bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of mRNA stability by the virion host shutoff function of herpes simplex virus
Authors: Oroskar A A , Read G S
Published: January 2020

vhs1 is a mutant of herpes simplex virus type 1 that is defective in the virion host shutoff function responsible for the degradation of cellular mRNAs and the concomitant shutoff of host protein synthesis. In this study, the effect of the vhs1 mutation on the metabolism of viral mRNAs was examined by measuring the half-lives and patterns of accumulation of 10 different viral mRNAs representing all kinetic classes. The vhs1 mutation had the effect of dramatically lengthening the cytoplasmic half-lives of all 10 mRNAs. In wild-type virus infections, the 10 mRNAs had similar half-lives, suggesting that little, if any, target mRNA selectivity was exhibited by the vhs function. The vhs1 mutation caused overaccumulation of a number of mRNAs. The effect was most dramatic for the alpha (immediate-early) mRNA for ICP27 and the beta (early) mRNAs encoding thymidine kinase, ICP8, and DNA polymerase. Whereas in wild-type infections these mRNAs increased to peak levels and subsequently declined in abundance, in vhs1 infections they continued to accumulate until late times. A significant but less dramatic overaccumulation was observed for several beta-gamma (delayed-early) and gamma (late) mRNAs. The results suggest that the vhs protein plays an important role in determining the half-lives of viral mRNAs belonging to all kinetic classes and in so doing is important in the normal downregulation at late times of alpha and beta gene expression.
Multiple steps are required for the induction of tumors by Abelson murine leukemia virus
Authors: Green P L , Kaehler D A , Bennett L M , Risser R
Published: January 2020

Helper virus-free Abelson murine leukemia virus (A-MuLV) was used to induce monoclonal pre-B-cell tumors in mice. The clonality, patterns of immunoglobulin heavy-chain gene rearrangement, tumorigenicity, and v-abl oncogene expression in individual preleukemic and leukemic colonies were compared. Our results indicate that A-MuLV preleukemic cells with low or undetectable tumorigenic potential give rise to leukemic cells with high tumorigenic potential by a process of subclone selection. The levels of v-abl oncogene product in preleukemic and leukemic cell populations were not significantly different. These results suggest that an additional event(s) unrelated to the level of the v-abl protein product is required for A-MuLV-transformed cells to become fully malignant.
Physical mapping and nucleotide sequence of a herpes simplex virus type 1 gene required for capsid assembly
Authors: Pertuiset B , Boccara M , Cebrian J , Berthelot N , Chousterman S , Puvion-Dutilleul F , Sisman J , Sheldrick P
Published: January 2020

In this report, we describe some phenotypic properties of a temperature-sensitive mutant of herpes simplex type 1 (HSV-1) and present data concerning the physical location and nucleotide sequence of the genomic region harboring the mutation. The effect of shifts from the permissive to the nonpermissive temperature on infectious virus production by the mutant A44ts2 indicated that the mutated function is necessary throughout, or late in, the growth cycle. At the nonpermissive temperature, no major differences were detected in viral DNA or protein synthesis with respect to the parent A44ts+. On the other hand, electron microscopy of mutant-infected cells revealed that neither viral capsids nor capsid-related structures were assembled at the nonpermissive temperature. Additional analyses employing the Hirt extraction procedure showed that A44ts2 is also unable to mature replicated viral DNA into unit-length molecules under nonpermissive conditions. The results of marker rescue experiments with intact A44ts2 DNA and cloned restriction fragments of A44ts+ placed the lesion in the coordinate interval 0.553 to 0.565 (1,837 base pairs in region UL) of the HSV-1 physical map. No function has previously been assigned to this region, although it is known to be transcribed into two 5' coterminal mRNAs which code in vitro for a 54,000-molecular-weight polypeptide (K. P. Anderson, R. J. Frink, G. B. Devi, B. H. Gaylord, R. H. Costa, and E. K. Wagner, J. Virol. 37:1011-1027, 1981). We sequenced the interval 0.551 to 0.565 and found an open reading frame (ORF) for a 50,175-molecular-weight polypeptide. The predicted product of this ORF exhibits strong homology with the product of varicella-zoster virus ORF20 and lower, but significant, homology with the product of Epstein-Barr virus BORF1. For the three viruses, the corresponding ORFs lie just upstream of the gene coding for the large subunit of viral ribonucleotide reductase. The ORF described here corresponds to the ORF designated UL38 in the recently published nucleotide sequence of the HSV-1 UL region (D. J. McGeoch, M. A. Dalrymple, A. J. Davison, A. Dolan, M. C. Frame, D. McNab, L. J. Perry, J. E. Scott, and P. Taylor, J. Gen. Virol. 69:1531-1574, 1988).
Purification and characterization of adult diarrhea rotavirus: identification of viral structural proteins
Authors: Fang Z Y , Glass R I , Penaranda M , Dong H , Monroe S S , Wen L , Estes M K , Eiden J , Yolken R H , Saif L
Published: January 2020

