bluuebunny/crossref_metadata_2025 huggingface dataset:
Genome RNA terminus conservation and diversity among vesiculoviruses
Authors: Nichol S T , Holland J J
Published: January 2020

Sequence analysis of the RNA genome termini of various vesiculovirus standard and defective interfering (DI) particles demonstrated that some virus regulatory sequences and domains of virus N protein are highly conserved while others show considerable divergence. Clearly, distinct RNA signal sequences and protein-coding regions of these virus genomes have quite different evolutionary pressures or constraints. Terminal regions of DI-particle RNA genomes of these viruses were found to possess self-complementary stems at the RNA termini, demonstrating the conservation of this DI-particle structural feature throughout the vesiculovirus group. A high degree of conservation of the 3'-terminal sequences of recent and historic isolates of vesicular stomatitis virus New Jersey was also demonstrated.
Modification of acute murine cytomegalovirus adrenal gland infection by adoptive spleen cell transfer
Authors: Shanley J D
Published: January 2020

Homozygous (nu/nu), athymic nude mice, infected with murine cytomegalovirus (MCMV), develop unremitting and ablative virus infection involving both the adrenal cortex and medulla. During acute infection, adoptive transfer of MCMV-immune, but not naive, spleen cells suppressed virus replication in the adrenal glands, but not the lungs or salivary gland. T lymphocytes, not macrophages or B cells, were responsible for limiting viral replication. The effect by donor cells was restricted by compatibility at the major histocompatibility locus. Restriction of MCMV replication in the adrenal gland was associated with T lymphocytes of the L3T4 phenotype. Thus, T-cell immunity is critical in regulating MCMV replication in the adrenal glands, and T lymphocytes restricted by class II major histocompatibility antigens mediate this effect.
Ribavirin cures cells of a persistent infection with foot-and-mouth disease virus in vitro
Authors: de la Torre J C , Alarcón B , Martínez-Salas E , Carrasco L , Domingo E
Published: January 2020

Ribavirin (1-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide) eliminates foot-and-mouth disease virus from persistently infected cell cultures. The latter are 10-fold more sensitive to ribavirin than lytically infected cells. In treated cells no viral RNA or proteins could be detected by dot-blot hybridization to cDNA probes, virus and RNA infectivity assays, or immunofluorescence. A potential application of the drug for the treatment of animals carrying the virus is suggested.
Complete nucleotide sequence of wild-type hepatitis A virus: comparison with different strains of hepatitis A virus and other picornaviruses
Authors: Cohen J I , Ticehurst J R , Purcell R H , Buckler-White A , Baroudy B M
Published: January 2020

The complete nucleotide sequence of wild-type hepatitis A virus (HAV) HM-175 was determined. The sequence was compared with that of a cell culture-adapted HAV strain (R. Najarian, D. Caput, W. Gee, S.J. Potter, A. Renard, J. Merryweather, G.V. Nest, and D. Dina, Proc. Natl. Acad. Sci. USA 82:2627-2631, 1985). Both strains have a genome length of 7,478 nucleotides followed by a poly(A) tail, and both encode a polyprotein of 2,227 amino acids. Sequence comparison showed 624 nucleotide differences (91.7% identity) but only 34 amino acid differences (98.5% identity). All of the dipeptide cleavage sites mapped in this study were conserved between the two strains. The sequences of these two HAV strains were compared with the partial sequences of three other HAV strains. Most amino acid differences were located in the capsid region, especially in VP1. Whereas changes in amino acids were localized to certain portions of the genome, nucleotide differences occurred randomly throughout the genome. The most extensive nucleotide homology between the strains was in the 5' noncoding region (96% identity for cell culture-adapted strains versus wild type; greater than 99% identity among cell culture-adapted strains). HAV proteins are less homologous with those of any other picornavirus than the latter proteins are when compared with each other. When the sequences of wild-type and cell culture-adapted HAV strains are compared, the nucleotide differences in the 5' noncoding region and the amino acid differences in the capsid region suggest areas that may contain markers for cell culture adaptation and for attenuation.
Integration of the DNA of filamentous bacteriophage Cflt into the chromosomal DNA of its host
Authors: Kuo T T , Chao Y S , Lin Y H , Lin B Y , Liu L F , Feng T Y
Published: January 2020

