bluuebunny/crossref_metadata_2025 huggingface dataset:
The open reading frame ORF S3 of equine infectious anemia virus is expressed during the viral life cycle
Authors: Saman E , Breugelmans K , Heyndrickx L , Merregaert J
Published: January 2020

The genome of equine infectious anemia virus (EIAV) contains several small open reading frames (ORFs), the importance of which in the development of the virus is not clear. We investigated the possibility that the largest of these ORFs (ORF S3) is expressed during the course of the viral infection. The ORF S3 information was expressed in Escherichia coli, and the antigen was used to raise monospecific antiserum. A 20-kDa protein expressed in cells producing EIAV was identified as the gene product of ORF S3. Furthermore, sera from EIAV-infected animals specifically recognized this protein, indicating that the ORF S3 antigen is expressed in vivo as well. A model for the expression of this new viral antigen is presented. The proposed splicing pattern is similar to that of the VEP-1 protein of maedi-visna-virus, which tempts us to speculate that ORF S3 defines the second exon of the EIAV Rev protein.
The U3 region is not necessary for 3' end formation of spleen necrosis virus RNA
Authors: Iwasaki K , Temin H M
Published: January 2020

Primary transcripts of retroviruses contain two poly(A) sites, one near the 5' and one near the 3' end of the transcript, but only the 3' poly(A) site is used for 3' end formation of viral RNA. It was hypothesized on the basis of experiments with U3-deleted vectors of spleen necrosis virus that the U3 region contains sequences required for this RNA 3' end formation: the titer of a U3-deleted vector was 150 times lower than that of the parental vector, and the addition of the simian virus 40 poly(A) signal sequence increased the titer of the U3-deleted vector (J. P. Dougherty and H. M. Temin, Proc. Natl. Acad. Sci. USA 84:1197-1201, 1987). However, we now show that the U3 region transcribed from the 3' long terminal repeat is not required for RNA 3' end formation and that the experiments of Dougherty and Temin led to an erroneous conclusion. We show here that the deletion of the U3 region did not decrease the steady-state level of viral RNA or shift the site of poly(A) addition. The added simian virus 40 poly(A) signal sequence was used preferentially over the poly(A) signal of spleen necrosis virus, and it increased the levels of RNA transcribed from vectors with and without deletion of the U3 region. Our results indicate that alteration of regulatory sequences in retroviral vectors can change the steady-state RNA levels and titers of the vectors in an unpredictable manner.
Parental influenza virion nucleocapsids are efficiently transported into the nuclei of murine cells expressing the nuclear interferon-induced Mx protein
Authors: Broni B , Julkunen I , Condra J H , Davies M E , Berry M J , Krug R M
Published: January 2020

The interferon-induced murine Mx1 protein, which is localized in the nucleus, most likely specifically blocks influenza virus replication by inhibiting nuclear viral mRNA synthesis, including the mRNA synthesis catalyzed by inoculum (parental) virion nucleocapsids (R. M. Krug, M. Shaw, B. Broni, G. Shapiro, and O. Haller, J. Virol. 56:201-206, 1985). We tested two possible mechanisms for this inhibition. First, we determined whether the transport of parental nucleocapsids into the nucleus was inhibited in murine cells expressing the nuclear Mx1 protein. To detect the Mx1 protein, we prepared rabbit antibodies against the Mx1 protein with a CheY-Mx fusion protein expressed in bacteria. The fate of parental nucleocapsids was monitored by immunofluorescence with an appropriate dilution of monoclonal antibody to the nucleocapsid protein. The protein synthesis inhibitor anisomycin was added to the cells 30 min prior to infection, so that the only nucleocapsids protein molecules in the cells were those associated with nucleocapsids of the parental virus. These nucleocapsids were efficiently transported into the nuclei of murine cells expressing the Mx1 protein, indicating that this protein most likely acts after the parental nucleocapsids enter the nucleus. The second possibility was that the murine Mx1 protein might act in the nucleus to inhibit viral mRNA synthesis indirectly via new cap-binding activities that sequestered cellular capped RNAs away from the viral RNA transcriptase. We show that the same array of nuclear cap-binding proteins was present in Mx-positive and Mx-negative cells treated with interferon. Interestingly, a large amount of a 43-kDa cap-binding activity appeared after interferon treatment of both Mx-positive and Mx-negative cells. Hence, the appearance of new cap-binding activities was unlikely to account for the Mx-specific inhibition of viral mRNA synthesis. These results are most consistent with the possibility that the Mx1 protein acts directly to inhibit the viral transcriptase in the nucleus.
Construction of fowlpox virus vectors with intergenic insertions: expression of the beta-galactosidase gene and the measles virus fusion gene
Authors: Spehner D , Drillien R , Lecocq J P
Published: January 2020