Adult diarrhea rotavirus (ADRV) is a newly identified strain of noncultivable human group B rotavirus that has been epidemic in the People's Republic of China since 1982. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western (immuno-) blot analysis to examine the viral proteins present in the outer and inner capsids of ADRV and compared these with the proteins of a group A rotavirus, SA11. EDTA treatment of double-shelled virions removed the outer capsid and resulted in the loss of three polypeptides of 64, 61, and 41, kilodaltons (kDa). Endo-beta-N-acetylglucosaminidase H digestion of double-shelled virions identified the 41-kDa polypeptide as a glycoprotein. CaCl2 treatment of single-shelled particles removed the inner capsid and resulted in the loss of one polypeptide with a molecular mass of 47 kDa. The remaining core particle had two major structural proteins of 136 and 113 kDa. All of the proteins visualized on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were antigenic by Western blot analysis when probed with convalescent-phase human and animal antisera. A 47-kDa polypeptide was most abundant and was strongly immunoreactive with human sera, animal sera raised against ADRV and against other group B animal rotaviruses (infectious diarrhea of infant rat virus, bovine and porcine group B rotavirus, and bovine enteric syncytial virus) and a monoclonal antibody prepared against infectious diarrhea of infant rat virus. This 47-kDa inner capsid polypeptide contains a common group B antigen and is similar to the VP6 of the group A rotaviruses. Human convalescent-phase sera also responded to a 41-kDa polypeptide of the outer capsid that seems similar to the VP7 of group A rotavirus. Other polypeptides have been given tentative designations on the basis of similarities to the control preparation of SA11, including a 136-kDa polypeptide designated VP1, a 113-kDa polypeptide designated VP2, 64- and 61-kDa polypeptides designated VP5 and VP5a, and several proteins in the 110- to 72-kDa range that may be VP3, VP4, or related proteins. The lack of cross-reactivity on Western blots between antisera to group A versus group B rotaviruses confirmed that these viruses are antigenically quite distinct.
Mutational analysis of bovine papillomavirus E6 gene
Authors: Vousden K H , Androphy E J , Schiller J T , Lowy D R
Published: January 2020

The bovine papillomavirus E6 gene can independently transform mouse C127 cells. To characterize E6 in greater detail, we created 16 site-directed mutations in E6, including substitution mutations in the cysteine codons of the four Cys-X-X-Cys motifs that are conserved in all papillomavirus E6 proteins. Proteins mutated in six of the seven cysteines tested, as well as those lacking the nonconserved C-terminus, were stable in transfected cells but were unable to induce morphological transformation, indicating that these amino acids play an important role in the function of E6.
Susceptibility to raf and raf/myc retroviruses is governed by different genetic loci
Authors: Klinken S P , Hartley J W , Fredrickson T N , Rapp U R , Morse H C
Published: January 2020

The susceptibility to tumors induced by raf and raf/myc retroviruses was investigated in BALB/c, C57BL/6, (BALB/c x C57BL/6)F1 and (BALB/c x C57BL/6) backcross mice. Newborn mice were susceptible to neoplasms generated by both viruses, but resistance to raf-induced leukemia developed rapidly in all mice as they matured. Older C57BL/6 mice were also resistant to raf/myc lymphomas, whereas BALB/c mice remained susceptible to the virus at all ages, indicating that different genes control susceptibility to raf and raf/myc tumors. From these data and the susceptibility of C x B recombinant inbred strains, it appears that very few genes (perhaps even a single gene) may govern susceptibility to raf/myc lymphomas and that resistance is the dominant trait.
Detection of murine cytomegalovirus DNA in circulating leukocytes harvested during acute infection of mice
Authors: Bale J F , O'Neil M E
Published: January 2020

We used virus assay and in situ hybridization with a cloned fragment of the murine cytomegalovirus (MCMV) genome to study MCMV infection of circulating leukocytes harvested from 3-week-old BALB/c, C57BL/6, and C3H mice infected with MCMV intraperitoneally. Infectious virus or MCMV DNA was detected in leukocytes on days 1 through 21 of infection in BALB/c mice and on days 3 through 7 in C57BL/6 mice. On days 5 and 7, MCMV DNA or infectious virus was detected in the leukocytes of 17 (94%) of 18 BALB/c mice and 10 (59%) of 17 C57BL/6 mice. In both strains infection peaked on days 5 and 7, when as many as 0.01 to 0.1% of the circulating leukocytes contained MCMV DNA. In C3H mice, however, infectious virus was rarely recovered from leukocyte fractions and MCMV DNA was detected in the circulating leukocytes of only one animal. Circulating leukocytes may have an important role in the dissemination of CMV infections in susceptible hosts.
DNA sequences that regulate expression of a vaccinia virus late gene (L65) and interact with a DNA-binding protein from infected cells
Authors: Miner J N , Hruby D E
Published: January 2020

To be efficiently expressed in vivo, the vaccinia virus late gene, L65, requires 5'-proximal cis-acting elements which bind a factor from infected cells. Deletion mutagenesis and vaccinia virus helper-dependent transient expression procedures were used to demonstrate that two distinct late promoter elements direct transcription from two different start sites (proximal [+1] and distal [-92]). The -128 to -112 region was essential for L65 distal promoter function, while sequences between -59 and +50 were sufficient for L65 proximal promoter function. The proximal DNA sequences interact with a protein, binding factor I (BF-I), which was isolated and partially purified from vaccinia virus-infected cells at late times postinfection. This activity is not detectable in uninfected cells or in purified virions. This factor binds specifically to two different sites within the proximal promoter, one 5' and one 3' to the transcription start site, but does not bind to the distal promoter element.
Antiviral antibodies stimulate production of reactive oxygen species in cultured canine brain cells infected with canine distemper virus
Authors: Bürge T , Griot C , Vandevelde M , Peterhans E
Published: January 2020

