bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA-binding activity of papillomavirus proteins
Authors: Mallon R G , Wojciechowicz D , Defendi V
Published: January 2020

We demonstrate DNA binding by papillomavirus (PV) open reading frame (ORF) proteins that correspond to the early transforming and trans-activating (E6 and E2) and late structural regions (L2 and L1) from bovine PV type 1 and human PV types 6b and 16. All PV proteins were synthesized in Escherichia coli and had a common 13-amino-acid leader sequence from the expression vector pRA10. Antibodies have been generated in rabbits against these PV proteins. The PV ORF proteins bind double-stranded DNA, and this activity is demonstrated to be inherent to the PV proteins. DNA-binding activity by PV proteins is optimal at 50 mM NaCl and at pH 7.0. For some PV proteins (e.g., bovine PV type 1 E2), DNA binding is enhanced at a lower pH (pH 6.0) and NaCl concentration (50 to 100 mM). DNA binding is inhibited by the appropriate antibodies. The possible significance of these findings is discussed in relation to the genetic and structural evidence on the function of these ORFs.
The membrane-binding domain and myristylation of p60v-src are not essential for stimulation of cell proliferation
Authors: Calothy G , Laugier D , Cross F R , Jove R , Hanafusa T , Hanafusa H
Published: January 2020

Previous studies showed that the amino-terminal domain of Rous sarcoma virus p60v-src involved in myristylation and membrane association of the protein is required for morphological transformation and anchorage independence. Analysis of src delection mutants revealed that the amino-terminal one-third of p60v-src, including the membrane-binding domain, is not essential for induction of cell proliferation. These results demonstrated that, in contrast to the cellular target(s) involved in morphological transformation and anchorage independence, the target(s) involved in mitogenic activity is accessible to nonmyristylated src proteins.
Structure and function of the transcriptional control region of nonpassaged BK virus
Authors: Rubinstein R , Pare N , Harley E H
Published: January 2020

We compared the nucleotide sequence in the transcriptional control region of BK virus isolates cloned directly from human urine (BK-WW) with that of prototype BK virus. BK-WW was found to have a 63-base-pair insertion and only one of the 68-base-pair enhancer repeat elements. In transient expression assays, BK-WW enhancer showed approximately one-half the activity given by the prototype enhancer.
Expression of the Epstein-Barr virus gp350/220 gene in rodent and primate cells
Authors: Whang Y , Silberklang M , Morgan A , Munshi S , Lenny A B , Ellis R W , Kieff E
Published: January 2020

The gene encoding the Epstein-Barr virus envelope glycoproteins gp350 and gp220 was inserted downstream of the cytomegalovirus immediate-early, Moloney murine leukemia virus, mouse mammary tumor virus, or varicella-zoster virus gpI promoters in vectors containing selectable markers. Host cell and recombinant vector systems were defined which enabled the isolation of rodent or primate cell clones which expressed gp350/220 in substantial quantities. Continued expression of gp350/220 required maintenance of cells under positive selection for linked markers and periodic cloning. gp350/220 expressed in various host cells varied slightly in electrophoretic mobility, probably reflecting differences in glycosylation. Insertion of a stop codon into the gp350/220 open reading frame, upstream of the putative membrane anchor sequence, resulted in efficient secretion of truncated gp350 and gp220 from rat pituitary (GH3) cells. gp350/220 expressed in mammalian cells is highly immunogenic and elicits virus-neutralizing antibodies when administered to mice.
Effectiveness of topically administered neutralizing antibodies in experimental immunotherapy of respiratory syncytial virus infection in cotton rats
Authors: Prince G A , Hemming V G , Horswood R L , Baron P A , Chanock R M
Published: January 2020

Initial studies of the prophylactic effect of parenterally administered respiratory syncytial virus (RSV)-neutralizing antibodies in cotton rats indicated that virus replication in lung tissues was restricted when animals with preexisting antibody titers in serum of 1:100 or more (as measured by plaque reduction) were challenged intranasally with 10(4) PFU of virus. Subsequently, a therapeutic effect of parenterally administered RSV antibodies (present in human gamma globulin) was demonstrated in both cotton rats and owl monkeys. Parenteral inoculation of RSV-infected cotton rats or owl monkeys with purified human immunoglobulin licensed for intravenous administration in humans (IVIG) effected a 10(-1.7) to 10(-2.7) reduction in the level of pulmonary virus at the height of infection. Because of these encouraging results, we examined topical administration of IVIG to determine whether it was also effective and whether it offered an advantage over the parenteral route with regard to simplicity and the dose required for full therapeutic effect. IVIG (0.025 g/kg) administered topically by the intranasal route to anesthetized cotton rats at the height of RSV infection effected a 10(2.2)-fold reduction in viral titers of pulmonary tissues and a complete clearance of detectable virus in 92% of the animals within 24 h. In contrast, 4 g of IVIG per kg was required to produce a comparable therapeutic effect when the material was administered parenterally. Thus, the therapeutic effect of IVIG was 160 times greater by the topical route than by parenteral inoculation.
Mechanism of recovery from acute virus infection: treatment of lymphocytic choriomeningitis virus-infected mice with monoclonal antibodies reveals that Lyt-2+ T lymphocytes mediate clearance of virus and regulate the antiviral antibody response
Authors: Moskophidis D , Cobbold S P , Waldmann H , Lehmann-Grube F
Published: January 2020

