bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of temperature-sensitive mutants of Pichinde virus
Authors: Shivaprakash M , Harnish D , Rawls W
Published: January 2020

The synthesis of viral proteins and S RNAs in cells infected with 12 temperature-sensitive (ts) mutants of Pichinde virus was characterized. The mutants could be divided into five groups on the basis of the patterns of radiolabeled proteins immunoprecipitated from infected-cell lysates. Markedly reduced nucleoprotein levels and undetectable amounts of glycoprotein precursor and L protein were synthesized at the nonpermissive temperature in cells infected with five of the mutants. Reduced but detectable amounts of the viral proteins were synthesized at the nonpermissive temperature in cells infected with a single mutant. Two mutants were associated with the intracellular accumulation of glycoprotein precursor, which was apparently not transported across the cell membrane in cells incubated at the nonpermissive temperature. The synthesis of viral proteins in cells infected with two mutants was indistinguishable from those produced by wild-type virus. Two additional mutants were associated with markedly reduced amounts of immunoprecipitable proteins in infected cells incubated at both the permissive and nonpermissive temperatures. Analysis of viral RNA with radiolabeled single-stranded cDNA probes representing complementary and genomic-sense sequences corresponding to the 3' region of S RNA revealed two basic patterns of viral RNA synthesis. At the nonpermissive temperature, the synthesis of complementary- and genomic-sense sequences and mRNA of the S RNA segment was markedly reduced in cells infected with representative members of these mutant groups, suggesting the presence of mutations altering transcriptase activity. Viral-complementary- and genomic-sense sequence and RNA synthesis, as well as nucleoprotein mRNA in cells, was detected in reduced amounts for mutants associated with reduced levels of proteins at both temperatures. Interestingly, RNA species larger than the S RNA segment were detected in cells infected with some of the mutants, especially those with putative transcriptase lesions. These molecules suggest a possible oligomeric intermediate in the synthesis of S RNA of Pichinde virus.
Isolation and characterization of simian immunodeficiency virus from mandrills in Africa and its relationship to other human and simian immunodeficiency viruses
Authors: Tsujimoto H , Cooper R W , Kodama T , Fukasawa M , Miura T , Ohta Y , Ishikawa K , Nakai M , Frost E , Roelants G E
Published: January 2020

Two isolates of simian retrovirus related to the human immunodeficiency virus (HIV) were obtained from apparently healthy mandrills, Papio (Mandrillus) sphinx, in western equatorial Africa. This virus, designated SIVMND (simian immunodeficiency virus from mandrills), appeared morphologically similar to HIV by electron microscopy, showed Mg2+-dependent reverse transcriptase activity, and induced cytopathic effect in human CD4-positive cells. Western blotting (immunoblotting) analyses revealed that the gag and pol products of SIVMND showed cross-reactivity with those of known HIVs and SIVs. Molecular clones covering full-length viral DNA were obtained from closed circular extrachromosomal DNA of SIVMND-infected cells. By clone-on-clone hybridization with known retroviruses of the HIV and SIV groups, SIVMND showed similar cross-hybridization with HIV-1, HIV-2, SIVAGM (African green monkey-derived SIV), and SIVMAC (rhesus macaque-derived SIV) in the gag and pol regions only at low stringency but not at high stringency, a result indicating that SIVMND is a new member of the HIV-SIV group. The existence of distinct SIVs in different monkey species suggest that recent interspecies transfer of HIV-SIV is unlikely in nature.
Replication and gene expression of hepatitis B virus in a transgenic mouse that contains the complete viral genome
Authors: Farza H , Hadchouel M , Scotto J , Tiollais P , Babinet C , Pourcel C
Published: January 2020

We have sought to address the problem of the host and tissue specificity of the hepatitis B virus (HBV) by using transgenic mice obtained after injection of head-to-tail dimers of the HBV genome. Viral DNA replication and protein synthesis were obtained in one of nine transgenic mice containing integrated HBV DNA. The RNAs encoding the HBV surface antigen and the core antigen were synthesized in the liver, the kidney, and the heart. In these organs, DNA replicative intermediates similar to those found during normal infection were associated with corelike structures. Large amounts of core polypeptides and capsids were detected in the nuclei in the absence of any pathological effect. These results show that the different steps of HBV multiplication can take place in nonliver nonhuman cells once the problem of entry into the host cell is overcome. In the absence of a small laboratory animal infectable by HBV, such transgenic mice should be helpful for the study of many aspects of viral multiplication.
Neutralizing antibodies to an immunodominant envelope sequence do not prevent gp120 binding to CD4
Authors: Skinner M A , Langlois A J , McDanal C B , McDougal J S , Bolognesi D P , Matthews T J
Published: January 2020

Animals immunized with the human immunodeficiency virus type 1 gp160 glycoprotein or certain recombinant envelope components develop potent virus-neutralizing activity. This activity is principally due to antibodies directed toward a hypervariable region of gp120 between cysteine residues 302 and 337 and is virus isolate specific. These antisera, as well as two neutralizing monoclonal antibodies directed against the same hypervariable sequence, do not appreciably block gp120 from binding CD4. In contrast, serum samples from infected humans possess high titers of antibodies that block gp120-CD4 binding; these titers approximately correlate with the serum neutralization titers. Our results suggest that there are at least two targets on the envelope glycoprotein for virus neutralization. The target responsible for the broader neutralizing activity of human serum may be a conserved region of gp120 involved in CD4 binding. The antibodies directed at the hypervariable region of the envelope inhibit a different step in virus infection which is subsequent to receptor binding. The extent to which these two different epitopes of gp120 may be involved in protection against human immunodeficiency virus infection is discussed.
Restricted replication of hepatitis A virus in cell culture: encapsidation of viral RNA depletes the pool of RNA available for replication
Authors: Anderson D A , Ross B C , Locarnini S A
Published: January 2020