Canine distemper is characterized mainly by respiratory, enteric, and nervous symptoms. Infection of the central nervous system results in demyelination, to which inflammation has been shown to contribute significantly. It has been proposed that macrophages play a major role as effector cells in this process. We report that cultured dog brain cells contain a population of macrophages capable of producing reactive oxygen species as measured by luminol-dependent chemiluminescence. In cultures infected with canine distemper virus, a burst of reactive oxygen is triggered by antiviral antibody. This response depends on the presence of viral antigens on the surfaces of infected cells and is mediated by the interaction of antigen-bound antibody with Fc receptors on the macrophages. Since there is no evidence in vitro or in vivo that oligodendrocytes, the cells forming myelin, are infected, our observation supports the hypothesis that "innocent bystander killing" is important in demyelination caused by canine distemper virus. Reactive oxygen species released from macrophages may contribute to destruction of myelin.
Genetic and biochemical characterization of the thymidine kinase gene from herpesvirus of turkeys
Authors: Martin S L , Aparisio D I , Bandyopadhyay P K
Published: January 2020

The thymidine kinase gene encoded by herpesvirus of turkeys has been identified and characterized. A viral mutant (ATR0) resistant to 1-beta-D-arabinofuranosylthymine was isolated. This mutant was also resistant to 1-(2-fluoro-2-deoxy-beta-D-arabinofuronosyl)-5-methyluracil and was unable to incorporate [125I]deoxycytidine into DNA. The mutant phenotype was rescued by a cloned region of the turkey herpesvirus genome whose DNA sequence was found to contain an open reading frame similar to that for known thymidine kinases from other viruses. When expressed in Escherichia coli, this open reading frame complemented a thymidine kinase-deficient strain and resulted in thymidine kinase activity in extracts assayed in vitro.
Mice immunized with recombinant vaccinia virus expressing dengue 4 virus structural proteins with or without nonstructural protein NS1 are protected against fatal dengue virus encephalitis
Authors: Bray M , Zhao B T , Markoff L , Eckels K H , Chanock R M , Lai C J
Published: January 2020

We have constructed vaccinia virus recombinants expressing dengue virus proteins from cloned DNA for use in experimental immunoprophylaxis. A recombinant virus containing a 4.0-kilobase DNA sequence that codes for three structural proteins, capsid (C), premembrane (pre-M), and envelope (E), and for nonstructural proteins NS1 and NS2a produced authentic pre-M, E, and NS1 in infected CV-1 cells. Mice immunized with this recombinant were protected against an intracerebral injection of 100 50% lethal doses of dengue 4 virus. A recombinant containing only genes C, pre-M, and E also induced solid resistance to challenge. Deletion of the putative C-terminal hydrophobic anchor of the E glycoprotein did not result in secretion of E from recombinant-virus-infected cells. Recombinants expressing only the E protein preceded by its own predicted N-terminal hydrophobic signal or by the signal of influenza A virus hemagglutinin or by the N-terminal 71 amino acids of the G glycoprotein of respiratory syncytial virus produced glycosylated E protein products of expected molecular sizes. These vaccinia virus recombinants also protected mice.
Identification of a region within the human immunodeficiency virus type 1 long terminal repeat that is essential for transactivation by the hepatitis B virus gene X
Authors: Twu J S , Rosen C A , Haseltine W A , Robinson W S
Published: January 2020

Hepatitis B virus (HBV) X-gene product activates transcription of the chloramphenicol acetyltransferase (CAT) gene under control of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). To identify a cis-acting regulatory sequence within the HIV-1 LTR which is responsive to the HBV X-gene trans-activating function, we examined the effects of HBV X-gene expression in cells with a series of LTR/CAT deletion mutants. A region of the HIV-1 LTR containing the previously identified kappa B-like enhancer element was found to be responsive to HBV X-gene activation, and this effect was independent of, and additive with, the effect of the HIV-1 tat-III protein on CAT expression. Since kappa B-like enhancer sequences are known to regulate transcription of a variety of viruses and cellular genes, our results suggest that the X gene could activate such a gene during HBV infection and replication.
Preferential binding of simian virus 40 T-antigen dimers to origin region I
Authors: Deb S P , Deb S
Published: January 2020

The sequence components that direct high-affinity binding of simian virus 40 (SV40) T antigen to SV40 origin region I are composed of two recognition pentanucleotides separated by a spacer. This region has binding sites for two T-antigen monomeric units. We extended the tripartite region I sequence by one and two sets of spacers and pentanucleotides and also shortened the region by one pentanucleotide. Our T-antigen-binding studies with these constructs show that the protein has a strong preference for binding to an even rather than an odd number of pentanucleotides separated by spacer sequences. Gel retardation assays reveal that the size of the complex formed between the 17-base-pair region I sequence and T antigen did not increase when the sequence was extended with one spacer-pentanucleotide sequence but did increase with two such units. DNase I footprinting and fragment assay experiments indicate that the protein did not protect a pentanucleotide that was not paired with another pentanucleotide. The unpaired pentanucleotide resumed its binding activity when it was paired with a spacer and another pentanucleotide sequence. We propose that T antigen binds to region I as a preformed dimer.
Identification of a promoter region for 3.6-kilobase mRNA of hepatitis B virus and specific cellular binding protein
Authors: Yaginuma K , Koike K
Published: January 2020