A DNA fragment from fowlpox virus cloned on a plasmid vector was modified to contain foreign DNA inserts within an intergenic region. In a first step, a 32-base-pair intergenic region from the fowlpox virus genome corresponding to the position of the thymidine kinase locus in the vaccinia virus genome was enlarged to 55 base pairs by site-directed mutagenesis. A unique restriction endonuclease site introduced upstream of the intergenic region was then used to insert various foreign DNA fragments. The lacZ gene encoding beta-galactosidase and the measles virus gene encoding the fusion protein were positioned downstream of two vaccinia virus p7.5 promoter elements in either a direct repeat or inverted repeat orientation. Foreign DNA inserts contained within the fowlpox virus sequence were transferred to the viral genome by homologous recombination occurring in cells infected with a fowlpox virus temperature-sensitive mutant and transfected with both wild-type viral DNA and plasmid DNA. Recombinant viruses were selected for the expression of beta-galactosidase activity by screening for blue plaques in the presence of a chromogenic substrate. Stable recombinants expressing both the lacZ gene and the unselected measles gene were obtained when the p7.5 promoter was present as an inverted repeat. However, when the p7.5 promoter was in the direct repeat orientation, viral recombinants which initially expressed both gene inserts readily deleted the lacZ gene flanked by the promoter repeat. The methods described enable precise insertion and deletion of foreign genes in the fowlpox virus genome and could be applied to other intergenic regions of the same virus as well as other poxviruses.
Point mutations in the Moloney murine leukemia virus enhancer identify a lymphoid-specific viral core motif and 1,3-phorbol myristate acetate-inducible element
Authors: Speck N A , Renjifo B , Hopkins N
Published: January 2020

The transcriptional enhancer of the Moloney murine leukemia virus (MoMLV) is organized as a 75-base-pair repeat, and in each copy of the repeat there are multiple binding sites for nuclear factors. We have introduced point mutations into each of the known nuclear factor-binding sites in the MoMLV enhancer, in both copies of the direct repeat, and have analyzed the transcriptional activity conferred by the mutated enhancers by transient-expression assays in both hematopoietic and nonhematopoietic cell lines. Mutation of individual binding sites in the MoMLV enhancer has moderate effects (less than 2-fold to 20-fold) on transcription in six independent cell lines. Several mutations decreased transcription from the MoMLV enhancer ubiquitously (the leukemia virus factor b site and the glucocorticoid response element), whereas others affected transcription specifically in lymphoid cell lines (core motif) or, more significantly, in fibroblasts (nuclear factor 1 site). The transcriptional activity of the MoMLV enhancer can be induced 8- to 10-fold by 1,3-phorbol myristate acetate in Jurkat T cells. Mutations in any of three adjacent binding sites (leukemia virus factor b and c sites and the core motif) within a 28-base-pair region in the center of the direct repeat sequence of the MoMLV enhancer completely attenuate the response to 1,3-phorbol myristate acetate.
Feline leukemia virus infection as a potentiating cofactor for the primary and secondary stages of experimentally induced feline immunodeficiency virus infection
Authors: Pedersen N C , Torten M , Rideout B , Sparger E , Tonachini T , Luciw P A , Ackley C , Levy N , Yamamoto J
Published: January 2020

Preexistent feline leukemia virus (FeLV) infection greatly potentiated the severity of the transient primary and chronic secondary stages of feline immunodeficiency virus (FIV) infection. Of 10 FeLV-FIV carrier cats, 5 died of experimentally induced FIV infection, compared with 2 deaths in 10 cats infected only with FeLV and 1 death in 7 cats infected only with FIV. FIV-infected cats with preexistent FeLV infections developed severe depression, anorexia, fever, diarrhea, dehydration, weight loss, and leukopenia 4 to 6 weeks after infection and were moribund within 2 weeks of the onset of signs, whereas cats infected only with FIV developed much milder self-limiting gross and hematologic abnormalities. Pathologic findings in dually infected cats that died were similar to those observed previously in cats dying from uncomplicated primary FIV infection but were much more widespread and severe. Coinfection of asymptomatic FeLV carrier cats with FIV did not increase the levels of FeLV p27 antigen present in their blood over that seen in cats infected with FeLV alone. The amount of proviral FIV DNA was much higher, however, in dually infected cats than in cats infected only with FIV; there was a greater expression of FIV DNA in lymphoid tissues, where the genome was normally detected, and in nonlymphoid tissues, where FIV DNA was not usually found. Dually infedted cats that recovered from the primary stage of FIV infection remained more leukopenic than cats infected with FIV or FeLV alone, and their CD4+/CD8+ T-lymphocyte ratios were inverted. One of these cats developed what was considered to be an opportunistic infection. It was concluded, therefore, that a preexistent FeLV infection in some way enhanced the expression and spread of FIV in the body and increased the severity of both the resulting transient primary and chronic secondary stages of FIV infection. This study also demonstrated the usefulness of the FIV model in studying the role of incidental infectious diseases as cofactors for immunodeficiency-causing lentiviruses.
Simian immunodeficiency virus inhibits bone marrow hematopoietic progenitor cell growth
Authors: Watanabe M , Ringler D J , Nakamura M , DeLong P A , Letvin N L
Published: January 2020

The pathogenic mechanisms underlying the depressed hematopoietic functions seen in human immunodeficiency virus-infected individuals were explored in rhesus monkeys infected with the simian immunodeficiency virus of macaques (SIVmac). Bone marrow hematopoietic progenitor cell colony formation, both granulocyte/macrophage (CFU-GM) and erythrocyte (BFU-E), was shown to be decreased in number in SIVmac-infected rhesus monkeys. SIVmac was readily isolated from bone marrow cells of infected monkeys and was shown to be harbored in macrophages rather than T lymphocytes. The in vitro infection of normal bone marrow cells by SIVmac inhibited colony formation. A striking in vivo correlation between increased SIVmac load in bone marrow cells and decreased hematopoietic progenitor cell colony growth was also shown. Finally, inhibition of SIVmac replication in bone marrow macrophages resulted in increased progenitor cell colony growth from bone marrow cells. These results suggest that the infection of bone marrow macrophages by the acquired immunodeficiency syndrome (AIDS) virus may contribute to depressed bone marrow hematopoietic progenitor cell growth. Moreover, inhibition of AIDS virus replication in these macrophages might induce significant improvement in hematopoietic function.
Very high frequency of reversion to guanidine resistance in clonal pools of guanidine-dependent type 1 poliovirus
Authors: de la Torre J C , Wimmer E , Holland J J
Published: January 2020