After intravenous infection of mice, lymphocytic choriomeningitis virus multiplied in spleens and livers, attaining highest concentrations on days 4 to 6. The subsequent clearance was as rapid, and 8 to 10 days after inoculation, infectivity was usually below detectability. During the effector phase of virus elimination, both cytotoxic T-cell (CTL) activity and the number of cells producing antiviral antibodies were high. Monoclonal antibodies directed against T lymphocytes and T-lymphocyte subsets were inoculated once intravenously 5, 6, or 7 days after infection of the animals, and the effects on antiviral immune responses, as well as on elimination of virus from the organs, were determined. Treatment with anti-Thy-1 and anti-Lyt-2 antibodies blocked elimination of the virus and profoundly diminished the activity of spleen CTLs but reduced the antibody response partially (anti-Thy-1) or increased it (anti-Lyt-2). In contrast, treatment with the anti-L3T4 antibody had essentially no effect on either virus elimination or CTL response but abolished antibody production. We conclude that Lyt-2+ (cytotoxic-suppressive) T lymphocytes are needed for elimination of the virus and also regulate the humoral response but that antiviral antibodies are not essential for control of the infection.
Comparison of the relative roles of the F and HN surface glycoproteins of the paramyxovirus simian virus 5 in inducing protective immunity
Authors: Paterson R G , Lamb R A , Moss B , Murphy B R
Published: January 2020

To compare the relative roles of the paramyxovirus simian virus 5 (SV5) major surface glycoproteins, fusion (F) and hemagglutinin-neuraminidase (HN), in inducing protective immunity, two recombinant vaccinia viruses were constructed. The F and HN polypeptides expressed by the recombinant viruses were indistinguishable from their authentic SV5 counterparts in electrophoretic mobility, glycosylation, and, for the F protein, cleavage of the precursor, F0, to the disulfide-linked subunits F1 and F2. Injection of rabbits and hamsters with live recombinant virus elicited an antibody response to either F or HN and provided a source of monospecific polyclonal antisera to the SV5 proteins. The vaccinia virus-SV5 F (vaccinia-F) recombinant induced higher levels of neutralizing antibody than did the vaccinia-HN recombinant, but animals inoculated with vaccinia-HN were better protected from challenge with SV5. Animals infected with both the vaccinia-HN and vaccinia-F viruses were nearly as well protected from challenge as were animals infected with SV5.
Two strains of Epstein-Barr virus (B95-8 and a P3HR-1 subclone) that lack defective genomes induce early antigen and cause abortive infection of Raji cells
Authors: Lin J C , Raab-Traub N
Published: January 2020

The heterogeneity of Epstein-Barr virus (EBV) obtained from P3HR-1 cells has permitted derivation of a distinct subclone of P3HR-1 (L. Heston, M. Rabson, N. Brown, and G. Miller, Nature (London) 295:160-163, 1982). We have analyzed the biologic properties and genomic structure of this subclonal virus (clone 13) compared with those of parental P3HR-1 and B95-8 viruses. Synthesis of EBV compared with those of parental P3HR-1 and B95-8 viruses. Synthesis of EBV proteins in Raji cells superinfected with virus derived from P3HR-1, clone 13, and B95-8 was analyzed both by fluorography of radiolabeled proteins and by immunoblotting. Highly concentrated preparations of clone 13 and B95-8 virus induced most of the spectrum of EBV proteins in Raji cells with the exception of the 145,000-, 140,000-, and 110,000-molecular-weight proteins, which were either undetectable or reduced. Moreover, both clone 13 and B95-8 viruses also induced the same patterns of early antigen diffuse components as the parental P3HR-1 virus did. However, only P3HR-1 virus could induce EBV DNA synthesis in superinfected Raji cells, as determined both by buoyant density centrifugation and by in situ cytohybridization with biotinylated recombinant EBV DNA probes. Defective heterogeneous molecules present in P3HR-1 virus have been implicated in early antigen induction after superinfection of Raji cells. Therefore, Southern blots of clone 13, P3HR-1, and B95-8 viruses were hybridized to recombinant EBV fragments representing the sequences contained within the defective molecules in P3HR-1. The parental P3HR-1 contained the previously described defective molecules. No evidence for defective molecules was found in clone 13 or B95-8 viruses. These data indicate that concentrated preparations of both clone 13 and B95-8 viruses can induce abortive infection in Raji cells, but while the defective molecules are not needed for induction of early antigen diffuse components, they may be required for the induction of viral DNA synthesis.
Avian sarcoma and leukosis virus pol-endonuclease recognition of the tandem long terminal repeat junction: minimum site required for cleavage is also required for viral growth
Authors: Cobrinik D , Katz R , Terry R , Skalka A M , Leis J
Published: January 2020

