bluuebunny/crossref_metadata_2025 huggingface dataset:
Rotavirus SA11 genome segment 11 protein is a nonstructural phosphoprotein
Authors: Welch S K , Crawford S E , Estes M K
Published: January 2020

We investigated properties of the rotavirus genome segment 11 protein. A rotavirus SA11 genome segment 11 cDNA which contains the entire coding region was sequenced and inserted into the baculovirus transfer vector pVL941. Recombinants containing gene 11 cDNA were selected, and the gene 11 product expressed in Spodoptera frugiperda cells infected with these recombinants was inoculated into guinea pigs to produce hyperimmune antiserum. Characterization of the antiserum showed that it recognized a primary translation product with a molecular weight of 26,000 (26K protein) in recombinant-infected insect cells, in SA11-infected monkey kidney cells, and in cell-free translation reactions programmed with SA11 mRNA. A modified 28K product was also detected but only in SA11-infected monkey kidney cells. The 26K 28K proteins were shown to be phosphorylated in infected monkey kidney cells, and the 26K protein was phosphorylated in insect cells. We were unable to identify what type of modification caused the molecular weight shift to 28,000 in infected monkey kidney cells. Large amounts of the gene 11 product were detected by immunofluorescence in discrete foci in the cytoplasm of infected monkey kidney cells. Viruses of all known serotypes were also detected by immunofluorescence by using hyperimmune antiserum to the SA11 gene 11 product. The antiserum reacted with particle-depleted cytosol fractions but did not react with purified virus particles by immunoprecipitation or immunoblotting; it also did not neutralize virus infectivity in plaque reduction neutralization assays. Therefore, we conclude that the primary gene 11 product is a nonstructural phosphoprotein which we designated NS26.
Effect of infection with the ts22 mutant of Semliki Forest virus on development of the central nervous system in the fetal mouse
Authors: Mabruk M J , Glasglow G M , Flack A M , Folan J C , Bannigan J G , Smyth J M , O'Sullivan M A , Sheahan B J , Atkins G J
Published: January 2020

The A7 strain of Semliki Forest virus induces rapid fetal death in pregnant mice, whereas the ts22 mutant derived from it is teratogenic for a proportion of fetuses. Both A7 and ts22 induce viremia and infect the central nervous systems and fetuses of pregnant mice. Using immunogold-silver staining, a cDNA probe for a Semliki Forest virus nonstructural sequence, and a riboprobe derived from the same sequence, we showed that the skin and musculoskeletal systems of fetuses from mothers infected with ts22 were often heavily infected but the central nervous systems were not labeled before day 17 of pregnancy. Damage to the neural tube, including open-neural-tube defects, was detected in fetuses following infection of the mother at days 8 and 10 of pregnancy with both A7 and ts22. For ts22, neural tube damage induced by fetal infection before day 17 of pregnancy appeared to be indirect and caused by virus infection of mesenchymal cells surrounding the developing neural tube.
Epstein-Barr virus latent membrane protein: induction of B-cell activation antigens and membrane patch formation does not require vimentin
Authors: Liebowitz D , Kieff E
Published: January 2020

The latent membrane protein (LMP) of Epstein-Barr virus (EBV) forms patches associated with the vimentin intermediate filament system in EBV-transformed lymphoblastoid cell lines, EBV-infected Burkitt's lymphoma cells, and LMP-transfected, EBV-negative Burkitt's lymphoma cells. By gene transfer, LMP induces the expression of vimentin and B-cell activation antigens in EBV-negative Burkitt's lymphoma cells. We have now expressed LMP in an EBV-positive Burkitt's lymphoma cell line, Daudi, which does not express any LMP or vimentin. In these Daudi transfectants, LMP still formed plasma membrane patches in the absence of vimentin. LMP did not resist nonionic detergent extraction in Daudi cells as it does in vimentin-expressing cells. LMP still retained functional activity as judged by induction of B-cell activation antigens. These data indicate that LMP can form plasma membrane patches and induce B-lymphocyte activation independent of vimentin association.
Molecular cloning, characterization, and expression of the Tipula iridescent virus capsid gene
Authors: Tajbakhsh S , Lee P E , Watson D C , Seligy V L
Published: January 2020

The capsid protein is the major structural component of the icosahedral Tipula iridescent virus (TIV) that replicates in cytoplasmic inclusion bodies of insect cells. TIV capsid protein purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was digested with trypsin and fractionated by reverse-phase high-pressure liquid chromatography. A mixed oligonucleotide constructed from the amino acid sequence of a capsid tryptic peptide was used for the identification and cloning of the corresponding gene. The single-copy capsid gene, located on a 2.47-kilobase-pair HindIII TIV genomic fragment, codes for a 464-amino-acid protein (50,831 daltons) with a predicted pI of 6.34. Analysis of total RNA from infected Estigmene acrea cells indicated that the 1.8-kilobase capsid transcript was maximally produced between 14 and 24 h after infection. Transcript mapping by primer extension indicated that the RNA start site was in the A+T-rich TGCTACTAAT sequence, 19 nucleotides upstream from the first ATG codon of the capsid open reading frame. Expression of the TIV capsid protein in infected E. acrea cells was demonstrated by in vivo labeling of total proteins with [35S]methionine, using anti-capsid antiserum as the probe. Capsid protein was also expressed in Escherichia coli cells by using a pUC19 plasmid containing a lacZ-capsid gene fusion.
A 3'-coterminal nested set of independently transcribed mRNAs is generated during Berne virus replication
Authors: Snijder E J , Horzinek M C , Spaan W J
Published: January 2020