The promoter region for transcription of the 3.6-kilobase mRNA of hepatitis B virus was identified by the chloramphenicol acetyltransferase assay by using HuH-7 hepatoma cells and was found to function directly in virus production by way of the transient expression system of HBV. The 5'-upstream sequence from nucleotides 1573 to 1657 (the transcription start site) was indispensable for promoter function, while the AT-rich sequence (from nucleotides 1581 to 1604) containing a directly repeated sequence TGTT connecting the same flanking sequence PyAAAGAC (where Py is a pyrimidine) at both sides was an essential element within this promoter region. A specific cellular factor which interacted with the essential element was detected in the HuH-7 cell extract. A similar binding factor was also observed in HepG2 and huH2-2 hepatoma cells. This factor may thus be responsible for regulating 3.6-kilobase mRNA, pregenome RNA transcription, or both.
Modulation of arachidonic acid metabolism by Rous sarcoma virus
Authors: Barker K , Aderem A , Hanafusa H
Published: January 2020

Arachidonic acid (C20:4) metabolites were released constitutively from wild-type Rous sarcoma virus-transformed chicken embryo fibroblasts (CEF). 3H-labeled C20:4 and its metabolites were released from unstimulated and uninfected CEF only in response to stimuli such as serum, phorbol ester, or the calcium ionophore A23187. High-pressure liquid chromatography analysis showed that the radioactivity released from [3H]arachidonate-labeled transformed cells was contained in free arachidonate and in the cyclooxygenase products prostaglandin E2 and prostaglandin F2 alpha; no lipoxygenase products were identified. The release of C20:4 and its metabolites from CEF infected with pp60src deletion mutants was correlated with serum-independent DNA synthesis and with the expression of the mRNA for 9E3, a gene expressed in Rous sarcoma virus-transformed cells which has homology with several mitogenic and inflammatory peptides. 3H-labeled C20:4 release was not correlated with p36 phosphorylation, which argues against a role for this protein as a phospholipase A2 inhibitor. CEF infected with other oncogenic viruses encoding a tyrosine kinase also released C20:4, as did CEF infected with viruses that contained mos and ras; however, infection with a crk-containing virus did not result in stimulation of 3H-labeled C20:4 release, suggesting that utilization of this signaling pathway is specific for particular transformation stimuli.
Functional limits of oriP, the Epstein-Barr virus plasmid origin of replication
Authors: Chittenden T , Lupton S , Levine A J
Published: January 2020

The Epstein-Barr virus (EBV) genome contains two cis-acting elements which are required for stable extrachromosomal plasmid maintenance in latently infected cells. The first consists of 20 30-base-pair (bp) repeats, each of which contains a DNA-binding site for EBV nuclear antigen 1 (EBNA-1), the trans-acting factor required for plasmid persistence. The second element is composed of a 65-bp dyad symmetry, containing four EBNA-1-binding sites. Deletion mutants were constructed which reduce the number of EBNA-1-binding sites in the 30-bp repeats, alter the number of EBNA-1-binding sites in the dyad region, or truncate the dyad element. The effect of the deletion mutations on plasmid maintenance was examined by transfecting recombinant plasmids, containing both the mutated EBV sequences and a drug resistance marker, into D98-Raji cells. The plasmids were tested for their ability to generate drug-resistant D98-Raji cell colonies and their capacity to be maintained in an extrachromosomal form without undergoing extensive rearrangements. EBV plasmids with 12 or 15 copies of the 30-bp repeats were wild type in both assays. Plasmids with just two or six copies of these repeated elements failed to generate drug-resistant colonies at a normal level, and normal episomal plasmids were not detected in the resulting colonies. Rare colonies of cells resulting from transfection of these two- or six-copy mutants contained rearranged, episomal forms of the input plasmids. The rearrangements most often produced head-to-tail oligomers containing a minimum of eight 30-bp repeated elements. The rearranged plasmids were shown to be revertant for plasmid maintenance in that they yielded wild-type or greater numbers of drug-resistant colonies and persisted at the wild-type or a greater episomal copy number. By use of an EBV plasmid that contained no 30-bp elements, no revertants could be isolated. One to five copies of a synthetic linker corresponding to a consensus 30-bp repeated element inserted into a plasmid with no 30-bp elements now permitted the generation of oligomeric, episomal forms of the mutant test plasmid. These experiments demonstrate a requirement for a minimal number (six to eight copies) of the 30-bp repeated element. Deletions in the 65-bp dyad region had little or no effect upon the ability to generate enhanced numbers of drug-resistant D98-Raji colonies, indicating that the 30-bp repeated element is predominantly required for this phenotype.(ABSTRACT TRUNCATED AT 400 WORDS)
Responsiveness of the Epstein-Barr virus NotI repeat promoter to the Z transactivator is mediated in a cell-type-specific manner by two independent signal regions
Authors: Lieberman P M , Hardwick J M , Hayward S D
Published: January 2020