Integration of retroviral DNA is a site-specific reaction involving an endonuclease encoded by the viral pol gene (pol-endo). In vitro the pol-endo from avian sarcoma and leukosis viruses (ASLVs) cleaves both DNA strands near the U5-U3 junction of tandem long terminal repeats (LTR-LTR junction) in single-stranded and replicative form (RF)-I substrates. We have reported previously that the sequences that are required for cleavage of single-stranded substrates by the alpha beta form of the pol-endo differ for the plus and minus strands (G. Duyk, M. Longiaru, D. Cobrinik, R. Kowal, P. deHaseth, A. M. Skalka, and J. Leis, J. Virol. 56:589-599, 1985). This is not the case with RF-I substrates, in which a maximum of 22 base pairs of U5 and 8 base pairs of U3 were required for alpha beta pol-endo cleavage in each strand. Insertion of a palindromic octanucleotide (CATCGATG) at the LTR-LTR junction abolished cleavage in RF-I but not in single-stranded DNA substrates. Deletion of the four nucleotides (TTAA) at the junction prevented cleavage in the plus strand of RF-I DNA, but did not affect cleavage of single-stranded DNA. Furthermore, the alpha beta form of ASLV pol-endo did not recognize heterologous LTR-LTR junction sequences from the reticuloendotheliosis virus or Moloney murine leukemia virus in either substrate form, despite their sequence and structural similarities to the ASLV junction. These results support a role for a sequence-specific interaction between the ASLV pol-endo and the LTR-LTR junction domains that are required for cleavage. By using the infectious Rous sarcoma virus clone pATV8-K, we introduced a set of deletions into the U5 region that would be incorporated into the LTR-LTR junction on viral replication. In the unintegrated provirus, the deletions started 43 base pairs from the LTR-LTR junction and extended various lengths toward the junction. Results of transfection studies with these clones indicated that the U5 sequences that are required for virus production in vivo correspond to those that are required for cleavage of RF-I DNA in vitro.
Relationship between simian virus 40 large tumor antigen expression and tumor formation in transgenic mice
Authors: Van Dyke T A , Finlay C , Miller D , Marks J , Lozano G , Levine A J
Published: January 2020

A line of transgenic mice containing the simian virus 40 (SV40) large tumor antigen gene under the control of the viral enhancer-promoter expressed this viral protein in the brains of these mice within the first 2 weeks after birth. Multiple foci of anaplastic cells formed in the choroid plexuses of these mice at 36 to 41 days after birth, and normal tissue coexisted with these transformed foci. Immunoperoxidase staining to detect the SV40 T antigen showed tumor-specific expression of nuclear T antigen at late times in tumor development, approximately 90 to 100 days and thereafter. The level of SV40 T antigen, on a per cell basis, appeared to be lower in the great majority of choroid plexus cells at earlier times in tumor development. These results suggest that low levels of tumor antigen (14 to 36 days) are present before detectable pathology (36 to 41 days) and the level of T antigen per cell is higher in rapidly growing late-stage tumors (older than 90 days).
Localization of Escherichia coli RNA polymerase-binding sites on bacteriophage S13 replicative form I DNA by protection of restriction enzyme cleavage sites
Authors: Ringuette M , Spencer J H
Published: January 2020

Protection of restriction endonuclease cleavage sites by Escherichia coli RNA polymerase bound to the replicative form I of bacteriophage S13 DNA has been used to identify a number of regions of RNA polymerase binding. Digestion with HincII, AluI, HinfI, or HaeIII, under conditions optimized for "open" complex formation, revealed 12 regions of RNA polymerase binding. Based on differential salt sensitivities, five of the regions were classified as strong or tight binding sites. These were located before genes A (two sites), B, and D and at the 5' end of gene F. The seven regions which exhibited weaker binding were located at the 5' end of gene C (two sites), in the middle of gene D, just before and at the 3' end of gene F, at the 5' end of gene G, and in the middle of gene H. The sites before genes B and D coincide with sites previously identified as promoters in bacteriophage phi X174. One of the sites before gene A, that at nucleotides 5175-5211, represents a new putative promoter site in bacteriophage S13 and phi X174 located before the previously identified A gene promoter at nucleotides 10-45.
Two phosphorylated subclasses of polyomavirus large T antigen that differ in their modes of association with the cell nucleus
Authors: Humphrey G W , Pigiet V
Published: January 2020

Two classes of polyomavirus large T antigen were distinguished, differing in their modes of association with the cell nucleus. A weakly associated class, the nucleoplasmic T antigen, representing 30 to 40% of the total, was solubilized when cells were lysed isotonic buffer at pH 7.2. A more tightly bound class retained in isolated nuclei, the retained T antigen, was extractable either at pH 9.0 or in 2 M NaCl. The retained T antigen contained an additional mole of phosphate, 4 mol of PO4 per mol of T antigen, compared with the nucleoplasmic T antigen (3 mol of PO4 per mol of T antigen). Limit digestion with staphylococcal V8 protease yielded equivalent amounts of five peptides ranging in size from 7.5 to 20 kilodaltons. Additional phosphorylation within a 12-kilodalton peptide accounted for most of the difference in phosphate content between retained and nucleoplasmic T-antigen classes.
High-frequency changes in transcriptional activity in polyomavirus-transformed cell lines
Authors: Bouchard L , Mathieu F , Bastin M
Published: January 2020

We applied the Luria and Delbruck fluctuation test to analyze high-frequency changes in the phenotype of rat cells transformed by a plasmid carrying the polyomavirus middle T (pmt) gene. All of the transformed cell lines analyzed were capable of switching to the normal state with rates ranging from 10(-3) to 10(-2) per cell per generation. Analysis of both middle T antigen and middle T transcripts indicated that the reversion occurred by a mechanism involving a transcriptional block of the pmt locus. Cell lines containing two separate loci reverted with a lower rate, suggesting that phenotypic switching in these cells involved two independent events affecting each locus. The flat revertants mutated to the transformed state with rates in the range of 10(-5) to 5 X 10(-5) per cell per generation. To determine whether changes in pmt expression would affect neighboring sequences, we transfected a hybrid plasmid carrying pmt linked to the neo marker and selected either for morphological transformants or for G418-resistant cells. Although their coordinate regulation was not absolute, both genes were usually subject to the same changes, reflected by loss and reacquisition of transcriptional activity.
The viral protein sigma 3 participates in translation of late viral mRNA in reovirus-infected L cells
Authors: Lemieux R , Lemay G , Millward S
Published: January 2020