By using poly(A)-selected RNA from Berne virus (BEV)-infected embryonic mule skin cells as a template, cDNA was prepared and cloned in plasmid pUC9. Recombinants covering a contiguous sequence of about 10 kilobases were identified. Northern (RNA) blot hybridizations with various restriction fragments from these clones showed that the five BEV mRNAs formed a 3'-coterminal nested set. Sequence analysis revealed the presence of four complete open reading frames of 4743, 699, 426, and 480 nucleotides, with initiation codons coinciding with the 5' ends of BEV RNAs 2 through 5, respectively. By using primer extension analysis and oligonucleotide hybridizations, RNA 5 was found to be contiguous on the consensus sequence. The transcription of BEV mRNAs was studied by means of UV mapping. BEV RNAs 1, 2, and 3 were shown to be transcribed independently, which is also likely--although not rigorously proven--for RNAs 4 and 5. Upstream of the AUG codon of each open reading frame a conserved sequence pattern was observed which is postulated to function as a core promoter sequence in subgenomic RNA transcription. In the area surrounding the core promoter region of the two most abundant subgenomic BEV RNAs, a number of homologous sequence motifs were identified.
Release of RNA polymerase from vero cell mitochondria after herpes simplex virus type 1 infection
Authors: Tsurumi T , Lehman I R
Published: January 2020

Infection of Vero cells with herpes simplex virus type 1 results in the appearance in soluble extracts of a DNA primase activity. The partially purified enzyme, Mr, approximately 100,000, is identical in resistance to alpha-amanitin, pH profile, Mg2+ dependence, salt sensitivity, and KmATP to the catalytic core of Vero cell mitochondrial RNA polymerase. Moreover, the products synthesized are those expected of an RNA polymerase rather than a DNA primase. Inasmuch as the enzyme is not present in soluble extracts of uninfected Vero cells, we presume that the specific appearance of RNA polymerase in extracts of herpesvirus-infected cells results from infection-induced disruption of the mitochondrial membrane, followed by release of the enzyme into the cytosol.
trans activation of the thymidylate synthase promoter of herpesvirus saimiri
Authors: Lang G , Fleckenstein B
Published: January 2020

Herpesvirus saimiri has been shown to possess a thymidylate synthase (TS) gene that is unusual in its transcriptional regulation. Although TS is believed to be required for viral DNA synthesis, the TS-specific 2.5-kb mRNA was found most abundantly during the late phases of asynchronous virus replication in permissive cultures. To study the kinetics of gene activation, the TS promoter and regulatory sequences were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene. No CAT expression or transcripts were found after transfection of fusion genes into permissive owl monkey kidney (OMK) cells. However, the promoter was strongly activated when CAT plasmids were cotransfected with intact herpesvirus saimiri virion DNA or were transferred to OMK cells that were lytically infected with herpesvirus saimiri or a related herpesvirus, herpesvirus ateles. CAT was expressed at reduced levels in cultures when viral DNA replication was inhibited by phosphonoacetic acid; this indicates that the gene is activated during the delayed-early phase. However, the highest amounts of mRNA were present in the late period of replication. Deletion analyses localized essential response elements for trans activation in the promoter upstream region between nucleotides -311 and -56; they consisted of related tandem repeats and perfect palindromes. A sequence with two overlapping palindromes of 16 and 18 bp was found to be a major target for activation of the herpesvirus saimiri TS promoter. These palindromes did not have any significant homologies with known sequences of herpesviruses or cellular DNA; the 18-bp palindrome had, however, a certain structural similarity with a conserved sequence of the E2-responsive cis sequence that is required for transcription regulation of early papillomavirus genes.
Pattern of polyomavirus replication from infection until tumor formation in the organs of athymic nu/nu mice
Authors: Demengeot J , Jacquemier J , Torrente M , Blangy D , Berebbi M
Published: January 2020

Inbred athymic nu/nu BALB/c mice were injected subcutaneously with the highly oncogenic polyomavirus A2 strain, and the sites of viral DNA replication were determined by whole mouse section hybridization (T. W. Dubensky, E. A. Murphy, and L. P. Villareal, J. Virol. 50:779-783, 1984) and Southern blot analysis. We show that infection is persistent in some epithelial tissues (skin, mammary, and salivary glands), in lymphoid organs (spleen and nodes), and in mesenchymal bone tissue. Only mammary glands and bones were targets for tumor formation. Although the same pattern of infection was observed in males and females, mammary adenocarcinomas were induced exclusively in females, while the frequency of osteosarcomas was similar in both sexes. No viral DNA or lytic lesion was detected in kidney, liver, or lung tissue. The restricted targeting of polyomavirus oncogenicity in nude mice, compared with newborn immunocompetent animals, inoculated via the same route with the same virus strain, therefore does not reflect selective tissue targeting of virus replication. These results further document the influence of the age, immunological status, and genetic background of the host on the pattern of viral infection and tumor formation.
Alignment of U3 region sequences of mammalian type C viruses: identification of highly conserved motifs and implications for enhancer design
Authors: Golemis E A , Speck N A , Hopkins N
Published: January 2020