The replication of hepatitis A virus (HAV) in BS-C-1 cells was examined under single-cycle growth conditions by using strand-specific probes for detection of viral RNA species. No measurable lag phase was demonstrated between accumulation of positive-strand HAV RNA and production of infectious virions, indicating that replication of virion RNA is rate limiting for the production of infectious virus. Intracellular viral RNA was further analyzed by using 2 M LiCl to fractionate the insoluble nonvirion 35S RNA and replicative intermediates (RI) from the soluble virions and double-stranded replicative forms, in conjunction with sucrose density gradient ultracentrifugation to separate the different forms of viral RNA. Throughout the productive phase of HAV infection, 95 to 97% of positive-strand HAV RNA was soluble in 2 M LiCl and was shown to be contained in mature virions. Of the LiCl-insoluble HAV RNA, more than 99% was positive-stranded 35S RNA, whereas 0.4% was negative stranded and had the sedimentation and partial RNase resistance characteristics of RI. The pattern of RNA accumulation in HAV-infected cells is thus very different from that seen in poliovirus-infected cells, where large pools of RI and mRNA are produced before RNA is sequestered into mature virions. The results of this study suggest that encapsidation of positive-strand HAV RNA inhibits transcription at all times during the growth cycle, thereby reducing the pool of replicating RNA and the final yield of infectious HAV.
Replication of simian virus 40 DNA after UV irradiation: evidence of growing fork blockage and single-stranded gaps in daughter strands
Authors: Mezzina M , Menck C F , Courtin P , Sarasin A
Published: January 2020

The molecular mechanisms of in vivo inhibition of mammalian DNA replication by exposure to UV light (at 254 nm) was studied in monkey and human cells infected with simian virus 40. Analysis of viral DNA by electron microscopy and sucrose gradients confirmed that the presence of UV-induced lesions severely blocks DNA synthesis, and thus the conversion of replicative intermediates (RIs) into fully replicated form I DNA is inhibited by UV irradiation. These blocked RI molecules present several special features when visualized by electron microscopy. (i) In excision repair-proficient monkey and human cells they are composed of a double-stranded circular DNA with a double-stranded tail whose size corresponds to the average interpyrimidine dimer distance, as determined by the dimer-specific T4 endonuclease V. (ii) In excision repair-deficient human cells from patients with xeroderma pigmentosum, UV-irradiated RIs present a Cairns-like structure similar to that observed for replicating molecules obtained from unirradiated infected cells. (iii) Single-stranded gaps are visualized in the replicated portions of UV-irradiated RI molecules; such regions are detected and clearly distinguishable from double-stranded DNA when probed by a specific single-stranded DNA-binding protein such as the bacteriophage T4 gene 32 product. Consistent with the presence of gaps in UV-irradiated RI molecules, single-strand-specific S1 nuclease digestion causes a shift in their sedimentation properties when analyzed in neutral sucrose gradients compared with undamaged molecules. These results are in agreement with and reinforce the model in which UV lesions are a barrier to the replication fork movement when present in the template for the leading strand; when lesions are in the template for the lagging strand they inhibit synthesis or completion of Okazaki fragments, leaving gaps opposite the lesion. Moreover, cellular DNA repair-linked endonucleolytic activity may induce double-stranded breaks in the blocked region of the replication forks, resulting in the tailed structures observed in viral DNA molecules obtained from excision repair-proficient cell lines.
Leader protein of foot-and-mouth disease virus is required for cleavage of the p220 component of the cap-binding protein complex
Authors: Devaney M A , Vakharia V N , Lloyd R E , Ehrenfeld E , Grubman M J
Published: January 2020

Suppression of host protein synthesis in cells infected by poliovirus and certain other picornaviruses involves inactivation of the cap-binding protein complex. Inactivation of this complex has been correlated with the proteolytic cleavage of p220, a component of the cap-binding protein complex. Since picornaviral RNA is not capped, it continues to be translated as the cap-binding protein complex is inactivated. The cleavage of p220 can be induced to occur in vitro, catalyzed by extracts from infected cells or by reticulocyte lysates translating viral RNA. Expression of polioviral protease 2A is sufficient to induce p220 cleavage, and the presence in 2A of an 18-amino-acid sequence representing a putative cysteine protease active site correlates with the ability of different picornaviruses to induce p220 cleavage. Foot-and-mouth disease virus (FMDV) infection induces complete cleavage of p220, yet the FMDV genome codes for a 2A protein of only 16 amino acids, which does not include the putative cysteine protease active site. Using cDNA plasmids encoding various regions of the FMDV genome, we have determined that the leader protein is required to initiate p220 cleavage. This is the first report of a function for the leader protein, other than that of autocatalytic cleavage from the FMDV polyprotein.
Transactivating potential of the 3' open reading frame of murine mammary tumor virus
Authors: van Klaveren P , Bentvelzen P
Published: January 2020