Cells latently infected with Epstein-Barr virus (EBV) can be activated to express lytic-cycle polypeptides by the introduction of the EBV-encoded Z transactivator, indicating that this protein has a pivotal role in virus reactivation. We examined the target specificity of the Z transactivator in short-term contransfection assays and found that the most responsive target to Z transactivation was the divergent NotI repeat promoter, located within the EBV BamHI H fragment. In contrast, target plasmids containing the cat gene linked to heterologous viral promoters were not activated by cotransfection with the Z gene. S1 nuclease analysis of RNA from chemically induced B95-8 cells and from Vero cells cotransfected with NotI repeat promoter-CAT and Z showed that Z transactivation increased the level of correctly initiated, stable RNA transcripts. The NotI repeat gene (ntr) gives rise to a highly abundant mRNA species after chemical induction of lytic virus replication, but no protein product had been previously identified. Using monospecific antiserum raised against a synthetic peptide from the BHLF1 open reading frame, we demonstrated that the ntr gene encodes a protein product that is found in nuclear patches colocalizing with nucleoli. A series of deletions introduced into the upstream sequences of the NotI-repeat-promoter revealed two separate Z-response regions. The minimal promoter region between -7 and -155 of the leftward RNA cap site and an upstream region between -644 and -902 were both independently capable of conferring Z responsiveness. However, the minimal region, which was activated by Z cotransfection in Vero cells, was poorly responsive in lymphocytes, whereas the response of the far-upstream region to Z cotransfection was lymphocyte specific. In its human host, EBV infects both epithelial and lymphocyte populations. This dual lifestyle may have led to the evolution of multiple Z-response signals that enable the Z transactivator to interact with both cell-specific promoter and enhancer factors.
T-cell-mediated clearance of mouse hepatitis virus strain JHM from the central nervous system
Authors: Sussman M A , Shubin R A , Kyuwa S , Stohlman S A
Published: January 2020

Clearance of the neurotropic JHM strain of mouse hepatitis virus from the central nervous system was examined by the transfer of spleen cells from immunized donors. A T cell with the surface phenotype of Thy1.2+ CD4+ CD8- asialo-GM1+ Mac-1- was found to be necessary for viral clearance. The surface phenotype and adherence to nylon wool suggest that these cells are activated helper-inducer T cells. Adoptive transfer to congenic histocompatibility strains demonstrated the necessity for compatibility at the D locus of the major histocompatibility complex. The expression of the CD4 surface marker and the requirement for major histocompatibility complex class I were further studied by the transfer of cells to recipients treated with anti-CD4 or anti-CD8 monoclonal antibodies. Treatment of recipients with either the anti-CD8 or the anti-CD4 antibodies inhibited virus clearance from the central nervous system. This suggests that the CD4+ cell acts as a helper and that virus is cleared from the central nervous system. This suggests that the CD4+ cell acts as a helper and that virus is cleared from the central nervous system by CD8+ cells that recognize viral antigen in the context of the H-2Db gene product.
Monoclonal antibody analysis of simian virus 40 small t-antigen expression in infected and transformed cells
Authors: Montano X , Lane D P
Published: January 2020

The monoclonal antibody PAb280 binds to small t antigen but not to large T antigen. Its binding site within the unique region of small t antigen was localized by studying its reaction with simian virus 40 mutants, other papovaviruses, and bacterial expression vectors coding for fragments of small t antigen. The antibody was used to define the cellular location of small t antigen by immunocytochemistry and by immunoprecipitation of subcellular extracts of infected cells. PAb280 reacts strongly with a cytoplasmic form of small t antigen that appears to be associated with the cytoskeleton and is not detected by antibodies directed to the common N terminus of small t and large T antigens. Immunoperoxidase staining of cells infected by the simian virus 40 defective strain SV402 with PAb280 and other anti-T antibodies demonstrated that this virus produced an N-terminal fragment of large T antigen as well as small t antigen. In cells infected by the virus, this fragment was located in the cell nucleus but was very unstable. These results suggest that the activity of the SV402 virus in transformation assays may not be entirely due to the action of small t antigen alone.
5-Azacytidine up regulates the expression of Epstein-Barr virus nuclear antigen 2 (EBNA-2) through EBNA-6 and latent membrane protein in the Burkitt's lymphoma line rael
Authors: Masucci M G , Contreras-Salazar B , Ragnar E , Falk K , Minarovits J , Ernberg I , Klein G
Published: January 2020

Nonproductive infection of B lymphocytes by Epstein-Barr virus (EBV) is associated with a highly restricted expression of viral genes. In growth-transformed lymphoblastoid cell lines, the products of these genes include a complex of at least six EBV nuclear antigens (EBNAs) (EBNA-1 through EBNA-6) and one membrane protein (latent membrane protein [LMP]). EBV-carrying Burkitt's lymphoma (BL) biopsies and derived cell lines that have retained a representative phenotype (group I BL lines) express only EBNA-1 (M. Rowe, D. T. Rowe, C. D. Gregory, L. S. Young, P. J. Farrell, H. Rupani, and A. B. Rickinson, EMBO J. 6:2743-2751, 1987). We have found that EBNA-2 through EBNA-6 and LMP can be up regulated by treating the group I BL line Rael with the DNA-demethylating agent 5-azacytidine (5-AzaC). The drug acted in a time- and dose-dependent manner. EBNA-2-positive cells were detected by anti-complement immunofluorescence staining just 12 h after addition of 4 microM 5-AzaC and reached a maximum number at 72 h, when up to 75% of the population was positive. EBNA-2, EBNA-3, EBNA-4, EBNA-4, EBNA-6, and LMP were demonstrated immunoblots starting at 48 h. The EBV-encoded early antigens and viral capsid antigens were also induced but at a lower level. EBNA-2 and the lytic cycle-associated antigens appeared with a different time course and in largely nonoverlapping cell subpopulations, as demonstrated by double fluorescence staining. Thus, EBNA-2 expression was not restricted to lytically infected cells, nor was EBNA-2 required for entry into the lytic cycle. The coding and regulatory sequences of EBNA-2 and LMP were found to be highly methylated in Rael cells and were, as expected, demethylated after 5-AzaC treatment. These findings suggest that DNA methylation may participate in the regulation of growth transformation-associated viral genes in BL cells.
Absence of mouse mammary tumor virus proviral amplification in chemically induced lymphomas of RF/J mice
Authors: Kraft R , Ishizaka S T , Okenquist S A , Childs G , Lilly F , Lenz J
Published: January 2020