We have carefully examined the frequency of guanidine-resistant revertants in six different clonal pools of guanidine-dependent mutants of type 1 poliovirus. The mutation frequency was (6.5 +/- 6.3) x 10(-4) (with all amino acid substitutions occurring at position 227). The minimal corrected base substitution frequency per single nucleotide site in the codon for amino acid 227 was (2.1 +/- 1.9) x 10(-4).
Processing of the Epstein-Barr virus-encoded latent membrane protein p63/LMP
Authors: Moorthy R , Thorley-Lawson D A
Published: January 2020

We have analyzed the processing of the Epstein-Barr virus-encoded latent membrane protein (p63/LMP) in lymphoblastoid cell lines, Burkitt's lymphoma cell lines, and rodent fibroblasts transfected with the p63/LMP gene. Pulse-chase analysis by immunoprecipitation, under denaturing conditions, reveals a half-life of 2 h. This is due to turnover in the plasma membrane with cleavage of the protein, resulting in a 25,000-molecular-weight (p25) fragment derived from the carboxy-terminal portion of LMP. This fragment is rich in proline and acidic amino acids and sheds into the cytoplasm, where it appears to accumulate, being present in a six- to sevenfold molar excess over p63/LMP in immunoprecipitation analyses. p25 is, like p63/LMP, also phosphorylated (pp25) on serine and threonine residues, in the same ratio and to approximately the same extent as the intact p63/LMP molecule. Amino acid sequence analysis and carboxy-terminal labeling suggest that p25 is derived through a single cleavage adjacent to the sequence LGAPGGGPDNGPQDPD.
Construction and characterization of two infectious molecular clones of encephalomyocarditis virus
Authors: Naviaux R K , Cohen S H , Vanden Brink K M , Jordan G W
Published: January 2020

We constructed and characterized two infectious molecular clones of encephalomyocarditis (EMC) virus. Both constructs, pDL and pDA, were assembled from five overlapping cDNA clones derived from the diabetogenic variant of EMC virus (EMC-D) and from two synthetic oligonucleotide cartridges. pDA contained a single point mutation at position 1720 within the "puff" region of capsid protein 1AB that was derived from the nondiabetogenic variant of EMC virus (EMC-B). This point mutation resulted in an amino acid substitution of arginine (EMC-B) for lysine (EMC-D). Our construction illustrates two novel findings: (i) that the problem of stably cloning long poly(C) tracts of EMC virus can be circumvented by the use of a shortened, synthetic, poly(dC-dG) oligonucleotide cartridge, and (ii) that a single point mutation in the puff region of the capsid protein 1AB leads to change in its electrophoretic mobility and to a change in the plaque size of recombinant virus.
Transcription of the S gene in transgenic mice is associated with hypomethylation at specific sites and with DNase I sensitivity
Authors: Pourcel C , Tiollais P , Farza H
Published: January 2020

The methylation status of hepatitis B virus (HBV) DNA was investigated in different organs from two strains of transgenic mice (E36 and E11) expressing the hepatitis B surface antigen (HBsAg) gene specifically in the liver. Specific sites in the S gene were shown to be methylated in all the organs of adult mice except in the liver. These sites were methylated in 14-day-old fetal liver and were progressively demethylated during development and after birth. In one strain in which HBsAg expression is lost upon transmission by females, extensive de novo methylation of the transgene was detected in the livers and bodies of 14-day-old fetuses from transgenic females. The extent of methylation was such that activation of the gene was no longer possible. DNase I-hypersensitive sites were detected in the enhancer region of HBV in the liver of HBsAg-positive mice but not in HBsAg-negative progeny of E36 females. These data indicated that in two independent transgenic lines, HBV sequences are reproducibly activated in the developing liver along with cellular liver-specific genes and that transcription is associated with demethylation at specific sites in the S gene and with DNase hypersensitivity.
Rotavirus genome segment 4 determines viral replication phenotype in cultured liver cells (HepG2)
Authors: Ramig R F , Galle K L
Published: January 2020

One-step growth determinations were performed with five strains of rotavirus in HepG2, a cell line derived from human liver. Three virus strains (SA11-C13, SA11-C14, and RRV) replicated in HepG2 cells and attained yields 10- to 100-fold above input titers. Two virus strains (B223 and SA11-4F) failed to replicate above input titer. Analysis of reassortants that segregated the genes of parental virus pairs able and unable to replicate revealed that the HepG2 cell growth phenotype segregated with genome segment 4. Immunofluorescence analysis of infected HepG2 cells showed that the production of detectable antigen correlated with the growth phenotype and also segregated with genome segment 4. Thus, we conclude that (i) some virus strains were capable of replication in cultured liver cells while other strains could not replicate under identical conditions and that (ii) the inability of some virus strains to replicate resulted from a segment 4-associated block in replication before protein synthesis. These results are discussed in terms of what is known of the functions of VP4.
Protein kinase A-dependent binding of a nuclear factor to the 21-base-pair repeat of the human T-cell leukemia virus type I long terminal repeat
Authors: Poteat H T , Chen F Y , Kadison P , Sodroski J G , Haseltine W A
Published: January 2020