The procaryotic chloramphenicol acetyltransferase (CAT) gene controlled by the murine mammary tumor virus (MMTV) promoter shows reduced activity in a rat mammary tumor cell line after infection with MMTV but to a considerably lesser extent than the CAT gene controlled by a heterologous promoter, indicating trans-acting regulation of promoter activity by MMTV. Cotransfection of vectors capable of expressing RNA from the 3' open reading frame (orf) of MMTV with the CAT-MMTV construct resulted in enhanced CAT activity, suggesting that orf carries a transactivating potential. Since transactivation was also found with a vector containing only orf and part of the viral env gene, it was concluded that a separate transcriptional unit exists for the orf message.
Soluble gp350/220 and deletion mutant glycoproteins block Epstein-Barr virus adsorption to lymphocytes
Authors: Tanner J , Whang Y , Sample J , Sears A , Kieff E
Published: January 2020

The Epstein-Barr virus (EBV) major outer envelope glycoprotein complex, gp350/220, was known to be a ligand for CR2, a B-lymphocyte plasma membrane protein. By Scatchard analysis, soluble EBV gp350/220 binds with high affinity (KD, 1.2 x 10(-8) M) to approximately the same number of B-lymphocyte surface sites as do CR2-specific monoclonal antibodies. Soluble gp350, gp220, or an amino-terminal, 576-amino-acid gp220 derivative binds similarly to B-lymphocyte receptors. Soluble gp350/220, gp220, or even a 470-amino-acid, amino-terminal gp220 derivative blocks EBV adsorption or infection. These experiments demonstrate that (i) gp350/220 is the predominant or exclusive EBV ligand for B lymphocytes; (ii) ligand-receptor blockade can prevent lymphocyte infection by EBV; and (iii) the amino-terminal, 470-amino-acid domain of gp350/220 contains the key ligand domain(s). Consistent with the ligand domain(s) being in the amino-terminal half of gp220 are the findings that the gp350/220-specific, EBV-neutralizing monoclonal antibody 72A1 blocks EBV adsorption by recognizing an epitope in the amino-terminal 470 (probably within the amino-terminal 162) amino acids and a deletion of amino-terminal amino acids 28 and 29 from gp350/220 inactivates ligand activity.
Mutational analysis of upstream AUG codons of poliovirus RNA
Authors: Pelletier J , Flynn M E , Kaplan G , Racaniello V , Sonenberg N
Published: January 2020

The 5' untranslated region of poliovirus type 2 Lansing RNA consists of 744 nucleotides containing seven AUG codons which are followed by in-frame termination codons, thus forming short open reading frames (ORFs). To determine the biological significance of these small ORFs, all of the upstream AUG codons were mutated to UUG. The point mutations were introduced into an infectious poliovirus cDNA clone, and RNA transcribed in vitro from the altered cDNA was transfected into HeLa cells to recover the virus. Mutation of AUG 7 resulted in a virus (called R2-5NC-14) with a small-plaque phenotype, whereas mutation of the other six AUG codons produced virus with a wild-type plaque morphology. To determine whether the small-plaque phenotype of R2-5NC-14 was due to altered translational efficiency of the viral mRNA, we constructed chimeric mRNAs containing the 5' noncoding region of poliovirus mRNA fused to the chloramphenicol acetyltransferase (CAT) coding sequence. mRNA containing a mutated AUG 7 codon showed decreased translational efficiency in vitro. The results indicate that the upstream ORFs of poliovirus RNA are not essential for viral replication and do not act as barriers to the translation of poliovirus mRNA. AUG 7 and flanking sequences may play a positive acting role in poliovirus RNA translation.
The herpes simplex virus type 1 alpha protein ICP27 can act as a trans-repressor or a trans-activator in combination with ICP4 and ICP0
Authors: Sekulovich R E , Leary K , Sandri-Goldin R M
Published: January 2020

The herpes simplex virus type 1 (HSV-1) alpha proteins ICP4, ICP0, and ICP27 are trans-acting proteins which affect HSV-1 gene expression. To investigate potential interactions between these alpha products and to determine the specificity of action of the alpha proteins in combination with each other compared with their activities individually, we performed a series of transient-expression assays. In these assays we used plasmids containing the alpha genes encoding ICP4, ICP0, and ICP27 either singly or in combination as effectors and HSV-1 genes of different kinetic classes and heterologous genes as targets. The HSV-1 targets consisted of promoter-regulatory domains from alpha (ICP0 and ICP27), beta (thymidine kinase and alkaline exonuclease), beta-gamma (glycoprotein D, glycoprotein B, and VP5), and gamma (glycoprotein C) genes, each fused to the chloramphenicol acetyltransferase (CAT) gene. The heterologous target genes consisted of the simian virus 40 early promoter with enhancer and the Rous sarcoma virus long terminal repeat promoter and enhancer each fused to the CAT gene. Target promoter activity was measured by the assay of CAT activity in extracts of transfected cells and by Northern (RNA) blot hybridization of CAT mRNA. The results of these experiments showed that ICP4 activated only HSV-1 target genes, whereas ICP0 activated all of the targets and ICP27 had little effect on any of the targets. ICP4 and ICP0 had a synergistic effect when inducing HSV-1 targets, but they did not have this effect on the heterologous targets pSV2-CAT or pRSV-CAT. In fact, lower levels of CAT activity and CAT mRNA were found in the presence of both effectors than with ICP0 alone. Most interestingly, although the effector plasmid containing the ICP27 gene had little effect on its own, two different and marked effects depending on the target were observed when ICP27 was combined with ICP4 or ICP0 or both. A trans-repression of the induction seen with ICP4 and ICP0 was found when ICP27 was present in the transfections with pSV2-CAT, pRSV-CAT, pICP0-CAT, pICP27-CAT, pTK-CAT, pgD-CAT, pgB-CAT, and pgC-CAT. This resulted in CAT activity levels which were similar to or lower than the basal level of expression of the target genes in the absence of effector plasmids. This trans-repression occurred over a wide range of concentrations of input ICP27 plasmid. In contrast to this repressive effect of ICP27, a trans-activation was seen when ICP4, ICP0, and ICP27 plasmids were combined in transfections with pAE-CAT and pVP5-CAT as targets. This trans-activation also occurred over a 10-fold range of input ICP27 plasmid. These results suggest that ICP27 can facilitate both down
Adenovirus vector expressing functional herpes simplex virus ICP0
Authors: Zhu X X , Young C S , Silverstein S
Published: January 2020