RF/J mice are susceptible to the induction of thymic lymphomas by the carcinogens 3-methylcholanthrene and N-methyl-N-nitrosourea. Given the association of mouse mammary tumor virus (MMTV) with certain thymomas, we examined genomic DNA from chemically induced lymphomas of RF/J mice for new MMTV proviruses. Of 13 tissue culture lines derived from 3-methylcholanthrene-induced tumors, 5 had acquired new proviruses. MMTV amplification coincided with the appearance of viral mRNAs and proteins. However, no primary tumors or animal-passaged tumors contained new proviruses. These observations indicate that MMTV does not have a role in the tumor induction process, although it may become activated and amplified in tissue culture lines derived from tumors.
Identification of cellular promoters by using a retrovirus promoter trap
Authors: von Melchner H , Ruley H E
Published: January 2020

A retrovirus vector has been developed that selects for instances in which the provirus integrates in close proximity to cellular promoters. Coding sequences for a selectable marker (histidinol dehydrogenase) were inserted into the 3' long terminal repeat (LTR) of an enhancerless Molony murine leukemia virus. Although 1.8 kilobase pairs in size, the elongated LTR did not appear to interfere with virus replication or integration. Thus, when the virus was passaged, the elongated LTRs efficiently duplicated, placing histidinol dehydrogenase-coding sequences in the 5' LTR just 30 nucleotides from the flanking cellular DNA. Selection for histidinol expression generated cell clones in which histidinol gene sequences in the 5' LTR were invariably expressed on transcripts initiating in nearby cellular sequences. The efficiency of transducing histidinol resistance was 2,500-fold lower than the efficiency of transducing neomycin resistance when the neomycin phosphotransferase gene was located within the retrovirus and expressed from an independent promoter. By tagging transcriptionally active sites, the vector provides a means to identify and isolate promoters active in different cell types. Furthermore, the virus may be useful as an insertional mutagen, since selection for cell populations containing proviruses only in expressed sites is expected to reduce the number of intergrants needed to screen for loss of gene function.
Polyadenylation at correct sites in genome RNA is not required for retrovirus replication or genome encapsidation
Authors: Swain A , Coffin J M
Published: January 2020

RNA transcripts polyadenylated at sites derived from flanking cellular DNA (readthrough transcripts) make up about 15% of the viral RNA in cells infected with avian leukosis virus. To test the functionality of such transcripts, a virus was created by introducing two mutations into the AAUAAA polyadenylation signal of Rous-associated virus 1, converting it to AAGGAA. The replication of this virus was not greatly affected at any level. However, less than 1% of viral transcripts produced during mutant virus replication were cleaved and polyadenylated at the correct site within viral long terminal repeat-related sequence. These results imply that readthrough transcripts, which are produced during normal viral replication, are polyadenylated and packaged into virions as normal transcripts and can serve as RNA genomes in the next round of replication. These results show that polyadenylation within virus-related sequences is not a necessary requirement for virus replication and that readthrough transcripts have the necessary properties to be intermediates in the process of transduction of cellular sequences.
Properties of a simian virus 40 mutant T antigen substituted in the hydrophobic region: defective ATPase and oligomerization activities and altered phosphorylation accompany an inability to complex with cellular p53
Authors: Tack L C , Cartwright C A , Wright J H , Eckhart W , Peden K W , Srinivasan A , Pipas J M
Published: January 2020

We have analyzed the biochemical properties of a nonviable simian virus 40 (SV40) mutant encoding a large T antigen (T) bearing an amino acid substitution (Pro-584-Leu) in its hydrophobic region. Mutant 5080 has an altered cell type specificity for transformation (transforming mouse C3H10T1/2 but not rat REF52 cells), is defective for viral DNA replication, and encodes a T that is unable to form a complex with the cellular p53 protein (K. Peden, A. Srinivasan, J. Farber, and J. Pipas, Virology 168:13-21, 1989). In this article, we show that 5080-transformed C3H10T1/2 cell lines express an altered T that is synthesized at a significantly higher rate but with a shorter half-life than normal T from wild-type SV40-transformed cells. 5080 T did not oligomerize beyond 5 to 10S in size compared with normal T, which oligomerized predominantly to 14 to 20S species. In addition, the 5080 T complex had significantly decreased ATPase activity and had a 10-fold-lower level of in vivo phosphorylation compared with that of normal T. Two-dimensional phosphopeptide analysis indicated several changes in the specific 32P labeling pattern, with altered phosphorylation occurring at both termini of the mutant protein compared with the wild-type T. Loss of p53 binding is therefore concomitant with changes in ATPase activity, oligomerization, stability, and in vivo phosphorylation of T and can be correlated with defective replication and restricted transformation functions. That so many biochemical changes are associated with a single substitution in the hydrophobic region of T is consistent with its importance in regulating higher-order structural and functional relationships in SV40 T.
Antibody-dependent cellular cytotoxicity detects type- and strain-specific antigens among human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus SIVmac isolates
Authors: Ljunggren K , Biberfeld G , Jondal M , Fenyö E M
Published: January 2020