We aligned published sequences for the U3 region of 35 type C mammalian retroviruses. The alignment reveals that certain sequence motifs within the U3 region are strikingly conserved. A number of these motifs correspond to previously identified sites. In particular, we found that the enhancer region of most of the viruses examined contains a binding site for leukemia virus factor b, a viral corelike element, the consensus motif for nuclear factor 1, and the glucocorticoid response element. Most viruses containing more than one copy of enhancer sequences include these binding sites in both copies of the repeat. We consider this set of binding sites to constitute a framework for the enhancers of this set of viruses. Other highly conserved motifs in the U3 region include the retrovirus inverted repeat sequence, a negative regulatory element, and the CCAAT and TATA boxes. In addition, we identified two novel motifs in the promoter region that were exceptionally highly conserved but have not been previously described.
Improved distribution of antigenic site specificity of poliovirus-neutralizing antibodies induced by a protease-cleaved immunogen in mice
Authors: Roivainen M , Montagnon B , Chalumeau H , Murray M , Wimmer E , Hovi T
Published: January 2020

Previous studies showed that the distribution of antigenic site specificity of neutralizing antibodies to type 3 poliovirus obtained with the inactivated poliovirus vaccine can be deficient as compared with that obtained following poliovirus infection. This observation was shown by the relatively low capacity of sera from inactivated-poliovirus-vaccine-immunized persons to neutralize poliovirus cleaved at antigenic site 1. We investigated possibilities for improving the situation in a mouse model. Balb/c mice were immunized with intact or trypsin-cleaved type 3 poliovirus (Saukett strain). Sera from mice immunized with the intact virus readily neutralized the intact virus but neutralized the cleaved virus only rarely. In contrast, cleaved-virus-immunized mice produced antibodies that were able to neutralize the cleaved virus as well as the intact one. Mice immunized with a 100-fold-higher dose of the intact virus produced significant levels of antibodies to the cleaved virus, too. Somewhat surprisingly, mice immunized with high doses of the cleaved virus produced antibodies specific for the intact loop between beta sheets B and C of VP1 (virion protein 1), which should be cleaved in the immunogen. This was shown by a higher titer of antibodies to intact Saukett virus than to the corresponding cleaved virus, as well as to a type 1/type 3 hybrid poliovirus in which only the BC loop amino acids were derived from type 3 poliovirus. The cleavage-induced enhanced availability of antigenic determinants residing outside the BC loop was also shown by increased neutralization titers of monoclonal antibodies specific for some of these other determinants. These results indicate that by using a trypsin-cleaved type 3 poliovirus as a parenteral immunogen, it is possible to change the distribution of antigenic site specificities of neutralizing antibodies to resemble that following poliovirus infection.
Identification and characterization of the herpes simplex virus type 2 gene encoding the essential capsid protein ICP32/VP19c
Authors: Yei S P , Chowdhury S I , Bhat B M , Conley A J , Wold W S , Batterson W
Published: January 2020

We describe the characterization of the herpes simplex virus type 2 (HSV-2) gene encoding infected cell protein 32 (ICP32) and virion protein 19c (VP19c). We also demonstrate that the HSV-1 UL38/ORF.553 open reading frame (ORF), which has been shown to specify a viral protein essential for capsid formation (B. Pertuiset, M. Boccara, J. Cebrian, N. Berthelot, S. Chousterman, F. Puvian-Dutilleul, J. Sisman, and P. Sheldrick, J. Virol. 63: 2169-2179, 1989), must encode the cognate HSV type 1 (HSV-1) ICP32/VP19c protein. The region of the HSV-2 genome deduced to contain the gene specifying ICP32/VP19c was isolated and subcloned, and the nucleotide sequence of 2,158 base pairs of HSV-2 DNA mapping immediately upstream of the gene encoding the large subunit of the viral ribonucleotide reductase was determined. This region of the HSV-2 genome contains a large ORF capable of encoding two related 50,538- and 49,472-molecular-weight polypeptides. Direct evidence that this ORF encodes HSV-2 ICP32/VP19c was provided by immunoblotting experiments that utilized antisera directed against synthetic oligopeptides corresponding to internal portions of the predicted polypeptides encoded by the HSV-2 ORF or antisera directed against a TrpE/HSV-2 ORF fusion protein. The type-common immunoreactivity of the two antisera and comparison of the primary amino acid sequences of the predicted products of the HSV-2 ORF and the equivalent genomic region of HSV-1 provided evidence that the HSV-1 UL38 ORF encodes the HSV-1 ICP32/VP19c. Analysis of the expression of the HSV-1 and HSV-2 ICP32/VP19c cognate proteins indicated that there may be differences in their modes of synthesis. Comparison of the predicted structure of the HSV-2 ICP32/VP19c protein with the structures of related proteins encoded by other herpes viruses suggested that the internal capsid architecture of the herpes family of viruses varies substantially.
Expression and secretion of the bovine coronavirus hemagglutinin-esterase glycoprotein by insect cells infected with recombinant baculoviruses
Authors: Parker M D , Yoo D , Babiuk L A
Published: January 2020

