursor proteins direct virus particle assembly. While Gag-Gag protein interactions which affect HIV assembly occur in the capsid (CA) domain of Pr55Gag, the nucleocapsid (NC) domain, which functions in viral RNA encapsidation, also appears to participate in virus assembly. In order to dissect the roles of the NC domain and the p6 domain, the C-terminal Gag protein domain, we examined the effects of NC and p6 mutations on virus assembly and RNA encapsidation. In our experimental system, the p6 domain did not appear to affect virus release efficiency but p6 deletions and truncations reduced the specificity of genomic HIV-1 RNA encapsidation. Mutations in the nucleocapsid region reduced particle release, especially when the p2 interdomain peptide or the amino-terminal portion of the NC region was mutated, and NC mutations also reduced both the specificity and the efficiency of HIV-1 RNA encapsidation. These results implicated a linkage between RNA encapsidation and virus particle assembly or release. However, we found that the mutant ApoMTRB, in which the nucleocapsid and p6 domains of HIV-1 Pr55Gag were replaced with the Bacillus subtilis MtrB protein domain, released particles efficiently but packaged no detectable RNA. These results suggest that, for the purposes of virus-like particle assembly and release, NC can be replaced by a protein that does not appear to encapsidate RNA.
Analysis of bovine herpesvirus 1 transcripts during a primary infection of trigeminal ganglia of cattle
Authors: Schang L M , Jones C
Published: January 2020

During an infection of nonneuronal cells, bovine herpesvirus 1 (BHV-1) gene expression proceeds in a well-defined cascade. Products of immediate-early (IE) genes are expressed first, and they activate expression of early (E) and late (L) genes. Although the same cascade is assumed to occur during an infection of neurons in trigeminal ganglia (TG) of cattle, no experimental data is available to support this hypothesis. Consequently, we analyzed BHV-1 gene expression in bovine TG at 1, 2, 4, 7, and 15 days postinfection (dpi). Infectious virus was detected in ocular swabs from 1 to 7 dpi but not 15 dpi. By reverse transcription (RT)-PCR, IE (bICP4), E (thymidine kinase, ribonucleotide reductase [RR]), L (glycoprotein C, and alpha trans-inducing factor), and dual-kinetic (bICP0 and bICP22) transcripts were analyzed. When cDNA synthesis was primed with random hexamers, IE and E transcripts were detected at the same time. However, full-length and poly(A)+ (FL&P) RR or bICP22 RNAs were detected before FL&P IE RNAs. Furthermore, FL&P IE transcripts were not detected until viral DNA increased in TG. IE transcripts were detected before E or L RNAs when rabbit kidney cells were infected with a low multiplicity of infection and the same RT-PCR detection method was used. These studies suggested that expression of full-length and polyadenylated IE transcripts in trigeminal ganglia was not efficient compared to that of RR and bICP22 transcripts.
The genes encoding the peripheral cannabinoid receptor and alpha-L-fucosidase are located near a newly identified common virus integration site, Evi11
Authors: Valk P J , Hol S , Vankan Y , Ihle J N , Askew D , Jenkins N A , Gilbert D J , Copeland N G , de Both N J , Löwenberg B , Delwel R
Published: January 2020

A new common region of virus integration, Evi11, has been identified in two retrovirally induced murine myeloid leukemia cell lines, NFS107 and NFS78. By interspecific backcross analysis, it was shown that Evi11 is located at the distal end of mouse chromosome 4, in a region that shows homology with human 1p36. The genes encoding the peripheral cannabinoid receptor (Cnr2) and alpha-L-fucosidase (Fuca1) were identified near the integration site by using a novel exon trapping system. Cnr2 is suggested to be the target gene for viral interference in Evi11, since proviruses are integrated in the first intron of Cnr2 and retroviral integrations alter mRNA expression of Cnr2 in NFS107 and NFS78. In addition, proviral integrations were demonstrated within the 3' untranslated region of Cnr2 in five independent newly derived CasBrM-MuLV (mouse murine leukemia virus) tumors, CSL13, CSL14, CSL16, CSL27, and CSL97. The Cnr2 gene encodes a seven-transmembrane G-protein-coupled receptor which is normally expressed in hematopoietic tissues. Our data suggest that the peripheral cannabinoid receptor gene might be involved in leukemogenesis as a result of aberrant expression of Cnr2 due to retroviral integration in Evi11.
Cryo-electron microscopy structure of yeast Ty retrotransposon virus-like particles
Authors: Palmer K J , Tichelaar W , Myers N , Burns N R , Butcher S J , Kingsman A J , Fuller S D , Saibil H R
Published: January 2020

The virus-like particles (VLPs) produced by the yeast retrotransposon Ty1 are functionally related to retroviral cores. These particles are unusual in that they have variable radif. A paired mass-radius analysis of VLPs by scanning transmission electron microscopy showed that many of these particles form an icosahedral T-number series. Three-dimensional reconstruction to 38-A resolution from cryo-electron micrographs of T = 3 and T = 4 shells revealed that the single structural protein encoded by the TYA gene assembles into spiky shells from trimeric units.
The autonomous growth of human papillomavirus type 16-immortalized keratinocytes is related to the endothelin-1 autocrine loop
Authors: Venuti A , Marcante M L , Flamini S , Di Castro V , Bagnato A
Published: January 2020