Reovirus late (uncapped) mRNA was previously shown to be efficiently translated in vitro extracts prepared from infected cells but not from uninfected cells. We demonstrated that different fractions from infected cells can stimulate translation of late viral mRNA when added to uninfected extracts. The activity of the different fractions correlated with their relative content of the sigma 3 capsid protein; the fraction prepared by high-salt wash of the ribosomes had the highest specific activity. The activity present in this fraction was abolished by preincubation with an anti-sigma 3 serum. Purified sigma 3 protein also stimulated the translation of late viral mRNA, confirming that it was the factor involved. Altogether, these results suggest that this protein plays the role of a late-viral-mRNA-specific initiation factor. The absence of an inhibitory effect of sigma 3 on the translation of other mRNAs indicates that this protein is not directly involved in the inhibition of host and early viral mRNA translation that occurs in infected cells but that a second mechanism is probably operative.
Rescue of type I collagen-deficient phenotype by retroviral-vector-mediated transfer of human pro alpha 1(I) collagen gene into Mov-13 cells
Authors: Stacey A , Mulligan R , Jaenisch R
Published: January 2020

A full-length cDNA clone corresponding to the human pro alpha 1(I) collagen gene was isolated and inserted into a retrovirus vector. Cell lines were obtained which produced recombinant viruses transducing the collagen cDNA (HUC virus). To test whether the transduced cDNA was functional, Mov-13 mouse cells were infected with the virus. These cells do not produce any type I collagen due to an insertional mutation of the pro alpha 1(I) gene which blocks transcription. While normal amounts of pro alpha 2(I) RNA were synthesized, no alpha 2(I) collagen chains were detectable in the mutant Mov-13 cells. Infection with HUC virus, however, resulted in the production of stable type I collagen, which was secreted into the medium. Analysis of pepsin-resistant proteins indicated that interspecies heterotrimers consisting of human alpha 1(I) and mouse alpha 2(I) collagen chains were secreted by the infected Mov-13 cells. Our results show that pro alpha (I) collagen chains from species as distant as human and mouse can associate to form stable type I collagen. The availability of a retrovirus vector transducing a functional pro alpha 1(I) collagen gene combined with the Mov-13 mutant system should enable us to study the effect of specific mutations on the synthesis, assembly, and function of type I collagen, not only in tissue culture but also in the animal.
Two distinct sequence elements mediate retroviral gene expression in embryonal carcinoma cells
Authors: Weiher H , Barklis E , Ostertag W , Jaenisch R
Published: January 2020

Moloney murine leukemia virus (M-MuLV) and M-MuLV-derived retroviral vectors are not expressed in early mouse embryos or in embryonal carcinoma cells. M-MuLV-derived mutants or M-MuLV-related variants which transduce the neomycin phosphotransferase gene can, however, induce drug resistance in embryonal carcinoma cells with high efficiency. In this study we investigated the sequences critical for retroviral gene expression in two different embryonal carcinoma cell lines, F9 and PCC4. We show that two synergistically acting sequence elements mediate expression in embryonal carcinoma cells. One of these is located within the U3 region of the viral long terminal repeat, and the second one is in the 5' untranslated region of the retrovirus. The latter element, characterized by a single point mutation, affects the level of stable RNA in infected cells, suggesting a regulatory mechanism similar to that of human immunodeficiency virus in human T cells.
Role of pseudorabies virus glycoprotein gI in virus release from infected cells
Authors: Mettenleiter T C , Schreurs C , Zuckermann F , Ben-Porat T
Published: January 2020

The Bartha vaccine strain of pseudorabies virus has a deletion in the short unique (Us) region of its genome which includes the genes that code for glycoproteins gI and gp63 (E. Petrovskis, J. G. Timmins, T. M. Gierman, and L. E. Post, J. Virol. 60:1166-1169, 1986). Restoration of an intact Us to the Bartha strain enhances its ability to be released from infected rabbit kidney cells and increases the size of the plaques formed on these cells (T. Ben-Porat, J. M. DeMarchi, J. Pendrys, R. A. Veach, and A. S. Kaplan, J. Virol. 57:191-196, 1986). To determine which gene function plays a role in virus release from rabbit kidney cells, deletions were introduced into the genomes of both wild-type virus and the "rescued" Bartha strain (Bartha strain to which an intact Us had been restored) that abolish the expression of either the gI gene alone or both gI and gp63 genes. The effect of these deletions on the phenotype of the viruses was studied. Deletion mutants of wild-type virus defective in either gI or gI and gp63 behave like wild-type virus with respect to virus release and plaque size on rabbit kidney cells. Deletion of gI from the rescued Bartha strain, however, strongly affects virus release and causes a decrease in plaque size. We conclude that gI affects virus release but that at least one other viral function also affects this process. This function is defective in the Bartha strain but not in wild-type virus; in its absence gI is essential to efficient release of the virus from rabbit kidney cells.
Multistage Friend erythroleukemia: independent origin of tumor clones with normal or rearranged p53 cellular oncogenes
Authors: Chow V , Ben-David Y , Bernstein A , Benchimol S , Mowat M
Published: January 2020