A cDNA fragment representing the hemagglutinin-esterase (HE) gene of bovine coronavirus (BCV) was inserted into the genome of Autographa californica nuclear polyhedrosis virus. Infection of insect cells with the recombinant virus resulted in the production of a 120-kilodalton disulfide-linked dimeric form of the BCV HE polypeptide. Deletion of the carboxy-terminal hydrophobic domain from the HE polypeptide resulted in secretion of a dimeric form of the truncated HE polypeptide. The acetylesterase activity of the BCV HE was detectable in insect cells expressing the BCV hemagglutinin and was inhibited by two monoclonal antibodies which also inhibit hemagglutination.
Phosphoprotein and nucleocapsid protein evolution of vesicular stomatitis virus New Jersey
Authors: Bilsel P A , Rowe J E , Fitch W M , Nichol S T
Published: January 2020

The entire phosphoprotein (P) and nucleocapsid (N) protein gene sequences and deduced amino acid sequences for 18 selected vesicular stomatitis virus isolates representative of the natural genetic diversity within the New Jersey serotype are reported. Phylogenetic analysis of the data using maximum parsimony allowed construction of evolutionary trees for the individual genes and the combined N, P, and glycoprotein (G) genes of these viruses. Virtually identical rates of nucleotide substitutions were found for each gene, indicating that evolution of these genes occurs at essentially the same rate. Although up to 19 and 17% sequence differences were evident in the P and N genes, respectively, no variation in gene length or evidence of recombinational rearrangements was found. However, striking evolutionary differences were observed among the amino acid sequences of vesicular stomatitis virus New Jersey N, P, and G proteins. The N protein amino acid sequence was the most highly conserved among the different isolates, indicating strong functional and structural constraints. Conversely, the P protein amino acid sequences were highly variable, indicating considerably fewer constraints or greater evolutionary pressure on the P protein. Much of the remarkable amino acid variability of the P protein resided in a hypervariable domain located between amino acids 153 and 205. The variability within this region would be consistent with it playing a structural role as a spacer to maintain correct conformational presentation of the separate active domains of this multifunctional protein. In marked contrast, the adjacent domain I of the P protein (previously thought to be under little evolutionary constraint) contained a highly conserved region. The colocalization of a short, potentially functional overlapping open reading frame to this region may explain this apparent anomaly.
Mutations which alter the level or structure of nsP4 can affect the efficiency of Sindbis virus replication in a host-dependent manner
Authors: Lemm J A , Durbin R K , Stollar V , Rice C M
Published: January 2020

Two mutants of Sindbis virus have been isolated which grow inefficiently at 34.5 degrees C in mosquito cells yet replicate normally in chicken embryo fibroblast cells at the same temperature. In addition, these mutants exhibit temperature-sensitive growth in both cell types and are RNA- at the nonpermissive temperatures (K.J. Kowal and V. Stollar, Virology 114:140-148, 1981). To clarify the basis of this host restriction, we have mapped the causal mutations for these temperature-dependent, host-restricted mutants. Functional mapping and sequence analysis of the mutant cDNAs revealed several mutations which mapped to the amino terminus of nsP4, the putative polymerase subunit of the viral RNA replicase. These mutations resulted in the following amino acid changes in nsP4: leucine to valine at residue 48, aspartate to glycine at residue 142, and proline to arginine at residue 187. Virus containing any of these mutations was restricted in its ability to replicate in mosquito but not chicken embryo fibroblast cells at 34.5 degrees C. In addition to its temperature-dependent, host-restricted phenotype, virus derived from one cDNA clone also exhibited decreased levels of nsP34 and nsP4 yet contained only a silent change in its genome. This C-to-U mutation occurred at nucleotide 5751, the first nucleotide after the opal termination codon separating nsP3 and nsP4. Our results suggest that this substitution decreases readthrough of the opal codon and diminishes production of nsP34 and nsP4. Such a decrease in synthesis rates might lead to levels of these products which are insufficient for viral RNA replication in mosquito cells at the higher temperature. This work provides the first evidence that nsP4 function can be strongly influenced by the host environment.
Processing of pseudorabies virus glycoprotein gII
Authors: Wölfer U , Kruft V , Sawitzky D , Hampl H , Wittmann-Liebold B , Habermehl K O
Published: January 2020

The glycoprotein complex gII of pseudorabies virus was isolated by immunoprecipitation with the monoclonal antibody M5, which was covalently linked to protein A-Sepharose. After sodium dodecyl sulfate-polyarylamide gel electrophoresis under reducing conditions and blotting onto poly(vinylidene difluoride) membrane, its subunits, gIIa, gIIb, and gIIc, were subjected to N-terminal sequencing. gIIa and gIIb start at position 59 and gIIc starts at position 503 according to the amino acid sequence deduced from the gene, indicating that there is one major protein (gIIa) which is cleaved into the two protein fragments gIIb and gIIc. Protein labeling with 14C-amino acids gave no indication that the three proteins (gIIa, gIIb, and gIIc) of the complex are present in equimolar ratios. It seems that gIIa is only a minor component of the complex, whereas gIIb and gIIc are contained in equimolar amounts.
Transcription of the baculovirus polyhedrin gene reduces the levels of an antisense transcript initiated downstream
Authors: Ooi B G , Miller L K
Published: January 2020