Some human papillomaviruses (HPVs) such as HPV type 16 (HPV16) and HPV18 are involved in cervical carcinoma, and they can immortalize and transform keratinocytes. Endothelin-1 (ET-1) is produced in keratinocytes and has been shown to act through ETA receptors as an autocrine growth factor for keratinocytes. This study examines whether HPV16 alters the ET-1-mediated autocrine loop in human keratinocytes, providing a selective growth advantage for transformed cells. ET-1 is released in similar amounts from normal and HPV-transfected keratinocytes. All HPV-transfected cell lines express high-affinity ETA receptors. A two-fold increase in ET-1 binding sites is present in HPV16-immortalized keratinocytes, and this effect seems to be linked to the overexpression of mRNA for this receptor rather than to differences in the surface/internalized ratio of the receptors. ET-1 induces significant increases in [3H]thymidine incorporation and cell proliferation. Furthermore, HPV-transfected keratinocytes can proliferate in the absence of any growth factor added to the growth medium, and the ETA receptor antagonist BQ123 prevents this proliferation. These data suggest a new mechanism in the growth control of HPV-transformed cells mediated by the upregulation of ET-1 autocrine loop.
Cyclophilin A-induced alterations of human immunodeficiency virus type 1 CA protein in vitro
Authors: Agresta B E , Carter C A
Published: January 2020

Cyclophilin A (CyP A), a cellular chaperone with cis-trans prolyl isomerase activity, is required for postassembly events in human immunodeficiency virus type 1 (HIV-1) replication. The requirement for CyP A maps to sequences in the capsid (CA) domain of the structural precursor, Gag. To determine the effects of interaction with CyP A on capsid (CA) protein structure, the binding interaction was investigated in vitro, using recombinant HIV-1 CA protein (which forms oligomers in solution) and human CyP A. The CA and CyP A proteins interacted to form a complex which was detected predominantly as a heterodimer on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Complex formation exhibited a pH optimum of 5. The CA protein in the complex was exclusively in a conformation whereby the N terminus was blocked to Edman degradation. This finding was unexpected since CyP A binds to the central region of the CA protein (residues 85 to 93). Examination of CA protein incubated with CyP A for alterations in structure indicated that CyP A preferentially interacted with the subpopulation of trypsin-susceptible subunits in the oligomers and significantly reduced their sensitivity to proteolysis. Like CA-CyP A complex formation, conversion to trypsin resistance also exhibited a pH optimum of 5. Both complex formation and the changes in tryptic susceptibility were only partially inhibited by cyclosporin A (CsA). This appeared to be due to a CA subpopulation able to bind CyP A despite the presence of CsA. Our results identify specific tryptic sites both proximal and distal to the CyP A binding region that are altered by CyP A binding and/or by CyP A's prolyl isomerase activity. Comparison with the X-ray structure of a complex containing CyP A bound to an amino-terminal fragment of the CA protein (CA1-151) (T.R. Gamble et al., Cell 87:1285-1294, 1996) indicates that the tryptic sites that become inaccessible are among the same residues that lose a significant amount of accessible surface area through CA-CA subunit contacts made in the presence of CyP A.
Priming with live respiratory syncytial virus (RSV) prevents the enhanced pulmonary inflammatory response seen after RSV challenge in BALB/c mice immunized with formalin-inactivated RSV
Authors: Waris M E , Tsou C , Erdman D D , Day D B , Anderson L J
Published: January 2020

To investigate enhanced disease associated with a formalin-inactivated (FI) respiratory syncytial virus (RSV) vaccine, we studied the pulmonary inflammatory response to RSV in BALB/c mice immunized with live RSV, FI-RSV, or combinations of the two. After RSV challenge, the number of granular cells, the ratio of CD4+/CD8+ lymphocytes, and the level of Th2-like cytokine mRNAs in the bronchoalveolar lavage specimens in mice immunized first with live RSV and then with FI-RSV were lower than that in FI-RSV-immunized mice and close to that in live RSV-immunized mice. These data suggest that prior live RSV infection prevents most of the enhanced inflammatory response seen in FI-RSV-immunized mice and might explain lack of enhanced disease in older FI-RSV-immunized children. A live RSV vaccine might similarly decrease the risk of enhanced disease with non-live RSV vaccines.
cis elements that contribute to geminivirus transcriptional regulation and the efficiency of DNA replication
Authors: Eagle P A , Hanley-Bowdoin L
Published: January 2020