The long terminal repeat (LTR) of the human T-cell leukemia virus type I (HTLV-I) contains an imperfect repeat of 21 nucleotides which governs the response to the virus trans-activator protein tax and to cyclic AMP. In a murine thymocyte cell line defective in the catalytic subunit of protein kinase A, the response of the HTLV-I LTR to cyclic AMP is abolished and the response to tax is substantially diminished. This report shows that a factor present in nuclear extracts of wild-type cells binds to the HTLV-I 21-nucleotide sequence and that this binding activity is missing from the extracts of protein kinase A-defective cells. Treatment of nuclear extracts of protein kinase A-defective cells with the bovine protein kinase A catalytic subunit restores the binding activity, whereas treatment of wild-type nuclear extracts with a protein phosphatase destroys the binding activity. The binding factor is referred to as protein kinase A-dependent factor (PKAF). These results indicate that in murine thymocytes the response of the HTLV-I LTR to cyclic AMP depends upon the binding of a phosphorylated protein to the 21-nucleotide repeat sequence and that the response to tax is partially dependent upon binding of the phosphorylated protein. The results suggest a model in which the phosphorylation of a transcription factor by protein kinase A regulates HTLV-I gene expression.
Low probability of double integration in transformation of nonpermissive cells by polyomavirus
Authors: Oh S Y , Amalfitano A , Friderici K , Chen M C , Fluck M M
Published: January 2020

The fate of polyomavirus genomes in stable transformants was examined in experiments in which rat or hamster cells were infected with mixed viral populations containing either two distinguishable wild types or a wild type plus a transformation-deficient mutant. The results demonstrate that in 90% of the cases in either situation, a single polyomavirus parental genome became integrated into the host genome. Both parents in the mixed infection were present in the same cells and both persisted in the infected cells until transformants appeared, eliminating the possibility that one virus would exclude the other in the early steps of the infection process. We believe that these results can be generalized to transformation events derived from normal single infections. Thus, contrary to previous results for the number of integration sites determined by restriction endonuclease and blot hybridization analysis, it appears that the probability for more than one integration event in transformation by polyomavirus is low (1 in 10). A reinterpretation of previous data is proposed.
Sequence-specific DNA binding by the v-erbA oncogene protein of avian erythroblastosis virus
Authors: Bonde B G , Privalsky M L
Published: January 2020

The v-erbA oncogene, a transduced copy of a thyroid hormone receptor, plays an important role in establishment of the transformed cell phenotype induced by avian erythroblastosis virus. The ability of thyroid hormone receptors to bind to specific sites on chromatin and to thereby modify the expression of adjacent target genes is a crucial element in their mechanism of action in the normal cell. The v-erbA protein also bound at high affinity to a set of DNA fragments recognized by the rat thyroid hormone receptor, but the relative affinity of the v-erbA protein for the different binding sites was distinct from that previously reported for the thyroid hormone receptors.
Large T antigens of many polyomaviruses are able to form complexes with the retinoblastoma protein
Authors: Dyson N , Bernards R , Friend S H , Gooding L R , Hassell J A , Major E O , Pipas J M , Vandyke T , Harlow E
Published: January 2020

Stable protein complexes between the large T antigens of mouse, monkey, baboon, or human polyomaviruses and the retinoblastoma protein were detected by an in vitro coimmunoprecipitation assay. All of the large T antigens tested were able to bind to both human and mouse retinoblastoma polypeptides, showing that these interactions have been conserved during evolution.
The BamHI J fragment (0.706 to 0.737 map units) of pseudorabies virus is transcriptionally active during viral replication
Authors: Cheung A K
Published: January 2020

The BamHI J fragment of the pseudorabies virus (PRV) genome has not been associated with any viral transcripts during viral replication. In this report, data are presented to show that a portion of BamHI-J is transcribed during a productive infection. Four oligo(dT)-cellulose-selected RNA species were detected by hybridization with probes derived from BamHI-J. These RNAs were partially colinear, and they were transcribed in the opposite orientation with respect to the immediate-early gene (IE180) of PRV. At least one of the transcripts overlapped (antisense) the coding sequence of IE180 by 450 nucleotides. Expression of these RNAs was sensitive to phosphonoacetic acid, indicating that they are transcripts of PRV late genes. There were several similarities between these RNAs and the latency-associated transcripts detected in the trigeminal ganglia of swine latently infected with PRV (A. K. Cheung, J. Virol. 63: 2908-2913, 1989).
An early postinfection signal mediated by monoclonal anti-beta 2 microglobulin antibody is responsible for delayed production of human immunodeficiency virus type 1 in peripheral blood mononuclear cells
Authors: Corbeau P , Devaux C , Kourilsky F , Chermann J C
Published: January 2020

We recently found (C. Devaux, J. Boucraut, G. Poirier, P. Corbeau, F. Rey, M. Benkirane, B. Perarneau, F. Kourilsky, and J.C. Chermann, submitted for publication) a latency in the human immunodeficiency virus (HIV) type 1 cytopathic effect in the human T-cell lymphotropic virus type I immortalized T-cell line MT4 that was mediated by anti-beta 2 microglobulin (beta 2m) monoclonal antibodies (MAb). Here we describe a delay in viral particle production in peripheral blood mononuclear cells (PBMC) that was mediated by three (B1-1G6, B2-62-2, and HC11-151-1) of four anti-beta 2m MAb tested, the nonefficient MAb (C21-48A) being specific for an epitope on beta 2m that was masked by association with the human leukocyte antigen class I heavy chain. Experiments were designed to determine the mechanism of interference. PBMC incubated with anti-beta 2m MAb before viral exposure were not protected from HIV infection. In addition, anti-beta 2m MAb were not efficient in preventing syncytium formation between HIV-infected PBMC and CD4-positive MT4 cells. In contrast, anti-beta 2m MAb treatment of freshly infected PBMC significantly delayed HIV production in these cells. The window of cell sensitivity to anti-beta 2m MAb treatment took place during a very early post-HIV-binding stage. The possible mechanism of anti-beta 2m MAb action is discussed.
Potential retroviral RNAs in Creutzfeldt-Jakob disease
Authors: Murdoch G H , Sklaviadis T , Manuelidis E E , Manuelidis L
Published: January 2020