A late 3.2-kilobase (kb) RNA initiated approximately 2 kb downstream of the 3' end of the Autographa californica nuclear polyhedrosis virus polyhedrin-coding sequence and traversed the polyhedrin gene in an antisense direction. This RNA was sense RNA for the two open reading frames flanking the polyhedrin gene. A mutant virus, vXpoly, which differs from wild-type virus only at the essential RNA initiation site in the polyhedrin promoter, exhibited higher levels of the 3.2-kb RNA than did wild-type virus during the polyhedrin transcriptional phase. Thus, transcription of the polyhedrin gene down regulates the levels of this 3.2-kb RNA.
Mutation Frequencies at Defined Single Codon Sites in Vesicular Stomatitis Virus and Poliovirus Can Be Increased Only Slightly by Chemical Mutagenesis
Authors: Holland John J. , Domingo Esteban , de la Torre Juan Carlos , Steinhauer David A.
Published: January 2020

Mutagenesis by a variety of chemical mutagens conferred only 1.1- to 2.8-fold increases in mutation frequencies at defined single base sites in vesicular stomatitis virus and poliovirus.
Analysis of a highly immunodominant epitope in the human immunodeficiency virus type 1 transmembrane glycoprotein, gp41, defined by a human monoclonal antibody
Authors: Bugge T H , Lindhardt B O , Hansen L L , Kusk P , Hulgaard E , Holmbäck K , Klasse P J , Zeuthen J , Ulrich K
Published: January 2020

A human monoclonal antibody, 41-7 [immunoglobulin G1(kappa)], directed against the transmembrane glycoprotein gp41 of the human immunodeficiency virus type 1 (HIV-1) has been produced by direct fusion of lymph node cells from an HIV-1-infected individual with a human B-lymphoblastoid cell line. The minimal essential epitope for 41-7 was mapped to a conserved seven-amino acid sequence, N-CSGKLIC-C, located within the N-terminal part of gp41. Antibodies blocking the binding of 41-7 could be detected in the serum of all HIV-1-infected individuals tested, irrespective of the stage of the infection. The epitope is located externally to the plasma membrane, and it is accessible to antibody in the native conformation of the glycoprotein. Despite this, no neutralizing activity of 41-7 could be demonstrated in vitro. These data indicate, directly and indirectly, that this immunodominant epitope on gp41, although exposed on the viral surface, elicits antibodies lacking antiviral activity and, hence, should be avoided in future vaccine candidates.
Penetration of the nervous systems of suckling mice by mammalian reoviruses
Authors: Flamand A , Gagner J P , Morrison L A , Fields B N
Published: January 2020

Penetration of the nervous systems of suckling mice by prototype strains of the three mammalian reovirus serotypes was studied after footpad inoculation of a dose (10(7) PFU) representing 3.5 x 10(3) 50% lethal doses (LD50) for reovirus type 3 Dearing and less than 1 LD50 for reoviruses type 1 Lang and type 2 Jones. Type 3 Dearing entered both motor and sensory neurons; infected neurons were clearly detectable by immunohistochemical staining 19 h after inoculation. By day 2, a second cycle of infection had occurred, and by day 4, several hundred motor and sensory neurons and interneurons were infected. By this time, infection also involved large areas of the brain stem and brain. There was evidence of both retrograde and anterograde movement of viral antigen within axons and dendrites. Unexpectedly, reovirus type 1 Lang followed neuronal pathways as well as being disseminated in the bloodstream. Reovirus type 2 Jones also entered neurons. While the number of motor neurons and interneurons infected with type 1 Lang or type 2 Jones remained limited within the first 4 days after inoculation, infection of sensory neurons increased with time and reached a level by day 4 comparable to that observed after infection with type 3 Dearing. Viral antigen was also found in the brain stem and brain, but this infection was limited. These three strains multiplied in nonneuronal tissues. Connective tissue in the footpad was massively infected by all three strains 19 h after inoculation. By this time, foci of infection were also present in muscle and skin. Viral antigen was repeatedly observed in the endothelium of blood vessels and in the meninges after infection with type 1 Lang. The titer of type 1 Lang increased in the blood with time, which was not observed after infection with strains of the other two serotypes. In this study, we found that prototype strains of the three reovirus serotypes exhibited different degrees of neurotropism, all being capable of entering neurons. Transmission of the infection occurred through synapses rather than from cell body to cell body. Thus reovirus, like herpesvirus and rabies virus, is a good marker for the identification of neuronal pathways, although its capacity to grow in neurons, unlike that of herpesvirus and rabies virus, is restricted to newborn animals.
Determinants of retrovirus gene expression in embryonal carcinoma cells
Authors: Akgün E , Ziegler M , Grez M
Published: January 2020

The expression of Moloney murine leukemia virus is restricted in embryonal carcinoma (EC) cells. To characterize specific mutations necessary for expression of retroviruses in EC cells, we analyzed the expression of retrovirus mutants and recombinants thereof in EC cell lines F9 and PCC4. DNA sequence comparison and functional studies allowed us to define three point mutations in the enhancer region of the viral mutants at positions -345, -326, and -166 and two point mutations within the 5'-untranslated region of the viral genome at positions +164 and +165 that were essential for retrovirus expression in EC cells. DNA fragments derived from either the wild type or mutant viruses were used to search for sequence-specific DNA-binding factors in nuclear extracts from undifferentiated PCC4 cells. A cellular factor was found to bind strongly to sequences within the enhancer region (-354 to -306) of wild-type viruses but only weakly to sequences derived from mutant viruses. This factor was named ECF-I (for EC cell factor I). Retroviral expression in EC cells correlates with decreased binding affinity for ECF-I.
Adeno-associated virus Rep protein inhibits human immunodeficiency virus type 1 production in human cells
Authors: Antoni B A , Rabson A B , Miller I L , Trempe J P , Chejanovsky N , Carter B J
Published: January 2020