The erythroleukemia induced by Friend virus complex in adult mice is a multistage malignancy characterized by the emergence, late in the disease, of tumorigenic cell clones. We have previously shown that a significant proportion of these clones have unique rearrangements in their cellular p53 oncogene. The clonal relationships among Friend tumor cells isolated in the late stages of Friend erythroleukemia were analyzed by examining the unique integration site of Friend murine leukemia virus and the unique rearrangement in their cellular p53 oncogene. The majority of clones isolated from individual mice infected with Friend virus were clonally related as judged by the site of Friend murine leukemia virus integration. However, Southern gel analysis of DNA from individual Friend cell clones indicated that all of the clones with a normal p53 gene from the same mice were clonally related, but were unrelated to the Friend cell lines with a rearranged p53 gene. These results suggest that Friend tumor cells with rearrangements in their p53 gene arise as the result of a unique transformation event, rather than by progression from already existing tumor cells with a normal p53 gene. They also suggest that such rearrangements in the p53 gene confer a strong selective advantage to these cells in vivo.
Temperature-sensitive cellular mutant for expression of mRNA from murine retrovirus
Authors: Inoue H , Kizaka S , Yutsudo M , Hakura A
Published: January 2020

The cellular mutant B812 isolated from a Fisher rat cell line shows temperature sensitivity of focus formation induced by various retroviruses such as recombinant murine retrovirus containing the middle T gene of polyomavirus (PyMLV), Kirsten murine sarcoma virus, Moloney murine sarcoma virus, and recombinant murine retrovirus containing the src gene of Rous sarcoma virus. B812 cells, however, show normal ability to proliferate and synthesize protein at the nonpermissive temperature, suggesting that their mutation is in a gene specifically concerned with the process of transformation by retroviruses. In this work, experiments with hybrids of mutant and wild-type cells showed that the temperature-dependent defect of this mutant was complemented by wild-type cells. To determine the step of transformation that is restricted at the nonpermissive temperature in B812, we examined the expressions of the oncogene (middle T antigen) in no. 7 (wild-type cells) and B812 cultures infected with PyMLV (the chimeric retrovirus containing the middle T gene of polyomavirus) at the permissive and nonpermissive temperatures. Middle T-associated protein kinase activity, the expression of middle T antigen, and PyMLV-specific mRNA were reduced at the nonpermissive temperature in B812 cultures infected with PyMLV. However, integration of PyMLV into the chromosomal DNA of the mutant was not affected at the nonpermissive temperature. These results suggest that B812 cells have a mutation affecting the expression of viral mRNAs from integrated proviral DNA at the nonpermissive temperature.
A human cytomegalovirus function inhibits replication of herpes simplex virus
Authors: Cockley K D , Shiraki K , Rapp F
Published: January 2020

Human embryonic lung (HEL) cells infected with human cytomegalovirus (HCMV) restricted the replication of herpes simplex virus type 1 (HSV-1). A delay in HSV replication of 15 h as well as a consistent, almost 3 log inhibition of HSV replication in HCMV-infected cell cultures harvested 24 to 72 h after superinfection were observed compared with controls infected with HSV alone. Treatment of HCMV-infected HEL cells with cycloheximide (100 micrograms/ml) for 3 or 24 h, conditions known to result in accumulation of HCMV immediate-early and early mRNA, was demonstrated effective in blocking HCMV protein synthesis, as shown by immunoprecipitation with HCMV antibody-positive polyvalent serum. Cycloheximide treatment of HCMV-infected HEL cells and removal of the cycloheximide block before superinfection inhibited HSV-1 replication more efficiently than non-drug-treated superinfected controls. HCMV DNA-negative temperature-sensitive mutants restricted HSV as efficiently as wild-type HCMV suggesting that immediate-early and/or early events which occur before viral DNA synthesis are sufficient for inhibition of HSV. Inhibition of HSV-1 in HCMV-infected HEL cells was unaffected by elevated temperature (40.5 degrees C). However, prior UV irradiation of HCMV removed the block to HSV replication, demonstrating the requirement for an active HCMV genome. HSV-2 replication was similarly inhibited in HCMV-infected HEL cells. However, replication of adenovirus, another DNA virus, was not restricted in these cells under the same conditions. Superinfection of HCMV-infected HEL cells with HSV-1 labeled with [3H]thymidine provided evidence that the labeled virus could penetrate to the nucleus of cells after superinfection. Evidence for penetration of superinfecting HSV into HCMV-infected cells was also provided by blot hybridization of HSV DNA synthesized in cells infected with HSV alone versus superinfected cell cultures at 0 and 48 h after superinfection. In addition, superinfection with vesicular stomatitis virus ruled out a role for interferon in restriction of HSV replication in this system.
Induction of the heat shock regulon of Escherichia coli markedly increases production of bacterial viruses at high temperatures
Authors: Wiberg J S , Mowrey-McKee M F , Stevens E J
Published: January 2020