ICP0, one of the five immediate-early (IE) gene products of herpes simplex virus (HSV), is a potent activator of transcription. To assess the biological activities of ICP0 and to explore its mechanisms of action, two helper-independent recombinant adenoviruses were constructed. In each recombinant, the E1 region was substituted with the ICP0-encoding genomic segment under the control of either the adenovirus major late promoter (MLP-0) or the HSV IE-0 promoter (0PRO-0). Infection of HeLa cells or 293 cells (a human embryo kidney cell line expressing adenovirus 5 E1a and -b functions) with the MLP-0 recombinant results in the synthesis of more IE-0 mRNA and ICP0 protein than did infection with the 0PRO-0 recombinant. Although 293 cells infected with MLP-0 accumulate 5- to 10-fold more IE-0 mRNA late in the infection than cells infected with HSV, the level of the protein product, ICP0, increased only slightly. In 293 cells, both recombinants could replicate, albeit at a slower rate and with lower final yields than wild-type adenovirus. Neither virus replicates its DNA in HeLa cells, and thus ICP0 cannot substitute for adenovirus E1a; however, the level of ICP0 that accumulates in MLP-0-infected HeLa cells was comparable to that of HSV-infected HeLa cells. In a functional test, we demonstrated that the adeno-ICP0 recombinant viruses can transactivate a transfected TK-CAT cassette, indicating that the ICP0 is biologically active.
Herpes simplex viruses lacking glycoprotein D are unable to inhibit virus penetration: quantitative evidence for virus-specific cell surface receptors
Authors: Johnson D C , Ligas M W
Published: January 2020

Herpes simplex virus (HSV) glycoprotein D (gD) plays an essential role in the entry of virus into cells. HSV mutants unable to express gD were constructed. The mutants can be propagated on VD60 cells, which supply the viruses with gD; however, virus particles lacking gD were produced in mutant-infected Vero cells. Virus particles with or without gD adsorbed to a large number (greater than 4 x 10(4] of sites on the cell surface; however, virions lacking gD did not enter cells. Cells pretreated with UV-inactivated virions containing gD (approximately 5 x 10(3) particles per cell) were resistant to infection with HSV type 1 (HSV-1) and HSV-2. In contrast, cells pretreated with UV-inactivated virions lacking gD could be infected with HSV-1 and HSV-2. If infectious HSV-1 was added prior to UV-inactivated virus particles containing gD, the infectious virus entered cells and replicated. Therefore, virus particles containing gD appear to block specific cell surface receptors which are very limited in number. Particles lacking gD are presumably unable to interact with these receptors, suggesting that gD is an essential receptor-binding polypeptide.
Role for DNA-protein interaction in activation of the herpes simplex virus glycoprotein D gene
Authors: Tedder D G , Pizer L I
Published: January 2020

On the basis of experiments with mutant virus and transfection with isolated genes, the herpes simplex virus immediate-early gene product ICP4 is known to positively regulate the transcription of viral early and late genes and negatively regulate expression from its own promoter. Binding of ICP4 to DNA sequences in several viral genes has been reported, yet the significance of ICP4-DNA interaction in transcriptional activation remains unclear. We have studied this problem by using the early glycoprotein D (gD) gene, which possesses a binding site at approximately -100 relative to the RNA initiation site. We linked this promoter and various mutant constructs to the chloramphenicol acetyltransferase gene in order to measure promoter activity in transient transfections both in the presence and in the absence of an ICP4-encoding plasmid. The natural promoter was activated 3.3-fold, and a deletion construct lacking the binding site was activated minimally (1.7-fold). Constructs containing multiple tandem repeats of the binding site (three or five inserts) demonstrated higher expression in the presence of ICP4 than did the natural promoter while retaining low levels of expression when unstimulated. Gel mobility shift assays and DNase I footprinting analyses indicated that ICP4 associated with multiple binding sites. In vitro transcription from a gD promoter construct containing multiple binding sites showed increased RNA synthesis in the presence of partially purified ICP4. These data provide the first direct evidence that binding of ICP4 to a specific DNA sequence in the gD gene contributes to activation of transcription.
Recombinant vaccinia virus/Venezuelan equine encephalitis (VEE) virus protects mice from peripheral VEE virus challenge
Authors: Kinney R M , Esposito J J , Mathews J H , Johnson B J , Roehrig J T , Barrett A D , Trent D W
Published: January 2020