The adeno-associated virus (AAV) rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40) required for AAV DNA replication and AAV gene regulation. In addition, the Rep proteins may have pleiotropic regulatory effects in heterologous systems, and in particular Rep78 may mediate a negative regulatory effect. We analyzed the effects of the AAV rep gene on human immunodeficiency virus type 1 (HIV-1) gene expression. The rep gene proteins of AAV type 2 (AAV2) inhibited the trans-activating ability of HIV-1. Constructs containing the AAV2 rep gene (pHIVrep) or a CAT gene (pBennCAT) expressed from the 5' HIV-1 long terminal repeat were inducible for Rep78 and Rep68 or CAT expression, respectively, when cotransfected with a plasmid containing the HIV-1 tat gene (pARtat). When equivalent amounts of pHIVrep and pBennCAT were cotransfected with increasing amounts of pARtat, expression of CAT activity was decreased. The pHIVrep construct was more inhibitory than plasmids expressing rep from the wild-type AAV2 p5 transcription promoter. rep expression from pHIVrep almost completely inhibited the replication of an HIV-1 proviral clone as measured by reverse transcriptase activity and p24 protein levels. Inhibition of HIV-1 production by Rep protein was also seen at the transcriptional level in that all HIV-1 transcripts were decreased when pHIVrep was present. The inhibitory effects of pHIVrep appear to be mediated primarily by Rep78 and perhaps Rep68. These results suggest that a trans-acting protein from a heterologous virus might be used to inhibit HIV-1 growth.
A 15-kilobase-pair region of the human cytomegalovirus genome which includes US1 through US13 is dispensable for growth in cell culture
Authors: Kollert-Jöns A , Bogner E , Radsak K
Published: January 2020

The genome of a temperature-sensitive, DNA-negative mutant of human cytomegalovirus was cloned in cosmids and analyzed by restriction endonuclease mapping and Southern blotting. The data presented show that in the mutant genome, nearly half of the short segment was deleted (14.3 to 15.1 kb; map position, 0.83 to 0.9), including the genes for a potential immediate early protein (US3) and a structural glycoprotein of 47 to 52 kDa (US6 through US11). The deleted DNA region was replaced by a 20.8- to 21.6-kb fragment that represented an inverted repetition of the retained portion of the short segment (map position, 0.92 to 1.0), suggesting that US20 through US36 were duplicated in the mutant. Northern (RNA) blots with appropriate probes of total cell RNA extracted from mutant-infected cells confirmed the absence of mRNAs originating from US3 or from US8 through US11. It is concluded that the deleted genes are dispensable for human cytomegalovirus replication in cell culture.
Characterization of the bovine herpesvirus 4 major immediate-early transcript
Authors: van Santen V L
Published: January 2020

The major immediate-early (IE) RNA of bovine herpesvirus 4 (BHV-4) has been identified and characterized by analyzing cytoplasmic polyadenylated RNA isolated from Madin-Darby bovine kidney cells infected with BHV-4(DN-599) in the presence of cycloheximide. Hybridization of cDNA to Southern blots of viral DNA, Northern (RNA) blot analysis, and S1 nuclease analyses showed that the major BHV-4 IE RNA is a spliced, 1.7-kb RNA, which is transcribed from right to left on the restriction map of the BHV-4 genome from DNA contained in the 8.3-kb HindIII fragment E. The major IE RNA contains three small exons at its 5' end, spliced to a 1.3-kb 3' exon. This RNA is present in much-reduced amounts when cells are infected in the absence of cycloheximide. However, late in infection, the major IE RNA gene region encodes abundant RNAs which differ in structure from the major IE RNA. Nucleotide sequence analysis of the gene encoding the major IE RNA revealed an open reading frame encoding 284 amino acids. A homology search of amino acid sequence data bases showed that a 141-amino-acid region near the amino terminus of the predicted amino acid sequence is similar to sequences near the amino terminus of herpes simplex virus type 1 IE110. This region of homology includes CXXC pairs, which could be involved in zinc finger structures. The region encoding this putative zinc finger domain is also found in RNAs transcribed from this IE region late in infection, but it is spliced to different sequences than those used in IE RNA. Thus, the major IE region of the BHV-4 genome could encode a family of proteins sharing a zinc finger domain.
Transcription factor AP-1 modulates the activity of the human foamy virus long terminal repeat
Authors: Maurer B , Serfling E , ter Meulen V , Rethwilm A
Published: January 2020

The human foamy virus (HFV) contains within the U3 region of its long terminal repeat (LTR) three perfect consensus sequences for the binding of the inducible transcription factor AP-1. Results of DNase I footprint protection and gel retardation assays demonstrated that proteins in extracts of HeLa and BHK-21 cells as well as bacterially expressed Jun and Fos proteins bind to these AP-1 sites. By conducting transient expression assays using chloramphenicol acetyltransferase plasmids carrying LTR sequences with point-mutated AP-1 sites, it was found that the three AP-1 sites contribute to the optimal activity of the HFV promoter. It is shown that induction of the HFV LTR by 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum factors is mediated through the AP-1 sites.
Isolation of cDNAs for DNA-binding proteins which specifically bind to a tax-responsive enhancer element in the long terminal repeat of human T-cell leukemia virus type I
Authors: Tsujimoto A , Nyunoya H , Morita T , Sato T , Shimotohno K
Published: January 2020