It was demonstrated for the first time that filamentous bacteriophage Cflt, which contains single-stranded DNA, can incorporate its genome into that of its host. Evidence in support of the incorporation was obtained from a Southern blot hybridization analysis of DNA isolated from Cflt-lysogenized cells. DNAs from different Cflt-lysogenized cells were purified, and the integration patterns were compared. Because all integration patterns were identical and only one fragment in Cflt replicative-form DNA was missing, it appears that the integration was site specific. Only one complement of viral DNA was integrated per host chromosome. To determine the attachment site on the viral DNA, the physical map of EcoRI, XhoI, SstII, and BglII on Cflt DNA was constructed. Based on this physical map and a Southern blot hybridization analysis of lysogen DNA with these restriction endonucleases, we demonstrated that DNA sequences from all regions of the Cflt genome were represented in the integrated viral sequences. The attachment site on the viral genome was located at 69.2 to 73.8 min on the Cflt DNA.
Translational requirement of La Crosse virus S-mRNA synthesis: in vivo studies
Authors: Raju R , Kolakofsky D
Published: January 2020

By using methods to isolate cytoplasmic RNAs which limit degradation, the effect of drugs which inhibit protein synthesis on the accumulation of La Crosse virus plus-strand S RNAs in vivo has been studied. Cycloheximide and puromycin treatment of infected cultures caused an abortive transcript of ca. 205 nucleotides (nt) to accumulate, whereas pactamycin led to the appearance of an RNA which was slightly shorter (ca. 200 nt). Both the 205- and 200-nt RNAs contained the same range of host primers at their 5' end, but their 3' ends mapped at ca. positions 175 and 165, respectively. Examination of the sequence in this region and at the mature mRNA termination site (position 886) suggests that the sequence YAAAAAT(A)GCAG is involved in transcription termination.
Hepatitis A virus cDNA and its RNA transcripts are infectious in cell culture
Authors: Cohen J I , Ticehurst J R , Feinstone S M , Rosenblum B , Purcell R H
Published: January 2020

A full-length cDNA copy of an attenuated, cell culture-adapted hepatitis A virus (HAV HM-175/7 MK-5) genome was constructed in the PstI site of plasmid vector pBR322. Transfection of monkey kidney cells with this plasmid failed to induce the production of hepatitis A virus (HAV). The HAV cDNA was excised from pBR322 and inserted, without the oligo(dG) X oligo(dC) tails, into an RNA transcription vector to yield plasmid pHAV/7. Transfection of monkey kidney cells with pHAV/7 DNA induced HAV infection. Transfection with RNA transcripts produced in vitro from pHAV/7 yielded about 10-fold more HAV than did transfection with pHAV/7 DNA. Marmosets inoculated with transfection-derived virus developed anti-HAV antibodies and had liver enzyme patterns that closely resembled the liver enzyme patterns seen in animals inoculated with virus from a comparable level of cell culture passage. Infectious RNA transcripts from HAV cDNA should be useful for studying the molecular basis of cell culture adaptation and attenuation as well as for studying specific viral functions.
Identification and characterization of the human cytomegalovirus immediate-early region 2 gene that stimulates gene expression from an inducible promoter
Authors: Hermiston T W , Malone C L , Witte P R , Stinski M F
Published: January 2020

The human cytomegalovirus (HCMV) XbaI E cloned DNA fragment of approximately 20 kilobases can complement an adenovirus mutant (dl312) defective in the E1a viral gene product (D. J. Spector and M. J. Tevethia, Virology 151:329-338, 1986). This viral DNA fragment contains three immediate-early (IE) genes between 0.709 and 0.751 map units (M. F. Stinski, D. R. Thomsen, R. M. Stenberg, and L. C. Goldstein, J. Virol. 46:1-14, 1983). Two of the IE genes, IE1 and IE2, were isolated and tested for a role in regulating viral gene expression. Since HCMV early and late promoters require additional characterization, the chloramphenicol acetyl transferase (cat) gene, driven by the adenovirus E2 promoter, was used as an indicator of gene expression. cat expression from this heterologous viral promoter was shown to be stimulated by HCMV at early times after infection. The IE1 gene product did not function independently in activating this promoter. The IE2 gene products could independently stimulate the expression of a plasmid of a plasmid when the cat gene was placed downstream of the inducible E2 promoter (E2CAT). Five proteins of different sizes have been predicted to originate from IE2, depending on mRNA splicing. The protein products specified by the IE2 gene were characterized with an antibody to a synthetic peptide according to the open reading frame of exon 2. Three of the five proteins are encoded by exon 2. Three viral proteins of 82, 54, and 28 kilodaltons (kDa) were detected. The exons contained in the region designated as IE2a have open reading frames that could code for two of the smaller proteins of 27 and 30 kDa. This region, when driven by the HCMV enhancer, could independently stimulate gene expression from E2CAT to a high level. A plasmid with the HCMV enhancer upstream of exons, that could code for the HCMV IE2 proteins of 48 and 51 kDa, as well as 27- and 30-kDa proteins, also stimulated E2CAT expression but at a lower level. The activity of this plasmid was augmented by the IE1 gene product, despite the fact that the latter gene product alone was inactive. It is proposed that the HCMV IE region 2 gene products are involved in the regulation of viral or host cell promoters either independently or in combination with other HCMV IE proteins.
Temporal distribution of transmissible mink encephalopathy virus in mink inoculated subcutaneously
Authors: Hadlow W J , Race R E , Kennedy R C
Published: January 2020