The A genomic component of the geminivirus tomato golden mosaic virus (TGMV) contains a 5' intergenic sequence that includes the overlapping AL61 promoter and positive-strand origin of DNA replication. The TGMV AL1 protein negatively regulates its own transcription and mediates origin recognition by binding to a repeated motif shared by the AL61 promoter and the viral origin. We examined a series of truncated or mutated 5' intergenic regions in transient expression and replication assay to identify other DNA sequences that contribute to TGMV promoter and origin function. These experiments revealed that negative regulation of the AL61 promoter is complex, involving multiple cis-acting sequences and the AL1 and AL4 proteins, which acted through different DNA elements. We also found that mutation of the TATA box motif in the AL61 promoter reduced overall transcriptional activity and AL1-mediated repression, confirming the importance of this sequence in promoter function. Mutation of a G-box consensus sequence was highly detrimental to AL61 transcription and abolished AL1 sensitivity, suggesting that AL1 interferes with transcriptional activation. Cotransfection experiments showed that the TATA box and G-box motif mutations also impaired viral DNA replication in the presence of a wild-type origin but had no effect in its absence, demonstrating that these transcriptional motifs also function as replication efficiency elements.
Cell-type-specific separate regulation of the E6 and E7 promoters of human papillomavirus type 6a by the viral transcription factor E2
Authors: Rapp B , Pawellek A , Kraetzer F , Schaefer M , May C , Purdie K , Grassmann K , Iftner T
Published: January 2020

Gene expression of human papillomaviruses (HPV) is tightly controlled by cellular factors and by the virally encoded E2 protein through binding to distinct sites within the regulatory noncoding region. While for the high-risk genital papillomaviruses a single promoter drives the expression of all early genes, a second promoter present in the E6 open reading frame of the low-risk HPV type 6 (HPV6) would allow an independent regulation of E6 and E7 oncogene expression. In this report, we provide the first evidence that E2 regulates both early promoters of HPV6 separately and we show that promoter usage as well as E2 regulation is cell type dependent. Among the different epithelial cell lines tested, only RTS3b cells allowed an expression pattern similar to that observed in naturally infected benign condylomas. While the E6 promoter was repressed by E2 to 50% of its basal activity, the E7 promoter was simultaneously stimulated up to fivefold. Activation of the E7 promoter was mediated predominantly by the binding of E2 to the most promoter-distal E2 binding site. Repression of the E6 promoter depended on the presence of two intact promoter-proximal binding sites. Mutation of both of these repressor binding sites reversed the effect of E2 on the E6 promoter from repression to activation. In contrast, in HT3 cells we observed an E2-mediated activation of the E6 promoter in the context of the wild-type noncoding region. This indicated that repression of the E6 promoter by binding of E2 to both promoter-proximal binding sites did not function in the cellular environment provided by HT3 cells. These data suggest that the separate regulation of the E6 and E7 promoters of HPV6 is mediated through successive occupation of binding sites with different affinities for E2 depending on the intracellular concentration of E2 and on the cellular environment provided by the infected cell.
Functional interactions between monomers of the retroviral envelope protein complex
Authors: Zhao Y , Lee S , Anderson W F
Published: January 2020

Retroviral vectors have been widely used in human gene therapy protocols. Entry into target cells is directed by the retroviral envelope protein, with receptor binding and postbinding fusion functions contributed mainly by the SU and TM subunits, respectively. We have generated mutants of the Moloney murine leukemia virus (MoMuLV) envelope protein with mutations in both the receptor binding domain of SU and throughout the TM subunit that are functionally inactive when expressed individually. However, the coexpression of these two classes of mutants partially restores envelope protein function and allows transduction. Several lines of evidence indicate that this complementation occurs in trans within envelope protein heterooligomers. The finding that the binding and postbinding functions of a retroviral envelope protein can be contributed by two different monomers should assist in the engineering of envelope proteins for tissue-specific gene delivery.
Multimerization of polyomavirus middle-T antigen
Authors: Senften M , Dilworth S , Ballmer-Hofer K
Published: January 2020

The oncogenic protein of polyomavirus, middle-T antigen, associated with cell membranes and interacts with a variety of cellular proteins involved in mitogenic signalling. Middle-T antigen may therefore mimic the function of cellular tyrosine kinase growth factor receptors, like the platelet-derived growth factor or epidermal growth factor receptor. Growth factor receptor signalling is initiated upon the binding of a ligand to the extracellular domain of the receptor. This results in activation of the intracellular tyrosine kinase domain of the receptor, followed by receptor phosphorylation, presumably as a consequence of dimerization of two receptor molecules. Similar to middle-T antigen, phosphorylation of growth factor receptors leads to recruitment of cellular signalling molecules downstream in the signalling cascade. In this study, we investigated whether middle-T antigen, similar to tyrosine kinase growth factor receptors, is able to form dimeric signalling complexes. We found that association with cellular membranes was a prerequisite for multimerization, most likely dimer formation. A chimeric middle-T antigen carrying the membrane-targeting sequence of the vesicular stomatitis virus G protein instead of the authentic polyomavirus sequence still dimerized. However, mutants of middle-T antigen unable to associate with 14-3-3 proteins, like d18 and S257A, did not form dimers but were still oncogenic. This indicates that both membrane association and binding of 14-3-3 are necessary for dimer formation of middle-T antigen but that only the former is essential for cell transformation.
The critical N-linked glycan of murine leukemia virus envelope protein promotes both folding of the C-terminal domains of the precursor polyprotein and stability of the postcleavage envelope complex
Authors: Li Z , Pinter A , Kayman S C
Published: January 2020