One of the gene products of human T-cell leukemia virus type I (HTLV-I), p40tax, activates its own viral transcription in trans through tax-responsive enhancers in viral long terminal repeats. Five species of cDNA clones for proteins that bind to the tax-responsive enhancer element in HTLV-I were isolated from the Jurkat cell library. The beta-galactosidase fusion protein prepared from the lysogen of a clone specifically recognized the cyclic AMP-responsive element in HTLV-I enhancer. The nucleotide sequence of a full-length cDNA clone (TAXREB67) had a coding capacity of 351 amino acids, which contained a basic motif followed by a leucine zipper structure near the carboxy terminus. Its mRNA was detected in human cell lines, including HTLV-I-infected or noninfected hematopoietic cell lines. The mRNA level in Jurkat cells was decreased temporarily by increasing cyclic AMP concentration but increased by increasing Ca2+ concentration. Polyclonal antibodies against the fusion protein specifically recognized a 52-kDa protein in Jurkat cells. Analyses of the function of this protein and its interactions with other cellular factors will be useful to help understand the regulatory mechanism through tax-responsive enhancers in HTLV-I.
Stable growth of simian virus 40 recombinants containing multimerized enhancers
Authors: Ondek B , Herr W
Published: January 2020

Multiple copies of each of three genetically defined simian virus 40 protoenhancers, A, B, and C, were able to substitute for the wild-type simian virus 40 enhancer. Although the recombinant viruses grew poorly, they could be propagated without the accumulation of enhancer rearrangements that might improve viability. Mutations that inactivate the multimerized B and C protoenhancers abolished virus growth, but, unexpectedly, a mutation that inactivates the octamer-enhanson within the B protoenhancer increased virus viability. This positive effect may reflect loss of repression of the B protoenhancer by the ubiquitous octamer-motif-binding protein Oct-1.
The N-terminal 513 amino acids of the envelope glycoprotein gB of human cytomegalovirus stimulates both B- and T-cell immune responses in humans
Authors: Liu Y N , Klaus A , Kari B , Stinski M F , Eckhardt J , Gehrz R C
Published: January 2020

Host defense against human cytomegalovirus (HCMV) involves both humoral and cell-mediated immunity. In this report, human immune responses to glycoproteins encoded by the HCMV gB homolog gene have been examined by using glycoproteins purified by immunoaffinity from HCMV virions and recombinant proteins expressed by vaccinia viruses containing either the entire gB open reading frame or a C-terminal deletion mutant, gBm165, coding for the N-terminal 513 amino acids of gB. Neutralizing antibodies, helper T cells, and cytotoxic T cells reactive with epitopes on the N-terminal portion of gB were detected in some seropositive individuals, suggesting that this region of gB may be important in eliciting protective immunity during natural infection for some individuals.
Down regulation of poliovirus receptor RNA in HeLa cells resistant to poliovirus infection
Authors: Kaplan G , Racaniello V R
Published: January 2020

A line of HeLa cells (SOFIA) was previously isolated that is resistant to poliovirus infection and does not express functional virus binding sites at the cell surface. The expression of the poliovirus receptor (PVR) gene in SOFIA cells was examined to determine the molecular basis for the failure of these cells to express PVRs. Southern blot analysis of genomic DNA revealed that the PVR gene in SOFIA cells did not contain gross alterations. However, PVR transcripts were not detected in Northern (RNA) blot analysis of SOFIA cell RNA. In vitro nuclear run-on analysis showed that transcription of PVR-specific RNA was reduced in SOFIA cells. Treatment of SOFIA cells with 5-azacytidine restored susceptibility to poliovirus infection, which correlated with the appearance of PVRs at the cell surface, as detected with anti-PVR monoclonal antibody D171. PVR RNA was detected in clones derived from 5-azacytidine-treated SOFIA cells. SOFIA cells were converted to poliovirus sensitivity at a rate of 5 to 7%, suggesting that down regulation of PVR expression involved few cellular targets. Resistance of SOFIA cells to poliovirus infection therefore appears to result from down regulation of PVR RNA, leading to lack of PVR expression at the cell surface. Methylation may play a role in regulating the expression of the PVR gene, which is not essential for survival of HeLa cells.
Genetic analysis of the Rous sarcoma virus subgroup D env gene: mammal tropism correlates with temperature sensitivity of gp85
Authors: Bova-Hill C , Olsen J C , Swanstrom R
Published: January 2020