Production of bacteriophages T2, T4, and T6 at 42.8 to 44 degrees C was increased from 8- to 260-fold by adapting the Escherichia coli host (grown at 30 degrees C) to growth at the high temperature for 8 min before infection; this increase was abolished if the host htpR (rpoH) gene was inactive. Others have shown that the htpR protein increases or activates the synthesis of at least 17 E. coli heat shock proteins upon raising the growth temperature above a certain level. At 43.8 to 44 degrees C in T4-infected, unadapted cells, the rates of RNA, DNA, and protein synthesis were about 100, 70, and 70%, respectively, of those in T4-infected, adapted cells. Production of the major processed capsid protein, gp23, was reduced significantly more than that of most other T4 proteins in unadapted cells relative to adapted cells. Only 4.6% of the T4 DNA made in unadapted cells was resistant to micrococcal nuclease, versus 50% in adapted cells. Thus, defective maturation of T4 heads appears to explain the failure of phage production in unadapted cells. Overproduction of the heat shock protein GroEL from plasmids restored T4 production in unadapted cells to about 50% of that seen in adapted cells. T4-infected, adapted E. coli B at around 44 degrees C exhibited a partial tryptophan deficiency; this correlated with reduced uptake of uracil that is probably caused by partial induction of stringency. Production of bacteriophage T7 at 44 degrees C was increased two- to fourfold by adapting the host to 44 degrees C before infection; evidence against involvement of the htpR (rpoH) gene is presented. This work and recent work with bacteriophage lambda (C. Waghorne and C.R. Fuerst, Virology 141:51-64, 1985) appear to represent the first demonstrations for any virus that expression of the heat shock regulon of a host is necessary for virus production at high temperature.
Vesicular stomatitis virus in Drosophila melanogaster cells: regulation of viral transcription and replication
Authors: Blondel D , Petitjean A M , Dezélée S , Wyers F
Published: January 2020

Vesicular stomatitis virus RNA synthesis was investigated during the establishment of persistent infection in Drosophila melanogaster cells. The transcription rate declined as early as 5 h after infection and was strongly inhibited after 7 h, leading to a decrease in viral mRNA levels and in viral protein synthesis rates. Full-length plus-strand antigenomes and minus-strand genomes were detected after a 3-h lag time and accumulated until 15 h after infection. Short encapsidated plus-strand molecules were also generated corresponding to the 5' end of viral defective antigenomes. Assembly and release of virions were not restricted, but their infectivity was extremely reduced. In persistently infected cells, an equilibrium was reached where the level of intracellular genomes maintained was constant and maximal even after the rate of all viral syntheses had decreased. These results are discussed with regard to the establishment of persistent infection.
Identification and gene mapping of a 14,700-molecular-weight protein encoded by region E3 of group C adenoviruses
Authors: Tollefson A E , Wold W S
Published: January 2020

Early region E3 of adenovirus type 5 should encode at least nine proteins as judged by the DNA sequence and the spliced structures of the known mRNAs. Only two E3 proteins have been proved to exist, a glycoprotein (gp19K) and an 11,600-molecular-weight protein (11.6K protein). Here we describe an abundant 14.7K protein coded by a gene in the extreme 3' portion of E3. To identify this 14.7K protein, we constructed a bacterial vector which synthesized a TrpE-14.7K fusion protein, then we prepared antiserum against the fusion protein. This antiserum immunoprecipitated the 14.7K protein from cells infected with adenovirus types 5 and 2, as well as with a variety of E3 deletion mutants. Synthesis of the 14.7K protein correlated precisely with the presence or absence of the 14.7K gene and with the synthesis of the mRNA (mRNA h) which encodes the 14.7K protein. The 14.7K protein appeared as a triplet on immunoprecipitation gels and Western blots (immunoblots).
Multiple endogenous xenotropic and mink cell focus-forming murine leukemia virus-related transcripts are induced by polyclonal immune activators
Authors: Krieg A M , Khan A S , Steinberg A D
Published: January 2020

Northern (RNA) analyses were used to study the kinetics of induction of endogenous mink cell focus-forming (MCF) and xenotropic murine leukemia virus (MuLV)-related sequences in NFS and C57BL/6 mice injected with the polyclonal immune activators lipopolysaccharide (LPS), concanavalin A, and 8-bromoguanosine. All three mitogens induced 8.4-, 7.2-, 3.0-, and 1.8-kilobase (kb) MCF-related transcripts coordinately in the spleens of injected mice. Xenotropic MuLV-related expression was also rapidly induced in spleens by the three polyclonal immune activators, but in a noncoordinate manner: a distinct set of transcripts with different kinetics of expression was induced by each mitogen. MCF-related induction after LPS injection was both rapid and sustained; it began within 30 min and persisted for at least 8 days in the spleens of both NFS and C57BL/6 mice. LPS also caused prolonged induction of xenotropic transcripts in spleens of C57BL/6 but not NFS mice. The gld mutation, which causes polyclonal immune activation, induced 8.4-, 10.0-, and 13-kb MCF-related transcripts in C3H/HeJ mice without altering expression of 7.2-, 5.6-, 4.0-, 3.0-, or 1.8-kb MCF-related transcripts. The data demonstrate that individual endogenous MuLV-related transcripts can be induced coordinately or independently and suggest that expression of these transcripts is linked to early stages of lymphocyte activation.
Infectious in vitro transcripts from cowpea chlorotic mottle virus cDNA clones and exchange of individual RNA components with brome mosaic virus
Authors: Allison R F , Janda M , Ahlquist P
Published: January 2020