Mice immunized with recombinant vaccinia virus (VACC) expressing Venezuelan equine encephalitis (VEE) virus capsid protein and glycoproteins E1 and E2 or with attenuated VEE TC-83 virus vaccine developed VEE-specific neutralizing antibody and survived intraperitoneal challenge with virulent VEE virus strains including Trinidad donkey (subtype 1AB), P676 (subtype 1C), 3880 (subtype 1D), and Everglades (subtype 2). However, unlike immunization with TC-83 virus, immunization with the recombinant VACC/VEE virus did not protect mice from intranasal challenge with VEE Trinidad donkey virus. These results suggest that recombinant VACC/VEE virus is a vaccine candidate for equines and humans at risk of mosquito-transmitted VEE disease but not for laboratory workers at risk of accidental exposure to aerosol infection with VEE virus.
Minimum requirements for immunogenic and antigenic activities of homologs of a synthetic peptide of influenza virus hemagglutinin
Authors: Tang X L , Tregear G W , White D O , Jackson D C
Published: January 2020

Synthetic peptides of increasing length and corresponding in sequence to the C-terminal end of the HA1 molecule of influenza virus were constructed and examined for their immunogenic and antigenic properties. Peptides containing at least the four C-terminal amino acids, when coupled to keyhole limpet hemocyanin, were capable of eliciting antibody in BALB/c mice that bound to the 24-residue parent peptide H3 HA1 (305 to 328). In the absence of a carrier, the C-terminal decapeptide was the shortest peptide capable of eliciting antibody. The specificity of this antibody was indistinguishable from that of a monoclonal antibody to the parent peptide which recognizes an epitope encompassed by the C-terminal seven residues. All peptides containing at least the C-terminal four residues were able to inhibit completely the binding of this monoclonal antibody to the parent peptide. Taken together, these results indicate that (i) the tetrapeptide is capable of eliciting specific antibody when coupled to a carrier, (ii) this tetrapeptide possesses all of the antigenic information necessary to occupy the paratope of a monoclonal antibody elicited by the longer parent peptide, and (iii) the decapeptide contains all of the information necessary to elicit a specific immune response and therefore carries an epitope recognized by T cells as well as one recognized by B cells.
Phosphorylation of the rev gene product of human immunodeficiency virus type 1
Authors: Hauber J , Bouvier M , Malim M H , Cullen B R
Published: January 2020

Replication of human immunodeficiency virus type 1 requires the functional expression in trans of the virally encoded rev gene product (previously called art/trs). Here we demonstrate that this protein can be metabolically labeled with 32Pi. The phosphate receptor in the rev protein is shown to be exclusively serine. Treatment of rev-expressing cells with phorbol ester, a specific activator of protein kinase C, led to significant but transient enhancement of the level of rev phosphorylation. These results indicate that the rev protein is posttranslationally modified in vivo and suggest that the level of this modification is subject to modulation by extracellular stimuli.
Vaccinia virus-encoded ribonucleotide reductase: sequence conservation of the gene for the small subunit and its amplification in hydroxyurea-resistant mutants
Authors: Slabaugh M , Roseman N , Davis R , Mathews C
Published: January 2020

The vaccinia virus gene that encodes the small subunit of ribonucleotide reductase was localized to the HindIII F fragment by using degenerate oligonucleotide probes. DNA sequencing revealed a leftward-reading open reading frame that predicted a protein of 37 kilodaltons whose amino acid sequence was much more homologous to the mouse and clam M2 sequences (approximately 80%) than to the corresponding herpesvirus (approximately 27%) or procaryotic (approximately 19%) gene products. Vaccinia virus mutants selected for the ability to grow in high concentrations of a specific inhibitor of ribonucleotide reductase, hydroxyurea, amplified the M2 gene and harbored tandem arrays (2 to 15 copies) of the gene within the HindIII F region. RNA isolated at early times after infection with wild-type virus and probed with an internal fragment of the M2 gene indicated one major (1.2 kilobases) and two minor (4.0 and 2.1 kilobases) transcripts. S1 nuclease analysis and primer extension experiments identified an RNA start site 12 nucleotides upstream of the putative initiation ATG codon.
Genetic mapping of the mouse c-fms proto-oncogene to chromosome 18
Authors: Hoggan M D , Halden N F , Buckler C E , Kozak C A
Published: January 2020

Chinese hamster X mouse somatic cell hybrids were analyzed by Southern blot hybridization with a probe specific for the cellular c-fms proto-oncogene. Results demonstrate that Fms, the genetic locus containing this sequence, maps to mouse chromosome 18. Mouse Fms is thus not linked to the same set of genes involved in growth regulation that human FMS is linked to.
Mutational analysis of the trans-activation-responsive region of the human immunodeficiency virus type I long terminal repeat
Authors: Hauber J , Cullen B R
Published: January 2020

We used site-directed mutagenesis to delineate sequences within the human immunodeficiency virus type I (HIV-I) long terminal repeat (LTR) required for trans-activation by the viral tat gene product. We demonstrated that sequences 3' to LTR position +44 are dispensable for trans-activation but that almost all of the mutations tested located between positions -17 and +44 greatly reduced trans-activation at both the transcriptional and posttranscriptional levels. However, displacement of the HIV-I LTR trans-activation-responsive region (TAR) 3' by insertion of up to 32 base pairs between the LTR TATA box and cap site had little effect on trans-activation. An analysis of the DNase I hypersensitivity profile of the HIV-I LTR in transfected cultures suggested the presence of at least two DNase I-hypersensitive sites, including one which extends into the viral TAR element; however, neither of these sites appeared to be significantly affected by tat coexpression. These results allow more precise delineation of the sequences important for TAR function and suggest that the TAR may be recognized by a host-specific DNA-binding protein rather than by the tat protein directly.
Subnuclear localization of the trans-activating protein of human T-cell leukemia virus type I
Authors: Slamon D J , Boyle W J , Keith D E , Press M F , Golde D W , Souza L M
Published: January 2020