The molecular nature of the related infectious agents that cause Creutzfeldt-Jakob disease (CJD) and scrapie is poorly understood, and an agent-specific nucleic acid genome has not yet been identified. Several biological manifestations of these agents resemble those seen in retrovirus-induced diseases. We therefore attempted to identify an agent-specific retrovirus-like RNA transcript in CJD infectious fractions. A series of synthetic oligonucleotides complementary to known mammalian retroviral primer binding sites were used in a primer extension assay. Substrate nucleic acids isolated from partially purified hamster brain CJD infectious fractions and from parallel normal brain fractions were compared with total starting brain RNA. This sensitive exogenous strong-stop reaction revealed that CJD infectious fractions contained a series of potential retroviral RNAs including apparent transcripts of endogenous hamster IAP genes. Most transcripts selectively recovered in the fractions were substantially protected from micrococcal nuclease digestion, and at least one substrate RNA, consistent with an intracisternal A particle, was packaged in a form that had the same buoyant density as CJD infectivity. Although a completely CJD-specific transcript was not identified, the copurification of potential retroviral transcripts with CJD infectivity suggests that models of disease involving retrovirus-like nucleic acid elements deserve further consideration.
Guinea pig cytomegalovirus immediate-early transcription
Authors: Yin C Y , Gao M , Isom H C
Published: January 2020

Guinea pig cytomegalovirus (GPCMV) immediate-early (IE) gene expression was analyzed. GPCMV IE RNA was defined as RNA obtained from GPCMV-infected guinea pig cells treated with cycloheximide for 1 h before infection and for 4 h postinfection. Mapping studies showed that GPCMV IE genes are located at several distinct sites on the GPCMV genome. A total of 17 GPCMV IE transcripts were identified, and 9 IE transcripts coded for by three specific regions of the genome (regions I, II, and III) were characterized in detail. A series of recombinant DNA clones were generated to identify the nine IE transcripts. Three of the IE transcripts from region I and three from region III were transcribed in the same direction from overlapping sequences. The 2.0-kilobase (kb) transcript encoded by the EcoRI E DNA fragment (region II) was the most abundant IE GPCMV transcript. The cloned GPCMV DNA subfragment that was used to identify the region II EcoRI E 2.0-kb transcript did not hybridize to GPCMV early or late RNA, indicating that this transcript is expressed only under IE conditions. Expression of RNAs from the IE genes was also measured during a natural GPCMV infection in the absence of cycloheximide. During the natural infection, the transcripts previously identified under IE cycloheximide block conditions were expressed, and the region II EcoRI E 2.0-kb transcript was the most abundant transcript at 1 h postinfection. In addition, a rise and fall in RNA levels was observed during the natural infection, demonstrating the transient nature of expression of these transcripts. We conclude that GPCMV IE gene expression is complex, involving a reasonably large number of genes, and demonstrates some similarities with IE transcription by other CMVs.
Association of p62c-yes with polyomavirus middle T-antigen mutants correlates with transforming ability
Authors: Kornbluth S , Cheng S H , Markland W , Fukui Y , Hanafusa H
Published: January 2020

A number of mutants of polyomavirus middle T antigen (MTag) were constructed into replication-competent avian retroviruses. To assess the ability of these MTag variants to transform and to associate with the avian p60c-src and p62c-yes proto-oncogene products, we used these viruses to infect chicken embryo fibroblasts. We found that the ability of individual mutant MTags to associate with p62c-yes correlated well with the ability of these mutants to transform, as has been previously shown for the association of MTag with p60c-src. All transformation-competent mutant MTags retained the ability to complex with p62c-yes. Two transformation-defective mutants, RX67 and RX68, which could weakly associate with p60c-src, were unable to associate with p62c-yes.dl1015, a transformation-defective mutant which could associate with p60c-src and with a phosphatidylinositol kinase activity, was also able to associate with p62c-yes. Therefore, some as yet unmeasured biochemical property is defective in this mutant.
Proteins of viral and cellular origin bind to the Aleutian disease virus (ADV) DNA 3'-terminal hairpin: presentation of a scheme for encapsidation of ADV DNA
Authors: Willwand K , Kaaden O R
Published: January 2020

We have observed the binding of viral and cellular proteins to the Aleutian disease virus (ADV) 3' terminus of replicative-form DNA. Gel retardation assays showed specific band shifts produced by whole-cell extracts from either ADV-infected or uninfected cells, as well as band reduction produced by ADV capsids. In all cases, binding was confined to the turnaround, T-shaped terminal form; no binding to the extended conformation of replicative-form DNA was detected. This indicates the importance of the T-shaped secondary structure in protein recognition. We have previously reported the binding of a 3'-terminal ADV DNA restriction fragment to the ADV capsid protein VP1 (K. Willwand and O.-R. Kaaden, Virology 166:52-57, 1988). Here we show that the region between nucleotides 14 and 102 on the ADV genome is required for binding. It is suggested that the VP1-DNA interaction mediates the binding of ADV DNA to empty viral capsids and that this is followed by displacement synthesis and packaging of ADV progeny DNA. A scheme for the possible mechanism of this process is presented.
Role of matrix protein in cytopathogenesis of vesicular stomatitis virus
Authors: Blondel D , Harmison G G , Schubert M
Published: January 2020