Information was sought on the temporal distribution of transmissible mink encephalopathy virus in royal pastel mink inoculated subcutaneously with 10(3.0) 50% intracerebral lethal doses of the Idaho strain. As determined by intracerebral assay in mink, extremely little replication of the virus occurred during the preclinical stage of infection. It seemed largely limited to lymph nodes draining the site of inoculation. Virus first appeared in the central nervous system (CNS) at 20 weeks, when all mink were still clinically normal. Early spongiform degeneration, limited to the posterior sigmoid gyrus of the frontal cortex, was first found at 28 weeks, or a few weeks before onset of clinical disease in most of the mink. Once virus reached the CNS, where greater concentrations occurred than elsewhere, it appeared in many extraneural sites (spleen, liver, kidney, intestine, mesenteric lymph node, and submandibular salivary gland). These seemingly anomalous findings, especially the limited extraneural replication of virus as a prelude to infection of the CNS, suggest that mink are not natural hosts of the virus. The results of this study support the generally held view that transmissible mink encephalopathy arises from chance or inadvertent infection of ranch mink with an exogenous virus, most likely feed-borne wild scrapie virus.
Removal of serine phosphates from simian virus 40 large T antigen increases its ability to stimulate DNA replication in vitro but has no effect on ATPase and DNA binding
Authors: Grässer F A , Mann K , Walter G
Published: January 2020

The effect of phosphorylation on the ability of simian virus 40 large T antigen to stimulate DNA synthesis in vitro was tested. Treatment of affinity-purified large T antigen with calf intestinal alkaline phosphatase resulted in the removal of 70 to 80% of the phosphate residues. Only serine-bound phosphate residues were affected. Phosphatase-treated large T antigen stimulated in vitro DNA synthesis fourfold over the untreated control. The stimulation was strongest at early times of DNA replication. At later times, DNA replication proceeded at equal rates with dephosphorylated and untreated large T antigen. The ATPase activity of large T antigen was not affected by phosphatase treatment. The origin-binding activity of large T antigen was tested over a wide range of large T antigen to DNA ratios, including DNA excess, and in the presence and absence of carrier DNA. Under no condition was an effect of dephosphorylation of large T antigen on its DNA-binding activity observed. These findings might indicate that phosphorylation at serine residues modulates the interaction of large T antigen with cellular factors. During DNA synthesis large T antigen was substantially rephosphorylated by kinases in the HeLa cell extract. As shown by two-dimensional peptide mapping, this phosphorylation occurred at all known in vivo sites. No phosphatase and protease activities were detectable in the HeLa cell extract.
Natural feline leukemia virus variant escapes neutralization by a monoclonal antibody via an amino acid change outside the antibody-binding epitope
Authors: Nicolaisen-Strouss K , Kumar H P , Fitting T , Grant C K , Elder J H
Published: January 2020