Human cell lines were infected with different strains of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) as well as with a simian immunodeficiency virus SIVmac isolate and used as targets in an antibody-dependent cellular cytotoxicity (ADCC) assay. Sera from HIV-1- or HIV-2-infected subjects provided the antibody, and lymphocytes from normal donors provided the effector cells. About 60% of HIV-1 antibody-positive sera mediated ADCC when tested against any given HIV-1 isolate-infected target cell (human T-cell lymphotropic virus type IIIB, B40, A2587), and about 75% of HIV-2 antibody-positive sera mediated ADCC when tested against target cells infected with HIV-2 isolates (lymphadenopathy-associated virus type 2 and SBL-6669) or simian immunodeficiency virus from macaques. Within each type, individual sera showed different reactivity patterns, and the probability that a serum was ADCC positive was higher when it was tested against several strains. When the ADCC reactivity of sera against different strains was compared, diversity as detected by ADCC appeared to be greater among HIV-1 strains than among HIV-2 strains. For HIV-1, 54 to 67% of the sera gave concordant ADCC reactions, whereas for HIV-2 and SIVmac, 91% of the sera gave concordant results. Almost no strain-specific differences were seen between SBL-6669 and lymphadenopathy-associated virus type 2. As we determined previously, HIV-1 and HIV-2 did not cross-react in ADCC. The results indicated that HIV-1 and HIV-2 antibody-positive sera mediate both strain- and type-specific ADCC. HIV-2 antibody-positive sera seem to mediate ADCC with broader reactivity and to a higher frequency compared with HIV-1 antibody-positive sera.
Two nuclear oncogenic proteins, P135gag-myb-ets and p61/63myc, cooperate to induce transformation of chicken neuroretina cells
Authors: Amouyel P , Laudet V , Martin P , Li R P , Quatannens B , Stéhelin D , Saule S
Published: January 2020

Several studies have shown that full transformation of primary rodent fibroblasts can be achieved in vitro through the cooperation of two oncogenes (usually one nuclear and one cytoplasmic) classified on the basis of different complementation groups. We have shown previously that cooperation between v-mil (cytoplasmic, serine-threonine kinase product), and v-myc (nuclear, DNA-binding product) is required to transform 7-day-old chicken neuroretina cells, which in usual culture medium do not rapidly proliferate. v-mil induces sustained growth of chicken neuroretina cells without transformation; v-myc fails to stimulate the proliferation of chicken neuroretina cells but is required to achieve transformation of the proliferating cells. Here, we present results indicating that the P135gag-myb-ets nuclear protein of avian erythroblastosis virus E26 is able to induce proliferation but not transformation of chicken neuroretina cells. v-myc is required in addition to P135gag-myb-ets to achieve chicken neuroretina cell transformation. In contrast, we found that the P135gag-myb-ets and P100gag-mil proteins are not able to cooperate in this system.
Semliki Forest virus E2 envelope epitopes induce a nonneutralizing humoral response which protects mice against lethal challenge
Authors: Grosfeld H , Velan B , Leitner M , Cohen S , Lustig S , Lachmi B E , Shafferman A
Published: January 2020

Along the 422 amino acids of the Semliki Forest virus (SFV) E2 envelope glycoprotein, we identified 13 peptide cassettes (ranging in size from 15 to 25 amino acids and designated A through N) that contain hydrophilic sequences flanked by amino acid sequences conserved in the E2 envelopes of the alphavirus family. Six peptide blocks containing either a single cassette or two to three contiguous cassettes (A, BC, DE, FG, HIK, and LMN) were produced in Escherichia coli as recombinant proteins fused to the N terminus of beta-galactosidase. All of the SFV E2 recombinant polypeptides except A-beta-galactosidase were recognized on Western blots (immunoblots) by anti-SFV polyclonal antisera. In addition, these five recombinant proteins induced in mice antibodies that interacted specifically with SFV E2 protein on Western blots as well as with the intact virions in an enzyme-linked immunosorbent assay. The six hybrid proteins were used to vaccinate mice and were tested for the ability to confer resistance against lethal doses of SFV. Peptides BC and HIK, located at amino acid positions 114 to 149 and 216 to 288, respectively, of E2, protected partially (40 to 60%) against SFV challenge. A third peptide, LMN, located between amino acid positions 289 and 352, rendered mice totally resistant to an SFV challenge of 250 50% lethal doses. The partially protective effects of the BC and HIK cassettes and the high efficacy of the LMN cassette were consistently demonstrated, independent of the adjuvant (complete Freund or alum), immunization protocol, and strain of mice used. None of the antisera raised against any given cassette could neutralize the virus in an in vitro tissue culture assay or in a plaque reduction neutralization test. Nevertheless, passive transfer experiments demonstrated that in the case of LMN, the protective effect was mainly of a humoral nature.
Sindbis virus ts103 has a mutation in glycoprotein E2 that leads to defective assembly of virions
Authors: Hahn C S , Rice C M , Strauss E G , Lenches E M , Strauss J H
Published: January 2020