Human T-cell leukemia virus type I is associated with human lymphoid malignancies. The p40xI protein encoded by the x gene of this virus is believed to play some role in virally mediated transformation. This gene is known to encode a transcriptional trans activator which previous studies have shown to be a nuclear protein. Further characterization of the intracellular kinetics of this protein showed that it migrated into the nucleus very soon after synthesis. Within the nucleus, p40xI was distributed almost equally between the nucleoplasm and the nuclear matrix. Given the proposed role of the nuclear matrix in RNA transcription, the association of p40xI with the matrix places it in an appropriate cellular compartment to exercise an effect on transcription.
A temperature-sensitive mutation in a herpes simplex virus type 1 gene required for viral DNA synthesis maps to coordinates 0.609 through 0.614 in UL
Authors: Marchetti M E , Smith C A , Schaffer P A
Published: January 2020

ts701 is a temperature-sensitive mutant of herpes simplex virus type 1 strain KOS induced by hydroxylamine mutagenesis (C.T. Chu, D. S. Parris, R. A. F. Dixon, F. E. Farber, and P. A. Schaffer, Virology 98:168-181, 1979). In the present study, the mutation rendering ts701 temperature sensitive was mapped to coordinates 0.609 through 0.614 in the UL region of the genome. At the nonpermissive temperature, ts701 (i) failed to induce the synthesis of viral DNA, (ii) exhibited a dramatically reduced ability to drive replication of a plasmid containing the herpes simplex virus origin of viral DNA synthesis, oriS, (iii) generated no viral polypeptides of the late (gamma 2) kinetic class, and (iv) produced virions with electron-translucent cores. Northern (RNA) blot hybridization demonstrated that two mRNAs--one of the beta kinetic class and one of the gamma kinetic class--hybridized to a 1.3-kilobase viral DNA fragment that rescued the mutation in ts701. Based on the phenotype and mapping of ts701, it is likely that its mutation lies in the gene specifying the 65,000-Mr DNA-binding protein (65KDBP) recently described by Marsden et al. (H.S. Marsden, M.E.M. Campbell, L. Haarr, M. C. Frame, D. S. Parris, M. Murphy, R. G. Hope, M. T. Muller, and C. M. Preston, J. Virol. 61:2428-2437, 1987).
Identification of the gene encoding the 65-kilodalton DNA-binding protein of herpes simplex virus type 1
Authors: Parris D S , Cross A , Haarr L , Orr A , Frame M C , Murphy M , McGeoch D J , Marsden H S
Published: January 2020

Hybrid arrest of in vitro translation was used to localize the region of the herpes simplex virus type 1 genome encoding the 65-kilodalton DNA-binding protein (65KDBP) to between genome coordinates 0.592 and 0.649. Knowledge of the DNA sequence of this region allowed us to identify three open reading frames as likely candidates for the gene encoding 65KDBP. Two independent approaches were used to determine which of these three open reading frames encoded the protein. For the first approach a monoclonal antibody, MAb 6898, which reacted specifically with 65KDBP, was isolated. This antibody was used, with the techniques of hybrid arrest of in vitro translation and in vitro translation of selected mRNA, to identify the gene encoding 65KDBP. The second approach involved preparation of antisera directed against oligopeptides corresponding to regions of the predicted amino acid sequence of this gene. These antisera reacted specifically with 65KDBP, thus confirming the gene assignment.
Both VP2 and VP3 are synthesized from each of the alternative spliced late 19S RNA species of simian virus 40
Authors: Good P J , Welch R C , Barkan A , Somasekhar M B , Mertz J E
Published: January 2020

The late 19S RNAs of simian virus 40 consist of a family of alternatively spliced RNAs, each of which contains open reading frames corresponding to all three of the virion proteins. Two approaches were used to test the hypothesis that each alternatively spliced 19S RNA species is translated to synthesize preferentially only one of the virion proteins. First, we analyzed the synthesis of virion proteins in simian virus 40 mutant-infected monkey cells that accumulate predominantly either only one spliced 19S RNA species or only the 19S RNAs. Second, we determined the virion proteins synthesized in a rabbit reticulocyte lysate programmed with specific, in vitro-transcribed 19S RNA species. These results indicated that VP2 and VP3, but not VP1, are synthesized from all 19S RNA species. Quantitative analysis of these data indicated that individual 19S RNA species containing a translation initiation signal upstream of the VP2 AUG codon were translated in a cell extract three- to fivefold less efficiently than were 19S RNA species lacking this signal and that the precise rate of synthesis of VP2 relative to VP3 varied somewhat with the sequence of the leader region. These data are consistent with the synthesis of VP2 and VP3 occurring by a leaky scanning mechanism in which initiation of translation at a specific AUG codon is affected by both (i) the intrinsic efficiency of ribosomes recognizing the sequences surrounding the AUG codon as an initiation signal and (ii) partial interference from 5'-proximal initiation signals and their corresponding open reading frames.
Expression of human sequences related to those of mouse mammary tumor virus
Authors: Franklin G C , Chretien S , Hanson I M , Rochefort H , May F E , Westley B R
Published: January 2020