Complete cDNA copies of genomic RNA1, RNA2, and RNA3 of cowpea chlorotic mottle virus (CCMV) were cloned 1 base downstream from a T7 RNA polymerase promoter. The mixture of capped in vitro transcripts from all three clones produced normal CCMV infections in barley protoplasts and cowpea plants. By using transcripts from these clones and from a similar set of biologically active clones of the related brome mosaic virus (BMV), all possible single component exchanges between the BMV and CCMV tripartite genomes were tested. Viral RNA replication was not observed with any heterologous combination of RNA1 and RNA2, which encode trans-acting viral RNA replication factors. However, substitution of the heterologous RNA3 into either genome produced viable hybrid viruses, both of which replicated in barley protoplasts and produced lesions on Chenopodium hybridum, a local lesion host for both parent viruses. In hybrid infections, BMV and CCMV coat proteins each readily packaged RNAs from the heterologous virus, but BMV RNAs were replicated to a higher level than CCMV RNAs, even when trans-acting RNA replication factors were provided by CCMV genes. Neither hybrid systemically infected the natural host of either parent virus, suggesting that host specificity determinants in BMV and CCMV are encoded by RNA3 and at least one other genomic RNA.
A retroviral RNA secondary structure required for efficient initiation of reverse transcription
Authors: Cobrinik D , Soskey L , Leis J
Published: January 2020

Genetic evidence is presented which suggests the existence of an important structural element in the 5' noncoding region of avian retrovirus RNA. The proposed structure, which we term the U5-leader stem, is composed of sequences in the middle of U5 and in the leader, flanking the primer-binding site. U5 and leader mutations which would disrupt this structure caused a partial replication defect. However, nucleotide substitutions in the leader, which would structurally compensate for a U5 deletion mutation, restored normal replication. Analysis of replication intermediates of viruses with the above mutations suggests that the U5-leader stem is required for efficient DNA synthesis in vivo and for initiation of DNA synthesis from the tRNA(Trp) primer in melittin-activated virions. However, this structure does not appear to be required for binding of the tRNA(Trp) primer to viral RNA. These results support a role for the U5-leader stem structure, independent of its primary sequence, in the initiation of retroviral replication.
Inducible expression of herpes simplex virus type 2 glycoprotein gene gG-2 in a mammalian cell line
Authors: Su H K , Courtney R J
Published: January 2020

The gG-2 glycoprotein gene of herpes simplex virus type 2 (HSV-2) was cloned into the mammalian expression vector pMSG under the control of the inducible mouse mammary tumor virus promoter. Transfection of this cloned gG-2 construct into NIH 3T3 cells resulted in the stable expression of gG-2 upon induction with dexamethasone. In addition, the 104,000-molecular-weight (104K) and 72K gG-2 precursors as well as the 34K secreted component were generated in the transformed cells. The synthesis of gG-2 in these transformed cells appeared to follow the same cleavage-processing pathway as gG-2 synthesis during an HSV-2 infection. These results indicate that the processing of gG-2 can occur in the absence of an HSV-2 infection.
Topoisomerase I is preferentially associated with isolated replicating simian virus 40 molecules after treatment of infected cells with camptothecin
Authors: Champoux J J
Published: January 2020

Detergent extraction of simian virus 40 (SV40) DNA from infected monkey CV-1 cells, after a brief exposure to the drug camptothecin, yields covalent complexes between topoisomerase I and DNA that band with reduced buoyant densities in CsCl. The following lines of evidence indicate that the enzyme is preferentially associated with SV40 replicative intermediates. First, the percentage of the isolated labeled viral DNA that exhibited a reduced buoyant density is inversely proportional to the length of the labeling period and approximately parallels the percentage of replicative intermediates for each labeling time (5 to 60 min). Second, after labeling for 60 min, the isolated low-density material was found to be enriched for replicative intermediates as measured by sedimentation in neutral sucrose. Third, analysis of extracted viral DNA by equilibrium centrifugation in CsCl-propidium diiodide gradients that separate replicating molecules from completed form I DNA revealed that camptothecin pretreatment specifically caused the linkage of topoisomerase I to replicating molecules. In addition, analysis of the low-density material obtained under conditions when only the newly synthesized strands of the replicative intermediates were labeled showed that the enzyme was associated almost exclusively with the parental strands. Taken together, these observations indicate that topoisomerase I is involved in DNA replication, and they are consistent with the hypothesis that the enzyme provides swivels to allow the helix to unwind. The observed bias in the distribution of topoisomerase I on intracellular SV40 DNA could be the result of rapid encapsidation of replicated molecules that precludes the association of topoisomerase I with the DNA or, alternatively, the result of a specific association of the enzyme with replicative intermediates.
Processing of gag precursor polyprotein of human T-cell leukemia virus type I by virus-encoded protease
Authors: Nam S H , Kidokoro M , Shida H , Hatanaka M
Published: January 2020

The biological activity encoded in the putative protease gene (pro) of human T-cell leukemia virus type I was investigated by using a vaccinia virus expression vector. The 53-kilodalton gag precursor polyprotein was processed into the mature p19, p24, and p15 gag proteins when the gag and protease-coding sequence was expressed under the control of a vaccinia virus promoter, suggesting that the protease may be synthesized through the mechanism of ribosomal frame shifting. The processing defect of a protease mutant could be complemented by cointroduction of a wild-type construct into the cell, demonstrating that the pro gene encodes the biologically active protease molecules which are capable of processing the gag precursor polyprotein in vivo in trans. A study involving the use of a variety of mutants constructed in vitro revealed that the protease consists of a nonessential carboxy-terminal region and a part essential for its activity, including the putative catalytic residue, aspartic acid. Furthermore, a cluster of adenine residues positioned at the overlapping region between the gag and pro genes was shown to be involved in the ribosomal frameshifting event for the synthesis of protease. To mimic the formation of the 76-kilodalton gag-pro precursor polyprotein formed by ribosomal slipping, the coding frames of the gag and pro gene were adjusted. The processing of the gag-pro precursor polyprotein depended on an intact protease gene, implying that a cis-acting function of human T-cell leukemia virus type I protease may be necessary to trigger the initial cleavage event that leads to the release of protease from the precursor protein.
Identification of Marek's disease virus nuclear antigen in latently infected lymphoblastoid cells
Authors: Wen L T , Tanaka A , Nonoyama M
Published: January 2020