The infectivity of Friend ecotropic murine leukemia virus was previously shown to be highly sensitive to modification in its envelope protein (Env) at only one of the eight signals for N-linked glycan attachment, the fourth from the N terminus (gs4). In the present study, a set of six single-amino-acid substitutions in or near gs4 was used to determine the function of this region of Env and the role played by the glycan itself. One mutant that lacked the gs4 glycan was fully infectious, while one that retained this glycan was completely noninfectious, indicating that the gs4 glycan per se is not required for Env function. Infectivity correlated with the level of mature Env complex incorporated into virus particles, which was determined by the severity of defects in transport of the envelope precursor protein (gPrEnv) from the endoplasmic reticulum into the Golgi apparatus, in cleavage of gPrEnv into the two envelope subunits (the surface protein [SU] and the transmembrane protein [TM]), and in the association of SU with cellular membranes. All of the mutants induced the wild-type level of superinfection interference, indicating that the gs4 region mutations did not interfere with proper folding of the N-terminal domain of SU. These results suggest that the gs4 region mediates folding of the C-terminal domains of gPrEnv and stability of the interaction between SU and TM. Although the gs4 glycan was not essential for infectivity, processing of all mutant Envs lacking this glycan was significantly impaired, suggesting that efficient folding of gPrEnv requires a glycan at this position. The conservation of a glycosylation site homologous to gs4 across a broad range of retroviruses suggests that this sequence may play a similar role in many retroviral Envs.
Induction of human papillomavirus type 18 late gene expression and genomic amplification in organotypic cultures from transfected DNA templates
Authors: Frattini M G , Lim H B , Doorbar J , Laimins L A
Published: January 2020

The genetic analysis of human papillomavirus (HPV) functions during the vegetative viral life cycle is dependent upon the ability to generate human keratinocyte cell lines which maintain episomal copies of transfected viral genomes. We have previously demonstrated that lipofection of normal human foreskin keratinocytes with recircularized cloned HPV-31 genomic sequences resulted in a high frequency of cell lines which maintained viral genomes as extrachromosomal elements (M.G. Frattini, H. Lim, and L.A. Laimins, Proc. Natl. Acad. Sci. USA 93:3062-3067, 1996). Following the growth of these cell lines in organotypic (raft) cultures, the differentiation-dependent expression of viral late genes, the amplification of viral genomes, and virion biosynthesis were observed. In the present study, we demonstrate that these methodologies are not restricted to HPV-31 but are applicable to other HPV types, including the oncogenic HPV-18. HPV-18 genomes were purified from bacterial vector sequences, religated, and transfected into normal human foreskin keratinocytes together with a neomycin-selectable marker. Following drug selection, resistant cells were expanded and examined for the state of the viral DNA. All cell lines examined were found to contain approximately 100 to 200 episomal copies of HPV-18 DNA per cell. Growth of these cell lines in raft cultures resulted in the differentiation-dependent expression of the E1 [symbol: see text] E4 and L1 capsid genes. In addition, viral genome amplification was observed in suprabasal cells following DNA in situ hybridization analysis of differentiated raft cultures. The induction of these late viral functions has previously been shown to be directly associated with differentiation-dependent virion biosynthesis. Our studies indicate the ability to perform a detailed genetic analysis of the various phases of the viral life cycle, including control of the differentiation-dependent late viral functions, using a second oncogenic HPV type.
Potentiation of human immunodeficiency virus type 1 Tat by human cellular proteins
Authors: Greenberg M E , Ostapenko D A , Mathews M B
Published: January 2020

The Tat protein is a potent activator of human immunodeficiency virus type 1 transcription. Tat has been shown to act by increasing both transcription initiation and elongation, but a detailed understanding of its interaction with the transcriptional machinery is lacking. With the aim of isolating cellular proteins that interact with Tat and play a role in transactivation, we have reexamined its function in a cell-free transcription assay. Monitoring the appearance of transactivation after addition of purified Tat at intervals to the reaction mix revealed a lag of approximately 10 min before Tat is able to effect transactivation. Incubation of Tat in nuclear or cytoplasmic extracts of human cells was sufficient to eliminate the lag, but nuclear extract from a rodent cell line was inactive. The accelerating effect of the human cell extract could be abrogated by dilution, heat inactivation, or chromatographic depletion. We infer that Tat is potentiated for transactivation through interaction with a protein factor(s) that is specific to human cells.
Superinfection in Bacteriophage S13 and Determination of the Number of Bacteriophage Particles Which Can Function in an Infected Cell
Authors: Tessman Ethel S. , Borrás Maria-Teresa , Sun Iris L.
Published: January 2020