The matrix (M) protein of vesicular stomatitis virus (VSV) plays an important structural role in viral assembly, and it also has a regulatory role in viral transcription. We demonstrate here that the M protein has an additional function. It causes visible cytopathic effects (CPE), as evidenced by the typical rounding of polygonal cells after VSV infection. We have analyzed a temperature-sensitive mutant of the M protein of VSV (tsG33) which is defective in viral assembly and which fails to cause morphological changes of the cells after infection at the nonpermissive temperature (40 degrees C). Interestingly, this defect in viral assembly as well as the CPE were reversible. Microinjection of antisense oligonucleotides which specifically inhibit M protein translation also inhibited the occurrence of CPE. Most importantly, when cells were transfected with a cDNA encoding the temperature-sensitive M protein of tsG33, no CPE was observed at the nonpermissive temperature. However, when these cells were shifted to the permissive temperature (32 degrees C), they rounded up and detached from the dish. These results demonstrate that M protein in the absence of the other viral proteins causes rounding of the cells, probably through a disorganization of the cytoskeleton. The absence of CPE at the nonpermissive temperature is correlated with an abnormal dotted staining pattern of M in these cells, suggesting that the mutant M protein may self-aggregate or associate with membranes rather than interact with cytoskeletal elements.
Aleutian disease parvovirus infection of mink and ferrets elicits an antibody response to a second nonstructural viral protein
Authors: Porter D D , Porter H G , Larsen A E
Published: January 2020

A second nonstructural protein of the Aleutian disease parvovirus was predicted from nucleotide sequence analysis and a detailed transcription map. Western immunoblotting analysis showed that infected mink and ferrets show an antibody response to this predicted protein.
Autonomous expression of RTVL-H endogenous retroviruslike elements in human cells
Authors: Wilkinson D A , Freeman J D , Goodchild N L , Kelleher C A , Mager D L
Published: January 2020

Northern (RNA) blot analysis of RNA from various human cell lines and tissues has demonstrated that elements belonging to the RTVL-H family of human endogenous retroviruslike sequences are expressed in several cell types. The highest levels of RTVL-H-related RNAs were observed in teratocarcinoma cell line NTera2D1, HeLa cells, two bladder carcinoma cell lines, and normal amniotic tissue. Expression was also observed in normal chorion and in some other cell lines. The RTVL-H transcription pattern varied among the different cell types, but several expressed a unit-length 5.6-kilobase transcript. Characterization of cDNA clones corresponding to transcripts present in NTera2D1 cells indicates that the complex transcription pattern observed in these cells is generated by the following: (i) transcription of both full-length and deleted genomic elements, which is initiated within the 5' long terminal repeat (LTR) and, in all but one case, polyadenylated in the 3' LTR; (ii) the splicing of both unit-length transcripts and transcripts from a deleted element; (iii) transcription involving solo LTR sequences; and (iv) transcription which, in one case, reads through the 3' LTR into flanking cellular sequences. Sequence data obtained from 25 cDNA clones revealed that at least 13 RTVL-H elements are expressed in NTera2D1 cells. The positions of several termination codons within the pol region are the same among nine different elements, indicating that an ancestral RTVL-H element bearing these mutations dispersed within the genome. We also found that RTVL-H expression varied among samples of amnion and chorion tissue isolated from different individuals. These findings demonstrate that regulated autonomous expression of RTVL-H sequences occurs in human cells.
Induction of immunoglobulin G Fc receptors by recombinant vaccinia viruses expressing glycoproteins E and I of herpes simplex virus type 1
Authors: Bell S , Cranage M , Borysiewicz L , Minson T
Published: January 2020

Glycoprotein E (gE) of herpes simplex virus type 1 (HSV-1) will bind immunoglobulin G (IgG) (Fc) affinity columns (R. B. Bauke and P. G. Spear, J. Virol. 32:779-789, 1979), but recent evidence suggests that the HSV-1 Fc receptor is composed of a complex of gE and glycoprotein I (gI) and that both gI and gE are required for Fc receptor activity (D. C. Johnson and V. Feenstra, J. Virol. 61:2208-2216, 1987; D. C. Johnson, M. C. Frame, M. W. Ligas, A. M. Cross, and N. D. Stow, J. Virol. 62:1347-1354, 1988). We have expressed gE and gI, either alone or in combination, on the surface of HeLa cells by using recombinant vaccinia viruses and have measured Fc receptor activity by Fc-rosetting or IgG-binding assays. Expression of gE alone resulted in the induction of Fc receptor activity, while expression of gI alone gave no detectable Fc binding. Coexpression of gE and gI resulted in higher levels of IgG binding than did expression of gE alone, despite the fact that under conditions of coexpression, the levels of surface gE were reduced. We propose that gE and gI together form a receptor of higher affinity than gE alone and that HSV-1 therefore has the potential to induce two Fc receptors of different affinities.
Activation of multiple genes by provirus integration in the Mlvi-4 locus in T-cell lymphomas induced by Moloney murine leukemia virus
Authors: Tsichlis P N , Lee J S , Bear S E , Lazo P A , Patriotis C , Gustafson E , Shinton S , Jenkins N A , Copeland N G , Huebner K
Published: January 2020