We have molecularly cloned a natural variant of feline leukemia virus subtype B. This isolate is unique in that it is not neutralized by a monoclonal antibody which neutralized all other feline leukemia virus isolates tested, including members of the A, B, and C subtypes. Western immunoblotting indicated that the monoclonal antibody was less able to bind to the gp70 of the resistant isolate (designated lambda B1) than to the gp70s of susceptible viruses. Nucleotide sequence analysis of the envelope gene of lambda B1 revealed a high degree of homology with the susceptible Snyder-Theilen, Gardner-Arnstein, and Rickard subtype B isolates, including the presence of a 5-amino-acid minimal binding epitope required for binding by the neutralizing monoclonal antibody. The only change within the vicinity of this epitope was in a single nucleotide, and this difference changed a proline residue to leucine three amino acids from the N terminus of the binding epitope. Competitive binding studies with synthetic peptides indicated that substitution of leucine for proline resulted in a 10-fold decrease in the ability of the peptide to compete for antibody binding to native antigen. The results are consistent with the interpretation that this amino acid change lowers the affinity of antibody binding, resulting in failure of the antibody to neutralize the variant virus.
Tight clustering of human hepatitis B virus integration sites in hepatomas near a triple-stranded region
Authors: Shih C , Burke K , Chou M J , Zeldis J B , Yang C S , Lee C S , Isselbacher K J , Wands J R , Goodman H M
Published: January 2020

The open circular genome of human hepatitis B virus (HBV) is known to contain a partially double-stranded DNA with a single-stranded gap region of variable length. This circular structure of the genome is maintained by base-pairing of the 5' ends of the two DNA stands, the long or L(-) strand and the short or S(+) strand. By cloning, mapping, and sequencing studies, we have localized three recombinational junctions of the integrated HBV in two hepatoma samples, HT14 and FOCUS. Breakpoints of recombination derived from these results and those of others appear to be clustered and coincidental with the identified 5' or the deduced 3' end of the long-strand DNA, respectively. Statistical analysis of these results supports the hypothesis that integration preferentially occurs in an extremely narrow region on the HBV genome. This site-specific recombinational mechanism appears to be conserved among different HBV subtypes. No extensive sequence homology was found between each pair of the recombining parental molecules; however, at the site of crossover, 2- to 3-base-pair junctional homology was consistently observed. Examination of the patterns of the integrated HBV DNAs allowed us to categorize these various patterns into four different groups according to their end specificity and strand polarity. The molecular form of relaxed circle is proposed to be one major substrate for HBV integration. The effect of free strand in the integration of HBV is emphasized in this model. Unlike any other known DNA animal viruses, the site specificity of HBV integration appears to be similar to that of the retroviruses.
The E6-E7 region of human papillomavirus type 18 is sufficient for transformation of NIH 3T3 and rat-1 cells
Authors: Bedell M A , Jones K H , Laimins L A
Published: January 2020

Plasmids containing the E6 and E7 open reading frames of human papillomavirus type 18 expressed from an autologous transcriptional control region were sufficient for transformation of NIH 3T3 and Rat-1 cells. Transformation by these sequences did not always involve morphological alterations even though anchorage-independent growth occurred at a high frequency. In these cells, the efficiency of transformation by the E6 and E7 construct was equivalent to or, in most cases, better than that observed with the entire viral genome. These data indicate that a major human papillomavirus type 18 transforming function is localized in either the E6 or E7 gene products or both.
Site-specific mutations at a picornavirus VP3/VP1 cleavage site disrupt in vitro processing and assembly of capsid precursors
Authors: Parks G D , Palmenberg A C
Published: January 2020

Most proteolytic cleavages within the picornavirus polyproteins are carried out by viral protease 3C. For encephalomyocarditis virus, the protease 3C-catalyzed processing occurs between Gln-Gly or Gln-Ser amino acid pairs which are flanked by proline residues, but the sequence-specific constraints on recognition and cleavage by the enzyme are not completely understood. To examine alternative cleavage site sequences, we constructed a cDNA plasmid which expresses the viral L-P1-2A capsid precursor in vitro and introduced site-specific mutations into the Gln-Gly pair at the VP3/VP1 junction. The altered protein substrates were tested for cleavage activity in assays with protease 3C. The encephalomyocarditis virus 3C processed Gln-Ala as efficiently as its natural sites but did not cleave Gln-Val, Gln-Glu, Lys-Gly, Lys-Ala, Lys-Val, Lys-Glu, or Pro-Gly combinations. Displacement of the flanking proline residue by an engineered insertion slowed but did not prevent cleavage at this site. Also, a mutant defective in processing at the VP3/VP1 junction was unable to form 14S pentameric assembly intermediates in vitro.
Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins
Authors: Kascsak R J , Rubenstein R , Merz P A , Tonna-DeMasi M , Fersko R , Carp R I , Wisniewski H M , Diringer H
Published: January 2020