Bacteriophage S13 shows exclusion of superinfecting homologous phage, but the exclusion is only partial. The superinfecting phage can form infectious replicative form deoxyribonucleic acid (RF), can direct protein synthesis, and can form progeny particles even at a superinfection time as late as 60 min after the first infection. Exclusion is also only partial for the closely related phage φX174. Seven min after the first infection, the exclusion mechanism begins to operate, requiring continuous phage-specified protein synthesis. The gene A protein (required for synthesis of progeny RF) appears to be involved in the exclusion mechanism. In superinfection experiments, it was found that at least 40 phage particles per cell can replicate and can carry out protein synthesis, though the number of sites for binding of RF to the membrane is only about 15 per cell. The results suggest that attachment of RF to a binding site is not required for protein synthesis. Evidence is presented that non-attached parental RF can serve as a template for single-stranded deoxyribonucleic acid synthesis.
Mechanism of Oncogenic Transformation by Rous Sarcoma Virus II. Effect of Rifampin on Rous Sarcoma Virus Infection
Authors: Richert Nancy J. , Balduzzi Piero
Published: January 2020

Rifampin partially inhibits focus formation and virus production in chick embryo fibroblasts infected with Bryan high-titer Rous sarcoma (RAV-1) virus. This inhibition occurs with exposure to rifampin during a critical period between day 1 and day 2 after infection. This suggests that the drug does not affect formation of the provirus or its transcription or translation after the “fixation” step, but it seems to affect one or more events which take place before fixation and activation of virus production.
Enlargement of
Escherichia coli
After Bacteriophage Infection II. Proposed Mechanism
Authors: Freedman M. L. , Krisch R. E.
Published: January 2020

Division of
Escherichia coli
was stopped and mean cellular volume was increased after infection with T-even phage. This host cell enlargement was temperature-dependent, cyanide-sensitive, and stable in the presence of hypertonic medium. Enlargement ceased at about the same time that energy metabolism ceased. Initially, enlargement was accompanied by a decrease in mean cell density. Tritiated 2, 6-diaminopimelic acid was accumulated and incorporated into cold acid-insoluble material at the preinfection rate. These findings suggest that the effect on host cell size is only in part an osmotic phenomenon and that it also reflects continued growth of the surface of the infected cell in the absence of cell division.
Enzymes and Nucleotides in Virions of Rous Sarcoma Virus
Authors: Mizutani Satoshi , Temin Howard M.
Published: January 2020

In addition to the previously described deoxyribonucleic acid (DNA) polymerase, DNA ligase, DNA exonuclease, and DNA endonuclease activities, purified virions of Schmidt-Ruppin strain of Rous sarcoma virus (SRV) have nucleotides and nucleotide kinase, phosphatase, hexokinase, and lactate dehydrogenase activities. The SRV virions have no glucose-6-phosphate dehydrogenase activity. All enzyme activities, but glucose-6-phosphate dehydrogenase and adenosine triphosphatase, were increased by disruption of the virions. The DNA polymerase, DNA ligase, and hexokinase activities had a higher specific activity in purified virion cores. It is suggested that during assembly virions of SRV may pick up cytoplasmic components which bind to virion proteins. The role of these components in viral replication is not known at present.
Spontaneous Virus Production by Clonal Lines of Simian Virus 40-Transformed Cells and Effects of Superinfection by Deoxyribonucleic Acid from Mutant Simian Virus 40 Strains
Authors: Dubbs D. R. , Kit Saul
Published: January 2020

Small amounts of infectious simian virus 40 (SV40) were recovered from parental cultures of SV40-transformed human embryonic lung (WI38 Va13A) cells, from 12 primary clones, from 17 secondary clones, and from 18 tertiary clones. The cloning experiments demonstrated that the capacity for spontaneous virus production is a hereditary property of WI38 Va13A cells. Infectious virus was not recovered from every clone at every passage. Repeated trials at different passage levels were necessary to detect virus production. Approximately one in 10
5
to 10
6
of the cells of the clonal lines initiated plaque formation when plated on the CV-1 line of African green monkey kidney cells. No increase in infectious center formation was observed after the clonal lines were treated with bromodeoxyuridine, iododeoxyuridine, or mitomycin C or after heterokaryon formation of treated cells with CV-1 cells. The clonal lines of WI38 Va13A cells were susceptible to superinfection by SV40 deoxyribonucleic acid (DNA). To determine whether only those cells which spontaneously produced virus supported the replication of superinfecting SV40 DNA, cultures were infected with DNA from a plaque morphology mutant and a temperature-sensitive mutant of SV40. After infection by SV40 DNA, approximately 100 to 4,400 times more transformed cells formed infectious centers than were spontaneously producing virus. To determine whether the resident SV40 genome or the superinfecting SV40 genome was replicating, infectious centers produced by SV40 DNA-infected WI38 Va13A cells on CV-1 monolayers were picked and the progeny virus was analyzed. Only the superinfecting SV40 was recovered from the infectious centers, indicating that in the majority of superinfected cells the resident SV40 was not induced to replicate.
Electron Microscopic Evidence for Linear Insertion of Bacteriophage MU-1 in Lysogenic Bacteria
Authors: Martuscelli J. , Taylor A. L. , Cummings D. J. , Chapman V. A. , DeLong S. S. , Cañedo L.
Published: January 2020