A new Marek's disease virus (MDV) nuclear antigen (MDNA) was identified in two MDV-transformed T-lymphoblastoid cell lines, MKT-1 and MSB-1, derived from chickens bearing tumors induced by MDV. This MDNA was not detected in MSB-1 cells maintained in iododeoxyuridine, which activates the latent MDV genome. Moreover, it was not found in chicken embryo fibroblasts undergoing productive and cytolytic infection with MDV. Expression of MDNA is not related to strain pathogenicity in chickens, because chicken embryo fibroblasts productively infected with the pathogenic RBIB strain or the nonpathogenic CV-1 strain of MDV did not express this antigen. DNA-protein immunoprecipitation studies revealed that MDNA bound to two sites in the 190,00-base-pair (bp) MDV genome. One of these loci identified by MDNA obtained from MKT-1 and MSB-1 cells corresponded to a 476-bp segment within the short unique region of BamHI-A MDV DNA. A second locus located in a 280-bp segment within the short inverted repeat region of BamHI-A was also identified by MDNA from MSB-1 cells but not by MDNA obtained from MKT-1 cells. Analyses of the nucleotide sequence by DNase digestion showed that MDNA protected a 60-bp segment spanning a 22-bp palindromic sequence of the short unique region and a 103-bp sequence encompassing a 32-bp palindrome in the short inverted repeat region of BamHI-A MDV DNA.
Analysis of mutant Moloney murine leukemia viruses containing linker insertion mutations in the 3' region of pol
Authors: Donehower L A
Published: January 2020

Twelve linker insertion mutations have been constructed in the 3' part of the pol gene of Moloney murine leukemia virus. This region of the Moloney murine leukemia virus genome encodes IN or p46pol, which is required for integration of the retroviral DNA into the host cell chromosome. Viral proteins synthesized by these mutants were used to pseudotype a neo-containing retroviral vector. Ten of twelve linker insertion mutant pseudotypes were unable to generate stable proviruses in infected mouse cells, as measured by the formation of G418-resistant colonies. Two mutants mapping at the 3' terminus of the IN-encoding region were competent for the formation of stable vector proviruses (hundreds of G418-resistant colonies per mutant pseudotype-infected plate). Representative linker insertion mutants were also tested for the ability to synthesize viral unintegrated DNA in newly infected cells. All assayed mutants were capable of synthesizing all normal forms of viral unintegrated DNA. The structure of integrated vector proviruses generated by defective and nondefective linker insertion mutants was also analyzed. All replication-competent mutants generated normal proviruses, while the few obtainable proviruses generated by replication-defective mutants were sometimes aberrant in structure. These results argue strongly (and confirm previous data) that the IN-encoding region of pol does not play a significant role in DNA synthesis, but is absolutely required for the formation of normal proviral DNA.
Proposed gag-encoded transcriptional activator is not necessary for Rous sarcoma virus replication or transformation
Authors: Carlberg K , Beemon K
Published: January 2020

It has been reported that gene expression directed by the long terminal repeat of Rous sarcoma virus (RSV) is trans activated by a protein encoded in an alternate reading frame within the RSV gag gene (S. Broome and W. Gilbert, Cell 40:537-546, 1985). We have made specific mutations to test the role of the putative transcriptional activator in RSV replication. Termination codons were created within the alternate reading frame coding for the trans activator, and the mutations were introduced into an infectious RSV plasmid. We were unable to demonstrate specific trans activation of the RSV long terminal repeat by either wild-type or mutant RSV plasmids in transient cotransfection assays. Experiments using mutant or wild-type RSV-infected chick embryo fibroblasts indicated that the proposed RSV transcriptional activator was not required for viral replication or transformation and did not increase steady-state levels of viral RNA.
Interplay of viral and cellular proteins along the long control region of human papillomavirus type 18
Authors: Garcia-Carranca A , Thierry F , Yaniv M
Published: January 2020

The long control region of human genital papillomavirus type 18 harbors transcription regulatory elements, such as the E6 promoter and a cell type-specific enhancer independent of the E2 protein. By performing DNase I footprint experiments in vitro with protein extracts from different cell lines and tissues we searched for cellular factors interacting with the totality of the control region. We detected a total of eight different protected sites; most of them were found with all the extracts. Two of these sites, one in the enhancer and the other in the sequences proximal to the E6 cap site, interact with a member of the activator protein 1 family. However, in the absence of fine mutational analysis, we cannot readily discern an exact functional role for the different binding sites. We also characterized two regions, which are protected only in the presence of E2, corresponding to the perfect palindromes ACCGN4CGGT and present either 500 nucleotides or tandemly repeated about 70 nucleotides upstream of the E6 cap site. This last protected area covers a large part of the putative TATA box of the E6 promoter and could explain its repression by bovine papilloma virus type 1 E2, which could interfere with binding of the TFII D ubiquitous transcription factor to the TATA sequence of the promoter.