Subgroup D avian sarcoma and leukosis viruses can penetrate a variety of mammalian cells in addition to cells from their natural host, chickens. Sequences derived from the gp85-coding domain within the env gene of a mammal-tropic subgroup D virus (Schmidt-Ruppin D strain of Rous sarcoma virus [SR-D RSV]) and a non-mammal-tropic subgroup B virus (Rous-associated virus type 2) were recombined to map genetic determinants that allow penetration of mammalian cells. The following conclusions were based on host range analysis of the recombinant viruses. (i) The determinants of gp85 that result in the mammal tropism phenotype of SR-D RSV are encoded within the 160 codons that lie 3' of codon 121 from the corresponding amino terminus of the gp85 protein. (ii) Small linear domains of the SR-D RSV gp85-coding domain placed in the subgroup B background did not yield viruses with titers equal to that of the subgroup D virus in a human cell line. (iii) Recombinant viruses that contained subgroup D sequences within the hr1 variable domain of gp85 showed modest-to-significant increases in infectivity on human cells relative to chicken cells. A recombinant virus that contained three fortuitous amino acid substitutions in the gp85-coding domain was found to penetrate the human cell line and give a titer similar to that of the subgroup D virus. In addition, we found that the subgroup D virus, the mutant virus, and recombinant viruses with an increased mammal tropism phenotype were unstable at 42 degrees C. These results suggest that the mammal tropism of the SR-D strain is not related to altered receptor specificity but rather to an unstable and fusogenic viral glycoprotein. A temperature sensitivity phenotype for infectivity of mammalian cells was also observed for another mammal-tropic avian retrovirus, the Bratislava 77 strain of RSV, a subgroup C virus, but was not seen for any other avian retrovirus tested, strengthening the correlation between mammal tropism and temperature sensitivity.
Mechanism of leukemogenesis induced by mink cell focus-forming murine leukemia viruses
Authors: Li J P , Baltimore D
Published: January 2020

The Friend or Moloney mink cell focus-forming (MCF) virus encodes a recombinant-type envelope glycoprotein, gp70, that is closely related to the membrane glycoprotein, gp55, of Friend spleen focus-forming virus (SFFV). We have shown previously that gp55 has the ability to activate cell growth by binding to the cellular receptor for erythropoietin. Here we show that gp70 encoded by either the Friend or Moloney MCF virus also binds to the erythropoietin receptor and that coexpression of the receptor and gp70 in an interleukin-3 (IL-3)-dependent cell line can activate IL-3-independent growth. Furthermore, when the cDNA for the human IL-2 receptor beta chain, which is related by sequence to the erythropoietin receptor, was introduced into this cell line, it became growth factor independent after infection either with SFFV or with one of the two MCF viruses but not with an ecotropic virus. Based on these observations, we propose a mechanism for the early stage of leukemogenesis induced by the MCF-type murine leukemia viruses.
A single-stranded gap in human immunodeficiency virus unintegrated linear DNA defined by a central copy of the polypurine tract
Authors: Charneau P , Clavel F
Published: January 2020

The structure of unintegrated human immunodeficiency virus type 1 (HIV-1) DNA from acutely infected human lymphoid cells was analyzed by nuclease S1 cleavage. We observed a unique, discrete single-stranded gap in unintegrated linear DNA molecules, located near the center of the genome. Oligonucleotide primer extension experiments determined that the downstream limit of this gap coincides with the last nucleotide of a central copy of the polypurine tract found in all sequenced lentivirus genomes. Other retroviruses have only one copy of the polypurine tract at the 5' boundary of the 3' long terminal repeat, which has been shown to determine initiation of retroviral DNA plus-strand synthesis. We conclude from our observations that the central repeat of the polypurine tract can create an additional site for plus-strand synthesis initiation in lentiviruses. The central single-stranded gap was not found in circular DNA molecules, the vast majority of them carrying only one long terminal repeat. This finding suggests that the generation of such circular molecules is associated with early DNA ligation events.
Immunologic priming with recombinant hepatitis A virus capsid proteins produced in Escherichia coli
Authors: Powdrill T F , Johnston J M
Published: January 2020

Hepatitis A virus capsid proteins (VP0, VP3, and VP1) have been synthesized in Escherichia coli for use in antigenic and immunogenic analyses. Rabbits immunized with each of these individual recombinant capsid proteins developed a rapid neutralizing antibody response when subsequently challenged with a subimmunogenic dose of whole virus.
Mapping the transcriptional transactivation function of simian virus 40 large T antigen
Authors: Zhu J Y , Rice P W , Chamberlain M , Cole C N
Published: January 2020

T antigen is able to transactivate gene expression from the simian virus 40 (SV40) late promoter and from several other viral and cellular promoters. Neither the mechanisms of transactivation by T antigen nor the regions of T antigen required for this activity have been determined. To address the latter point, we have measured the ability of a set of SV40 large T antigen mutants to stimulate gene expression in CV-1 monkey kidney cells from the SV40 late promoter and Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter. Transactivation, although reduced, was retained by an N-terminal 138-amino-acid fragment of T antigen. Mutants with alterations at various locations within the N-terminal 85 amino acids transactivated the RSV LTR promoter less well than did wild-type T antigen. Most of these were also partially defective in their ability to transactivate the SV40 late promoter. Two mutants with lesions in the DNA-binding domain that were unable to bind to SV40 DNA were completely defective for transactivation of both promoter, while a third mutant with a lesion in the DNA-binding domain which retained origin-binding activity transactivated both promoters as well as did wild-type T antigen. Only a low level of transactivation was seen with mutant T antigens which had lesions in or near the zinc finger region (amino acids 300 to 350). Mutations which caused defects in ATPase activity, host range/helper function, binding to p53, binding to the retinoblastoma susceptibility protein, or nuclear localization had little or no effect on transactivation. These results suggest that N-terminal portion of T antigen possesses an activation activity. The data are consistent with the idea that the overall conformation of T antigen is important for transactivation and that mutations in other regions that reduce or eliminate transactivation do so by altering the conformation or orientation of the N-terminal region so that its ability to interact with various targets is diminished or abolished.