Sindbis virus mutant ts103 is aberrant in the assembly of virus particles. During virus budding, proper nucleocapsid-glycoprotein interactions fail to occur such that particles containing many nucleocapsids are formed, and the final yield of virus is low. We have determined that a mutation in the external domain of glycoprotein E2, Ala-344----Val, is the change that leads to this phenotype. Mapping was done by making recombinant viruses between ts103 and a parental strain of the virus, using a full-length cDNA clone of Sindbis virus from which infectious RNA can be transcribed, together with sequence analysis of the region of the genome shown in this way to contain the ts103 lesion. A partial revertant of ts103, called ts103R, was also mapped and sequenced and found to be a second-site revertant in which a change in glycoprotein E1 from lysine to methionine at position 227 partially suppresses the phenotypic effects of the change at E2 position 344. An analysis of revertants from ts103 mutants in which the Ala----Val change had been transferred into a defined background showed that pseudorevertants were more likely to arise than were true revertants and that the ts103 change itself reverted very infrequently. The assembly defect in ts103 appeared to result from weakened interactions between the virus membrane glycoproteins or between these glycoproteins and the nucleocapsid during budding. Both the E2 mutation leading to the defect in virus assembly and the suppressor mutation in glycoprotein E1 are in the domains external to the lipid bilayer and thus in domains that cannot interact directly with the nucleocapsid. This suggests that in ts103, either the E1-E2 heterodimers or the trimeric spikes (consisting of three E1-E2 heterodimers) are unstable or have an aberrant configuration, and thus do not interact properly with the nucleocapsid, or cannot assembly correctly to form the proper icosahedral array on the surface of the virus.
Expression and immunogenicity of the extracellular domain of the human immunodeficiency virus type 1 envelope glycoprotein, gp160
Authors: Berman P W , Riddle L , Nakamura G , Haffar O K , Nunes W M , Skehel P , Byrn R , Groopman J , Matthews T , Gregory T
Published: January 2020

The envelope glycoprotein of human immunodeficiency virus type 1 is synthesized as a precursor, gp160, that subsequently is cleaved to yield mature gp120 and gp41. In these studies, the gene encoding gp160 was mutagenized so as direct the synthesis of a truncated protein consisting of the extracellular domains of both gp120 and gp41. The variant protein, termed sgp160, consisted of 458 amino acids of gp120 and 172 amino acids of gp41. To facilitate protein purification, the normal polyglycoprotein processing site between gp120 and gp41 was deleted through the use of site-directed mutagenesis. This allowed for the synthesis of a molecule that could be purified by affinity chromatography, using acid elution, without dissociation of the gp120 polypeptide from the gp41 polypeptide. The conformation of the sgp160 variant appeared to be functionally relevant, as reflected by its ability to bind to CD4 with an affinity comparable to that of the variant rgp120. The structure of the sgp160-containing polypeptide differed from that of rgp120 in that it tended to form high-molecular-weight aggregates that could be dissociated to monomers and dimers in the presence of reducing agents. Antibodies against the sgp160 protein reacted with authentic virus-derived gp160, gp120, and gp41; neutralized viral infectivity; and inhibited the binding of rgp120 to CD4. Rabbit antibodies to the sgp160 protein differed from those raised against rgp120 in that they were enriched for populations that blocked CD4 binding but did not prevent human immunodeficiency virus type 1-induced syncytium formation.
Defect of adenovirus type 12 replication in hamster cells: absence of transcription of viral virus-associated and L1 RNAs
Authors: Jüttermann R , Weyer U , Doerfler W
Published: January 2020

The nonpermissive interaction of hamster cells with human adenovirus type 12 (Ad12) is characterized by a total block of Ad12 DNA replication and late transcription, whereas most of the early functions of Ad12 DNA can be transcribed. Ad2 can replicate in hamster cells. The replication and late transcription defects of Ad12 DNA can be complemented to a certain extent by the E1B functions of Ad2 DNA. This complementation fails, however, to lead to the synthesis of the late Ad12 proteins and to the assembly of infectious virions. It will now be demonstrated that the Ad12 L1 (late genes of group 1) and virus-associated (VA) RNAs are not transcribed in hamster cells. Synthesis of these RNAs in productively infected human cells or Ad2-infected hamster cells is readily detectable by S1 nuclease protection experiments and Northern (RNA) blotting. Similarly, the Ad2-transformed hamster cell line BHK-Ad2E1 fails to complement L1 and VA RNA syntheses after superinfection with Ad12. However, Ad12 infection of the Ad5-transformed hamster cell line BHK297-C131 leads to the transcription of the Ad12 L1 and VA segments. This difference in complementation by the two transformed hamster cell lines might be accounted for by functions in the segment of Ad5 DNA extending between map units 30 and 40 and persisting in the Ad5-transformed hamster cells or by hamster host cell functions which might be operative in cell line BHK297-C131 but not in BHK-Ad2E1 or BHK-21 hamster cells.
A single-amino-acid substitution eliminates the stringent carbohydrate requirement for intracellular transport of a viral glycoprotein
Authors: Pitta A M , Rose J K , Machamer C E
Published: January 2020

In this report, we have investigated the contribution of primary sequence to the carbohydrate requirement for intracellular transport of two closely related glycoproteins, the G proteins of the San Juan and Orsay strains of vesicular stomatitis virus. We used site-directed mutagenesis of the coding sequence to eliminate the two consensus sites for glycosylation in the Orsay G protein. Whereas the nonglycosylated San Juan G protein required at least one of its two asparagine-linked oligosaccharides for transport to the plasma membrane at 37 degrees C, a fraction of the Orsay G protein was transported without carbohydrate. Of the 10 amino acid differences between these two proteins, residue 172 (tyrosine in San Juan, aspartic acid in Orsay) played the major role in determining the stringency for the carbohydrate requirement. The rates at which the glycosylated and nonglycosylated Orsay G proteins were transported to the cell surface were the same, although a smaller fraction of the nonglycosylated protein was transported. These results suggest that the carbohydrate does not promote intracellular transport directly but influences a polypeptide folding or oligomerization step which is critical for transport.