Sequences related to those of the mouse mammary tumor virus (MuMTV) genome have been cloned from human DNA by screening a library prepared from the DNA of a human breast cancer cell line with MuMTV gag-pol DNA. Nine distinct groups of (MuMTV-related) sequences were identified among 100 lambda recombinants by cross-hybridization experiments with subcloned fragments containing gag-pol-related DNA. The largest group, of 64 recombinants, contains the MuMTV-related sequences cloned by others. The other eight groups contain MuMTV-related sequences that have not been described previously. The gag-pol regions of one recombinant from each of the nine groups were hybridized to RNA prepared from five human breast cancer cell lines, from placenta, and from two cell lines derived from other malignancies. RNAs were detected by probes for seven of the groups. The RNAs ranged in size from 1.2 to 12 kilobases. Probes for six of the groups detected large RNAs that could represent transcripts of full-length proviral DNA. Two of the probes detected RNA in one breast cancer cell line only. Most of the RNAs were detected in more than one cell line.
Relative concentrations of serum neutralizing antibody to VP3 and VP7 proteins in adults infected with a human rotavirus
Authors: Ward R L , Knowlton D R , Schiff G M , Hoshino Y , Greenberg H B
Published: January 2020

Two outer capsid rotavirus proteins, VP3 and VP7, have been found to elicit neutralizing-antibody production, but the immunogenicity of these proteins during human rotavirus infection has not been determined. The relative amounts of serum neutralizing antibody against the VP3 and VP7 proteins of the CJN strain of human rotavirus were, therefore, determined in adult subjects before and after infection with this virus. Reassortant strains of rotavirus that contained the CJN gene segment for only one of these two neutralization proteins were isolated and used for this study. The geometric mean titer of serum neutralizing antibody to a reassortant virus (CJN-M) that contained VP7 of CJN and VP3 of another human rotavirus was 12.7 times less than that of antibody to CJN before infection and 20.3 times less after infection. This indicated that most neutralizing antibody was against the VP3 rather than the VP7 protein of CJN. This result was confirmed with other reassortants between CJN and animal rotavirus strains (EDIM and rhesus rotavirus). These findings suggest that VP3 is the primary immunogen that stimulates neutralizing antibody during at least some rotavirus infections of humans.
Early steps in the assembly of vesicular stomatitis virus nucleocapsids in infected cells
Authors: Dillon P J , Gupta K C
Published: January 2020

The assembly of nucleocapsids is an essential step in the replicative cycle of vesicular stomatitis virus (VSV). In this study, we have examined the early events of vesicular stomatitis virus nucleocapsid assembly in BHK-21 cells. Nuclease-resistant intracellular nucleocapsids were isolated at various stages of assembly and analyzed for RNA and protein contents. The smallest ribonucleoprotein complex formed during nucleocapsid assembly contains the 5'-terminal 65 nucleotides of nascent viral RNA complexed with the viral proteins N and NS. Elongation of the assembling nucleocapsids proceeds unidirectionally towards the 3' terminus by the sequential addition of viral proteins which incrementally protect short stretches of the growing RNA chain. Pulse-chase studies show that the assembling nucleocapsids can be chased into full-length nucleocapsids which are incorporated into mature virions. Our results also suggest an involvement of the cytoskeletal framework during nucleocapsid assembly.
Analysis of the primary structure of the long terminal repeat and the gag and pol genes of the human spumaretrovirus
Authors: Maurer B , Bannert H , Darai G , Flügel R M
Published: January 2020

The nucleotide sequence of the human spumaretrovirus (HSRV) genome was determined. The 5' long terminal repeat region was analyzed by strong stop cDNA synthesis and S1 nuclease mapping. The length of the RU5 region was determined and found to be 346 nucleotides long. The 5' long terminal repeat is 1,123 base pairs long and is bound by an 18-base-pair primer-binding site complementary to the 3' end of mammalian lysine-1,2-specific tRNA. Open reading frames for gag and pol genes were identified. Surprisingly, the HSRV gag protein does not contain the cysteine motif of the nucleic acid-binding proteins found in and typical of all other retroviral gag proteins; instead the HSRV gag gene encodes a strongly basic protein reminiscent of those of hepatitis B virus and retrotransposons. The carboxy-terminal part of the HSRV gag gene products encodes a protease domain. The pol gene overlaps the gag gene and is postulated to be synthesized as a gag/pol precursor via translational frameshifting analogous to that of Rous sarcoma virus, with 7 nucleotides immediately upstream of the termination codons of gag conserved between the two viral genomes. The HSRV pol gene is 2,730 nucleotides long, and its deduced protein sequence is readily subdivided into three well-conserved domains, the reverse transcriptase, the RNase H, and the integrase. Although the degree of homology of the HSRV reverse transcriptase domain is highest to that of murine leukemia virus, the HSRV genomic organization is more similar to that of human and simian immunodeficiency viruses. The data justify classifying the spumaretroviruses as a third subfamily of Retroviridae.
Mutations in the phosphorylation sites of simian virus 40 (SV40) T antigen alter its origin DNA-binding specificity for sites I or II and affect SV40 DNA replication activity
Authors: Schneider J , Fanning E
Published: January 2020