Temperate bacteriophage Mu-1 was used to generate a lysogenic derivative of the F′
lac
episome of
Escherichia coli
. Intact, covalently circular molecules of F′
lac
and lysogenic F′
lac
Mu
+
deoxyribonucleic acid (DNA) were isolated and examined by electron microscopy. The mean contour lengths of F′
lac
and F′
lac
Mu
+
molecules were 37.6 ± 0.4 μm and 53.2 ± 0.4 μm, respectively. The mean difference, 15.6 μm, is similar to the mean contour length of 12.9 ± 0.1 μm obtained for linear DNA molecules released by osmotic shock from mature phage Mu-1 virions. These results provide direct physical evidence that phage Mu-1 integrates by linear insertion of its genome into the DNA of lysogenic host bacteria. Chemical and physical analyses of phage Mu-1 DNA indicate that it is similar to
E. coli
DNA in respect of gross base composition, buoyant density, and melting temperature.
Repair Replication of HeLa Cell Deoxyribonucleic Acid in Cells Infected with Newcastle Disease Virus or Mengovirus or Treated with Puromycin
Authors: Hand Roger , Tamm Igor
Published: January 2020

We examined repair replication of HeLa cell deoxyribonucleic acid (DNA) in cells infected with mengovirus or Newcastle disease virus or treated with puromycin. Cellular DNA was damaged by ultraviolet light and then pulse-labeled with
3
H-thymidine. Autoradiographic analysis of non-S-phase DNA synthesis (repair replication) showed that there was no inhibition of this process at a time when overall cellular DNA synthesis was severely inhibited by either virus infection or puromycin treatment.
Quantitative Binding of
125
I-Concanavalin A to Normal and Transformed Cells
Authors: Arndt-Jovin Donna J. , Berg Paul
Published: January 2020

We have measured the quantitative binding of the radioactively labeled agglutinin
125
I-concanavalin A to normal mammalian cells and simian virus 40- and polyoma virus-transformed cells from tissue culture. Parallel measurements of the amount of
125
I-concanavalin A necessary to cause agglutination of the cells in suspension were carried out. The transformed and nontransformed cells used for these experiments show large differences in their ability to be agglutinated by
125
I-concanavalin A. However, these cell lines have the same number of specific binding sites and similar affinities for the agglutinin whether transformed, trypsinized, or nontransformed. We conclude that the differential capacity of concanavalin A to agglutinate transformed cells relative to normal cells does not result from differences in the number of binding sites between the two types of cells.
Inhibition of Host Deoxyribonucleic Acid Synthesis by T4 Bacteriophage in the Absence of Protein Synthesis
Authors: Duckworth Donna Hardy
Published: January 2020

The requirement for phage protein synthesis for the inhibition of host deoxyribonucleic acid synthesis has been investigated by using a phage mutant unable to catalyze the production of any phage deoxyribonucleic acid. It has been concluded that the major pathway whereby phage inhibit host syntheses requires protein synthesis. The inhibition of host syntheses by phage ghosts is not affected by inhibitors of protein synthesis.
Entry of Vesicular Stomatitis Virus into L Cells
Authors: Heine Jochen W. , Schnaitman Carl A.
Published: January 2020

Early stages of the entry of vesicular stomatitis (VS) virus into L cells were followed by electron microscopy with the aid of ferritin antibody labeling. Cells which were infected at 0 C and incubated for 10 min at 37 C were reacted first with antiviral-antiferritin hybrid antibody and then with ferritin or fluorescein-labeled apoferritin. Extensive ferritin labeling of the cell surface was detected by both electron and fluorescence microscopy. The labeled regions of the cell surface were continuous with and indistinguishable from the rest of the host cell membrane, suggesting incorporation of viral antigens into the cell surface during viral penetration. Fusion of parental viral membrane with host cell membrane was further demonstrated by examining the localization of
3
H-labeled viral structural proteins in cells infected at 0 C and incubated for short periods at 37 C. Viral nucleoprotein was found in a soluble fraction of the cells which was derived primarily from the cytoplasm, whereas a particulate fraction from the cells was enriched in viral envelope proteins. Cytoplasmic membrane was isolated from these cells, and this membrane contained viral envelope proteins. These results suggest that penetration by VS virus occurs by fusion of the viral and cellular membranes followed by release of nucleo-protein into the cytoplasm.
Separation of the Infectious Ribonucleic Acid of Potato Spindle Tuber Virus from Double-Stranded Ribonucleic Acid of Plant Tissue Extracts
Authors: Lewandowski Leon J. , Kimball Paul C. , Knight C. A.
Published: January 2020