Moloney murine leukemia virus-induced rat T-cell lymphomas harbor proviruses integrated near c-myc and near Mlvi-1/Mis-1/Pvt-1, another locus of common integration which maps 270 kilobases 3' of c-myc. In this report, we present the characterization of a new locus of common integration in Moloney murine leukemia virus-induced T-cell lymphomas (Mlvi-4) which maps 30 kilobases 3' of c-myc, between c-myc and Mlvi-1. The Mlvi-4 locus, whose chromosomal map location is conserved in rats, mice, and humans, is also the target of chromosomal rearrangements in a variety of animal and human tumors. Evidence presented elsewhere shows that provirus integration in Mlvi-4 enhances the expression of c-myc and Mlvi-1 by cis-acting mechanisms operating over long distances of genomic DNA. In this manuscript, we show that provirus integration in the Mlvi-4 locus activates, by promoter insertion, one additional gene which maps immediately 3' to the cluster of the Mlvi-4 proviruses and which is transcribed in the same orientation as c-myc, giving rise to 3- and 10-kilobase mRNA transcripts. The Mlvi-4 gene is also expressed in normal thymus and spleen at very low levels, giving rise to 3- and 5.5-kilobase messages. Although Mlvi-4 is expressed in normal thymus, it is not expressed in Moloney murine leukemia virus-induced T-cell lymphomas corresponding to several stages of T-cell differentiation, but lacking a provirus in this locus. This suggests that Mlvi-4 may be expressed only in a subpopulation of T cells. We conclude that provirus insertion in Mlvi-4 activates c-myc and two additional genes, Mlvi-1 and Mlvi-4, whose expression is restricted to, and may be developmentally regulated in, T cells. Since Mlvi-4 is the target of genetic changes in a great variety of human and animal neoplasms, these results are critical for our understanding of oncogenesis.
Mutational analysis of the human immunodeficiency virus type 1 env gene product proteolytic cleavage site
Authors: Bosch V , Pawlita M
Published: January 2020

The structural requirements for proteolytic cleavage of the human immunodeficiency virus type 1 env gene product, gp160, to gp120 and gp41 have been assessed by specific mutagenesis of the sequence Lys Ala Lys Arg Arg Val Val Glu Arg Glu Lys Arg located between amino acids 500 and 511, i.e., at the putative C terminus of gp120. The basic amino acids underlined have been mutated, individually and in combination, to neutral amino acids, and the cleavability of the mutated env gene products was examined after expression in CV-1 cells. The results show that the replacement of Arg-511 (cleavage presumably occurs C terminal to this amino acid) with Ser completely abolishes recognition and cleavage by the cellular protease(s), i.e., the remaining basic amino acids in the vicinity do not serve as alternative substrates. However, Arg-508 and Lys-510 are important features of the recognition site since, when they are individually changed to neutral amino acids, cleavage is severely impaired. The basic amino acids 500, 502, and 504 are, individually, not important for cleavage, since their individual replacement by neutral amino acids does not impair cleavage. However, when all four basic amino acids 500, 502, 503, and 504 are changed to neutral amino acids, cleavage is almost completely abolished. This shows that the sequence Arg Glu Lys Arg at the cleavage site is alone not sufficient for cleavage but that a contribution of other amino acids is required, whether the other amino acids provide a basic character or a certain structure in the vicinity of the cleavage site. When noncleavable or poorly cleavable mutant env genes are expressed from the infectious plasmid pNL4-3 in CD4+ human lymphoblastoid cells, noninfectious virus, incapable of spread throughout the culture, is produced.
The average number of molecules of Epstein-Barr nuclear antigen 1 per cell does not correlate with the average number of Epstein-Barr virus (EBV) DNA molecules per cell among different clones of EBV-immortalized cells
Authors: Sternås L , Middleton T , Sugden B
Published: January 2020

Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein required to support latent replication of Epstein-Barr virus (EBV). To assess the likelihood that EBNA-1 regulates the amount of EBV DNA in a cell, we measured the average numbers of EBNA-1 molecules and EBV DNA molecules per cell in different clones of cells. The amount of EBNA-1 protein present in recently established lymphoblastoid cell lines was measured with affinity-purified anti-EBNA-1 antibodies, and viral DNA was measured by nucleic acid hybridization. The average levels of EBNA-1 protein varied little between these cell lines, whereas the average amount of viral DNA present varied substantially; consequently, these numbers were not correlated. There is no apparent relationship between amounts of EBNA-1 and viral DNA.
Promoter evolution in BK virus: functional elements are created at sequence junctions
Authors: Markowitz R B , Tolbert S , Dynan W S
Published: January 2020

The archetypal strain of BK virus directed very little early gene expression compared with rearranged laboratory strains of the virus. One laboratory strain that was analyzed in detail contained newly created AP-1 binding enhancer modules spanning the junction between adjacent sequence repeats. Introduction of these sequences into the archetype activated the previously quiescent early promoter.
Alpha/beta interferons fail to induce antiviral activity from within the nucleus
Authors: Riviere I , de Maeyer-Guignard J
Published: January 2020