A series of mutants of simian virus 40 was constructed by oligonucleotide-directed mutagenesis to study the role of phosphorylation in the functions of large T antigen. Each of the previously mapped phosphorylated serine and threonine residues in large T antigen was replaced by an alanine or cysteine residue or, in one case, by glutamic acid. Mutant DNAs were assayed for plaque-forming activity, viral DNA replication, expression of T antigen, and morphological transformation of rat cells. Viable mutants were isolated, suggesting that modification of some residues is not essential for the biological functions of T antigen. Two of these mutants replicated more efficiently than did the wild type. Seven mutants were partially or completely deficient in viral DNA replication but retained cell transformation activity comparable with that of the wild-type protein. Biochemical analysis of the mutant T antigens demonstrated novel origin DNA-binding properties of several mutant proteins. The results are consistent with the idea that differential phosphorylation defines several functional subclasses of T-antigen molecules.
Nucleotide sequences of cDNAs for human papillomavirus type 18 transcripts in HeLa cells
Authors: Inagaki Y , Tsunokawa Y , Takebe N , Nawa H , Nakanishi S , Terada M , Sugimura T
Published: January 2020

HeLa cells expressed 3.4- and 1.6-kilobase (kb) transcripts of the integrated human papillomavirus (HPV) type 18 genome. Two types of cDNA clones representing each size of HPV type 18 transcript were isolated. Sequence analysis of these two types of cDNA clones revealed that the 3.4-kb transcript contained E6, E7, the 5' portion of E1, and human sequence and that the 1.6-kb transcript contained spliced and frameshifted E6 (E6*), E7, and human sequence. There was a common human sequence containing a poly(A) addition signal in the 3' end portions of both transcripts, indicating that they were transcribed from the HPV genome at the same integration site with different splicing. Furthermore, the 1.6-kb transcript contained both of the two viral TATA boxes upstream of E6, strongly indicating that a cellular promoter was used for its transcription.
Sequence duplication and internal deletion in the integrated human papillomavirus type 16 genome cloned from a cervical carcinoma
Authors: Choo K B , Lee H H , Pan C C , Wu S M , Liew L N , Cheung W F , Han S H
Published: January 2020

Integrated human papillomavirus type 16 (HPV16) sequences were cloned from a cervical carcinoma and analyzed by restriction mapping and nucleotide sequencing. The viral integration sites were mapped within the E1 and E2 open reading frames (ORFs). The E4 and E5 ORFs were entirely deleted. An internal deletion of 376 base pairs (bp) was found disrupting the L1 and L2 ORFs. Sequencing analysis showed that an AGATGT/ACATCT inverted repeat marked the deletion junction with two flanking direct repeats 14 and 8 bp in length. A 1,330-bp sequence duplication containing the long control region (LCR) and the E6 and E7 ORFs was also found. The duplication junction was formed by two 24-bp direct repeats with 79% (19 of 24) homology located within the LCR and the E2 ORF of the prototype viral genome, respectively. This observation leads us to propose that the initial viral integration involved an HPV16 dimer in which the direct repeats in tandem units recombined, resulting in reiteration of only a portion of the original duplication. A guanosine insertion between nucleotides 1137 and 1138 created a continuous E1 ORF which was previously shown to be disrupted. Results from this study indicate that sequence reiteration and internal deletion in the integrated, and possibly in the episomal, HPV16 genome are influenced by specific nucleotide sequences in the viral genome. Moreover, reiteration of the LCR/E6/E7 sequences further supports the hypothesis that the E6/E7 ORFs may code for oncogenic proteins and that regulatory signals in the LCR may play a role in cellular transformation.
Construction and characterization of poliovirus subgenomic replicons
Authors: Kaplan G , Racaniello V R
Published: January 2020

Poliovirus RNAs containing in-frame deletions within the capsid-coding region were produced by in vitro transcription of altered poliovirus type 1 cDNA by using bacteriophage T7 RNA polymerase. Three RNAs were transcribed that contained deletions of 2,317 nucleotides (bases 747 to 3064), 1,781 nucleotides (bases 1,175 to 2,956), and 1,295 nucleotides (bases 1,175 to 2,470). All three subgenomic RNAs replicated after transfection into HeLa cells, demonstrating that sequences encoding the capsid polypeptides are not essential for viral RNA replication in vivo. Viral RNA containing the largest deletion (R1) replicated approximately three times better than full-length RNA produced in vitro. Northern blot (RNA blot) hybridization analysis of total cellular RNA from HeLa cells at different times after transfection with R1 demonstrated the presence of increasing amounts of the expected 5.1-kilobase subgenomic RNA. Analysis by immunoprecipitation of viral proteins induced after transfection of R1 RNA into HeLa cells revealed the presence of proteins 2Apro, 2C, and 3Dpol and its precursors, suggesting that the polyprotein cleavages are similar to those occurring in virus-infected cells. Replication of P2/Lansing virion RNA was inhibited by cotransfection with the R1 replicon, as demonstrated by hybridization analysis with a serotype-specific oligonucleotide probe. A higher level of inhibition of RNA replication was observed when P2/Lansing RNA was cotransfected into HeLa cells with truncated R1 transcripts (R1-PvuII) that were missing 395 3' nucleotides and a poly(A) tail. These internally and terminally deleted RNAs inhibited the replication of subgenomic replicons R1, R2, and R3 and caused a reduction in plaque size when cotransfected with P1/Mahoney or P2/Lansing viral RNA, suggesting that individual cells had received both RNAs. No inhibition of plaque size was observed when replicon RNAs were used that were missing 1,384 or 1,839 3' nucleotides or contained plasmid-derived sequences downstream of the 3' poly(A). The trans-acting inhibitory effect of R1-PvuII on the replication of poliovirus P2/Lansing RNA did not involve entry of RNA into cells and appeared to reduce viral translation and RNA synthesis late in the infection cycle.