The infectious ribonucleic acid (RNA) of potato spindle tuber virus (PSTV) can be separated by hydroxyapatite chromatography from double-stranded RNA detectable in low amounts in both infected and uninfected plant tissue extracts. The chromatographic behavior of ribonuclease-sensitive PSTV RNA resembles that of transfer RNA.
Reinitiation Within One Cell Cycle of the Deoxyribonucleic Acid Synthesis Induced by Simian Virus 40
Authors: Hirai K. , Lehman J. M. , Defendi V.
Published: January 2020

The infection of secondary cultures of Chinese hamster cells with simian virus 40 (SV40) induces the appearance of cells with polyploid deoxyribonucleic acid (DNA) content or chromosomal component within one cell generation. The mechanism of this phenomenon was studied by the use of 5-bromodeoxyuridine (BUdR) incorporation as a DNA density marker. When cultures were treated with
14
C-BUdR and colcemide and harvested at 48 hr postinfection, only hybrid and light DNA molecules were found in control cultures, whereas in infected cultures there were also heavy molecules. The proportion of heavy DNA synthesized during the experimental period varied from 13 to 25%. It was determined by DNA-DNA hybridization that the heavy DNA consisted of cellular DNA. In radioautographic experiments, it was shown that, under the conditions used, a fraction of the infected cell population twice replicated its complete DNA content. Analysis of the kinetics indicated that the heavy DNA resulted from the reinitiation of DNA synthesis after the initial replication of the entire cell DNA. It was concluded that, after infection with SV40, a fraction of the Chinese hamster cell population undergoes two cycles of DNA synthesis without intervening mitosis.
Isolation and Characterization of Temperature-Sensitive Mutants of Vesicular Stomatitis Virus, New Jersey Serotype
Authors: Pringle C. R. , Duncan I. B. , Stevenson Mary
Published: January 2020

Forty-eight temperature-sensitive (
ts
) mutants have been isolated from a wild-type strain of the New Jersey serotype of vesicular stomatitis virus (VSV) after exposure to the base analogue mutagen 5-fluorouracil. Of these mutants, 47 have been classified into 6 nonoverlapping complementation groups containing 21, 17, 4, 3, 2, and 1 mutant, respectively (1 mutant remaining unallocated). The ribonucleic acid (RNA) phenotype of 23 of these mutants has been established. Four of the six groups contain one or more mutants unable to synthesize detectable amounts of viral RNA under restrictive conditions (39 C). No complementation was observed in mixed infection with
ts
mutants from the five established complementation groups of the Indiana serotype of VSV.
Deoxyribonucleic Acid Hybridization Analysis of the Defective Bacteriophage Carried by Strain 15 of
Escherichia coli
Authors: Grady Leo J. , Cowie Dean B. , Campbell Wayne P.
Published: January 2020

Evidence from several laboratories indicates that strain 15 of
Escherichia coli
is lysogenic for a defective phage. When lysates from induced cultures were centrifuged in CsCl, three bands were obtained. In order of decreasing density, these bands contained tailless particles, complete phages, and a second band of complete phages, in a ratio of 65.7:28.6:5.7. Reassociation rate measurements were used to establish that the molecular weights of the deoxyribonucleic acid (DNA) species from the phages in the first two bands are similar. A smaller genome is postulated in the complete phages from the minor band. Hybridization experiments revealed extensive homology between the DNA species from all three phage bands, thus suggesting that the complete and tailless particles are not different at the genetic level. The DNA from each phage band was also shown to hybridize almost completely with DNA from either
E. coli
15T
−
or a reportedly cured derivative of 15T
−
. In contrast, only about 25% of each phage DNA was able to react with DNA from
E. coli
strains B and K-12 C-600.
Superinfection with Bacteriophage T4: Inverse Relationship Between Genetic Exclusion and Membrane Association of Deoxyribonucleic Acid of Secondary Bacteriophage
Authors: Sauri C. J. , Earhart C. F.
Published: January 2020

The majority of the deoxyribonucleic acid (DNA) of superinfecting T4 bacteriophage which is injected and not hydrolyzed does not attach to host cell membrane. Low levels of association of secondary phage DNA with membrane appear to be related to temporal genetic exclusion.
Serological Relationships of
Salmonella
A Bacteriophages Isolated from Salmonellae in Different Kauffmann- White Groups
Authors: Bullas Leonard R. , Nutter Robert L.
Published: January 2020

Six different
Salmonella
group A phages from salmonellae in Kauffmann- White groups B, C
1
, C
2
, and D were examined serologically. Those phages which were specific for a particular somatic antigen were found to be serologically very similar. Antiserum against a phage with one specificity was able to neutralize a different phage with the same specificity but unable to neutralize, in the normal way, a phage with a different specificity. Phages mixed with heterologous phage antiserum responded with an “inhibition response” in which there appeared to be a neutralization of the phage infectivity for the first 10 min, followed by a reversal of the neutralization until, by 20 or 25 min, there was no apparent neutralization. This response was interpreted to indicate that the adsorption antigens, probably situated on the tail fibers, were different for phages with different specificities but sufficiently similar so that heterologous antibodies could react with the antigens; but the antigen-antibody complex was quickly disassociated, resulting in a modification of the antibody molecules but no change in the specificity sites of the antigen. A subgrouping of the
Salmonella
A phages based on their antigenic specificity is suggested.
Pathway of Infection of Mosquito Iridescent Virus
Authors: Stoltz D. B. , Summers M. D.
Published: January 2020