Electrophoretically pure murine alpha/beta interferons (IFN-alpha/beta) were microinjected directly into the nuclei of mouse L cells, each nucleus receiving 10 fl containing about 20,000 (IFN) molecules, an amount sufficient to induce the antiviral state when added to the culture medium of control cells. Three, six or 24 h after intranuclear delivery, the cells were challenged with vesicular stomatitis virus or Semliki Forest virus and the appearance of cytopathic effects was scored for each individual cell. The scoring of more than 1,000 intranuclearly injected cells in nine different experiments showed unambiguously that the intranuclear delivery of IFN-alpha/beta did not induce the antiviral state. The results argue strongly against the physiological importance of high-affinity nuclear binding sites for native IFN that have been recently described (V. M. Kushnaryov, H. S. MacDonald, G. P. Lemense, J. Debruin, J. J. Sedmak, and S. E. Grossberg, Cytobios 53:185-197, 1988). Together with earlier results of other groups describing the lack of IFN activity after intracytoplasmic injection (Y. Higashi and Y. Sokawa, J. Biochem. 91:2021-2028, 1982; G. Huez, M. Silhol, and B. Lebleu, Biochem. Biophys. Res. Commun. 110:155-160, 1983), these results lend weight to the hypothesis that the binding of IFN-alpha/beta to the plasma membrane receptor is sufficient to set into motion the complex mechanism of transmembrane signalling without requiring internalization of the bound IFN molecules.
T- and B-lymphocyte responses to human immunodeficiency virus (HIV) type 1 in macaques immunized with hybrid HIV/hepatitis B surface antigen particles
Authors: Michel M L , Mancini M , Riviere Y , Dormont D , Tiollais P
Published: January 2020

Recombinant human immunodeficiency virus (HIV)/hepatitis B surface antigen (HBsAg) subviral particles of dual antigenicity and immunogenicity were obtained by fusing 84 amino acids of the HIV type 1 external envelope glycoprotein within the pre-S2 part of the hepatitis B middle protein (M.-L. Michel, M. Mancini, E. Sobczak, V. Favier, D. Guétard, E.-M. Bahraoui, and P. Tiollais, Proc. Natl. Acad. Sci. USA 85:7957-7961, 1988). We now describe the humoral and cellular immune response of rhesus monkeys immunized with these hybrid particles. Macaque antisera raised by subcutaneous injections of the HIV/HBsAg particles were shown to be specific for HIV in peptide-binding assays. Moreover, we were able to generate in these vaccinated animals a T-cell-proliferative response to both parts of the hybrid particle, i.e., HIV and HBsAg. These results establish the presence of a T-cell epitope in this HIV segment, which has been shown previously (L.A. Lasky, G. Nakamura, D. H. Smith, C. Fennie, C. Shimasaki, E. Patzer, P. Berman, T. Gregory, and D. J. Capon, Cell 50:975-985, 1987) to be an important domain involved in the binding of the virus to its cellular receptor, the CD4 molecule. This work demonstrates the feasibility of using the HBsAg subviral particle as a carrier protein for the presentation of foreign immunogenic epitopes to the immune system.
Characterization of a transpositionally active Ty3 element and identification of the Ty3 integrase protein
Authors: Hansen L J , Sandmeyer S B
Published: January 2020

Ty3 is a Saccharomyces cerevisiae retrotransposon associated with tRNA genes. Two Ty3 elements have been cloned and characterized. The complete nucleotide sequence for one element, Ty3-2, was reported previously (L. J. Hansen, D. L. Chalker, and S. B. Sandmeyer, Mol. Cell. Biol. 9:5245-5256, 1988). However, this element is incapable of autonomous transposition. The complete DNA sequence of a transpositionally competent Ty3 element, Ty3-1, is presented here. Its sequence translates into two overlapping open reading frames, TYA3-1 and TYB3-1, which encode proteins with homology to the proteins specified by the retroviral gag and pol genes, respectively. Comparison of the Ty3-1 nucleotide sequence to Ty3-2 suggests that the TYB3-2 open reading frame of Ty3-2 is truncated by the deletion of a single nucleotide, which causes a frameshift mutation. Restoration of the reading frame with insertion of a single adenine by site-directed mutagenesis converted Ty3-2 into a transpositionally active element, Ty3-2(+ A). Western blot analysis with antibodies made against synthetic peptides identified integrase (IN) proteins in viruslike particle preparations from cells expressing Ty3 elements. Cells expressing Ty3-1 and Ty3-2 (+A) produce antibody-reactive proteins with approximate molecular masses of 61 and 58 kilodaltons (kDa), while cells expressing Ty3-2 produce reactive proteins of approximately 52 and 49 kDa. Together, these data show that the 61- or 58-kDa protein, or both, provides the integrase function of Ty3.
Activation of the E2F transcription factor in adenovirus-infected cells involves E1A-dependent stimulation of DNA-binding activity and induction of cooperative binding mediated by an E4 gene product
Authors: Raychaudhuri P , Bagchi S , Neill S D , Nevins J R
Published: January 2020

Previous experiments have demonstrated that the DNA-binding activity of the E2F transcription factor is increased upon adenovirus infection and that both the E1A and E4 genes are required for activation. In this study, we demonstrated that this enhanced binding of E2F to the E2 promoter is the result of two events. (i) There is stimulation of the DNA-binding activity of the E2F factor; this stimulation is E1A dependent but independent of E4. (ii) There is also induction of a stabilized interaction between E2F molecules bound to adjacent promoter sites; induction of stable E2F binding requires E4 gene function. This two-step activation process was also demonstrated in vitro. A heat-stable fraction from extracts of adenovirus-infected cells, which contains the 19-kilodalton E4 protein, was capable of stimulating stable E2F binding in an ATP-independent manner and appeared to involve direct interaction of the E4 protein with E2F. An extract from virus-infected cells devoid of the E4 19-kilodalton protein stimulated E2F DNA binding without forming the stable complex. This reaction required ATP. We conclude that activation of E2F during adenovirus infection is a two-step process involving a change in both the DNA-binding activity of the factor and the capacity to stabilize the interaction through protein-protein contacts.