Mosquito iridescent virus (MIV) is ingested in large amounts by first- and second-instar
Aedes taeniorhynchus
larvae without causing a high rate of infection. Electron microscope studies have been undertaken to determine the fate of ingested virus. Preliminary observations suggest that most, if not all, ingested particles are degraded shortly after entering the midgut. MIV and other virus particles employed in this study were apparently unable to penetrate the peritrophic membrane; consequently, none was observed inside, or in contact with, midgut epithelial cells.
Identification of Virus-Induced Proteins in Cells Productively Infected with Simian Virus 40
Authors: Fischer Hans , Sauer Gerhard
Published: January 2020

The number and molecular weight of the structural polypeptides of highly purified simian virus 40 (SV40) were determined by polyacrylamide gel electrophoresis. Six different polypeptides were found, two of which (VP1 and VP2) comprise the bulk of the viral capsid proteins. The pattern of protein synthesis in productively infected CV-1 cells was studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Identification of virus-induced proteins in the infected CV-1 cells was achieved in double-labeling experiments by electrophoresis with purified labeled SV40 capsid proteins. Four of these proteins (VP1 and VP4) could be classified as components of the virion because their synthesis occurred after the onset of viral deoxyribonucleic acid (DNA) replication and because they were inhibited by arabinofuranosylcytosine (ara-C). Appearance of two other virus-induced proteins was not prevented by ara-C; one of them did not comigrate in the electrophoresis with purified virion polypeptides, and both could be detected before the onset of viral DNA synthesis. These latter two proteins were classified on the basis of these criteria as nonvirion capsid proteins (NCVP1 and NCVP2).
Effect of Nalidixic Acid on the Growth of Deoxyribonucleic Acid Bacteriophages
Authors: Baird Judith P. , Bourguignon Gerard J. , Sternglanz Rolf
Published: January 2020

The effect of nalidixic acid on the growth of various deoxyribonucleic acid (DNA) bacteriophages has been investigated by one-step growth experiments. The
Escherichia coli
bacteriophages T5, λ, T7 and φR are strongly inhibited by nalidixic acid, whereas T4 and T2 are only partially inhibited. The
Bacillus subtilis
bacteriophages SP82, SP50, and φ29 are relatively unaffected by nalidixic acid. There is no correlation between those bacteriophages which can grow in the presence of nalidixic acid and the presence of an unusual base in the phage DNA.
Early Interaction of Rhinoviruses with Host Cells
Authors: Lonberg-Holm K. , Korant B. D.
Published: January 2020

The rate of attachment of type 2 virions to suspensions of HeLa cells is much greater than that of type 14, but the number of receptor sites per cell is similar for each type. The receptor sites may be partly saturated with excess virions; attachment is greatly reduced after about 10
4
particles have been taken up per cell. A lack of saturation of type 14 receptors by excess type 2 indicates that their receptor sites are separate on the cell surface. Excess of type 2 blocks attachment of type 1A, however, and excess of type 14 blocks type 51. Attachment of the human rhinoviruses is temperature-dependent with a Q
10
of 2.7. The eclipse reaction is also temperature-dependent. At 34.5 C, the irreversible eclipse of cell-associated rhinovirus type 2 requires only a few minutes, whereas the rate of eclipse of cell-associated type 14 is considerably slower. The eclipse product of type 2 rhinovirus has been recovered from infected cells. It sediments at about 90% of the rate of the infective virions and is missing virus polypeptide 4 (the smallest of the capsid polypeptides). Upon being subjected to CsCl gradient centrifugation, virus polypeptide 2 is also lost but the product still contains ribonucleic acid and bands at about 1.45 g/cc.
Binding of Deoxyribonucleic Acid-Dependent Deoxyribonucleic Acid Polymerase to Poxvirus
Authors: Tan K. B. , McAuslan B. R.
Published: January 2020

Poxvirus has a deoxyribonucleic acid polymerase activity that remains associated with the virus despite repeated centrifugation through sucrose gradients. Highly purified poxvirus preparation can adsorb deoxyribonucleic acid polymerase from cytoplasmic extracts of cells containing such an activity. These results indicate that caution must be used in assuming that an enzyme associated with a purified virus is necessarily an integral part of the virion.
Inhibition of Protein Synthesis in L Cells Infected with Vesicular Stomatitis Virus
Authors: Wertz Gail W. , Youngner Julius S.
Published: January 2020

The inhibition of protein synthesis in L cells by vesicular stomatitis virus (VSV) requires the synthesis of new protein subsequent to virus infection. However, two mechanisms may be involved in the inhibition of cell protein synthesis by VSV: an initial, multiplicity-dependent, ultraviolet-insensitive inhibition and a progressive, ultraviolet-sensitive inhibition.