Antibody response in mice to scrapie-associated fibril proteins (protease-resistant proteins [PrPs]) was generated to different epitopes depending on the source of antigen. Mice responded differently to PrPs isolated from scrapie-infected animals of homologous (mouse) versus heterologous (hamster) species. An enzyme-linked immunosorbent assay established to monitor this antibody response in mice immunized with PrPs was unable to detect such a response in scrapie-infected mice. A monoclonal antibody (MAb), 263K 3F4, derived from a mouse immunized with hamster 263K PrPs reacted with hamster but not mouse PrPs. MAb 263K 3F4 also recognized normal host protein of 33 to 35 kilodaltons in brain tissue from hamsters and humans but not from bovine, mouse, rat, sheep, or rabbit brains. This is the first demonstration of epitope differences on this host protein in different species. The defining of various epitopes on PrP through the use of MAbs will lead to a better understanding of the relationship of PrPs to their host precursor protein and to the infectious scrapie agent.
The duck hepatitis B virus pre-C region encodes a signal sequence which is essential for synthesis and secretion of processed core proteins but not for virus formation
Authors: Schlicht H J , Salfeld J , Schaller H
Published: January 2020

Analysis of the serum of duck hepatitis B virus (DHBV)-infected ducks has revealed the presence of C-terminally truncated viral core proteins (e antigens). These proteins are glycosylated and therefore were not released from infected cells by lysis but rather by active secretion, indicating that the DHBV core protein can be synthesized alternatively as a cytoplasmic or a secretory protein. Transient expression of cloned wild-type DHBV DNA and of a specifically designed viral mutant in a human hepatoma cell line (Hep-G2) showed that the DHBV core gene promoter is active in differentiated human liver cells and that synthesis and secretion of the processed core proteins are dependent on the expression of the pre-C region, a small open reading frame which precedes the core gene. In addition, these experiments showed that the mechanism of core protein processing and secretion is conserved between DHBV and the human hepatitis B virus and therefore might be important for the hepatitis B virus life cycle in general. In spite of this, intrahepatic injection of the pre-C mutant into uninfected ducks resulted in viremia without concomitant e-antigen synthesis, indicating that virus formation is independent of pre-C expression.
Fixation of the unmethylated or the 5'-CCGG-3' methylated adenovirus late E2A promoter-cat gene construct in the genome of hamster cells: gene expression and stability of methylation patterns
Authors: Müller U , Doerfler W
Published: January 2020

The late E2A promoter of adenovirus type 2 (Ad2) DNA can be inactivated by in vitro methylation of three 5'-CCGG-3' sequences at positions +23, +5, and -215 relative to the cap site in this promoter. This inactivation has been documented in transient expression experiments both in Xenopus laevis oocytes and in mammalian cells (K.-D. Langner, L. Vardimon, D. Renz, and W. Doerfler, Proc. Natl. Acad. Sci. USA 81:2950-2954, 1984; K.-D. Langner, U. Weyer, and W. Doerfler, Proc. Natl. Acad. Sci. USA 83:1598-1602, 1986). In the present study, in vitro-methylated or unmethylated promoter-gene assemblies were permanently fixed by integration in the hamster genome. In individually established cell lines, the degree of promoter methylation was correlated to gene activity. The pAd2E2AL-CAT construct, in which the late E2A promoter controls expression of the procaryotic chloramphenicol acetyltransferase (cat) gene, was fixed in BHK21 hamster cells by cotransfection with and selection for the pSV2-neo construct (P. J. Southern and P. Berg, J. Mol. Appl. Genet. 1:327-341, 1982) in which the early simian virus 40 promoter controls the gene for neomycin phosphotransferase. The pAd2E2AL-CAT construct was transfected in the unmethylated or in the 5'-CCGG-3' methylated form. The pSV2-neo plasmid was cotransfected in the unmethylated form. The stability of in vitro-imposed methylation patterns and cat gene expression were followed and correlated in a number of established cell lines which contained the constructs integrated in a non-rearranged configuration. The foreign DNA did not persist in the episomal state but was integrated, frequently in multiple tandems of the plasmid DNA. Among 19 cell lines established after transfecting the unmethylated pAd2E2AL-CAT construct, the late E2A promoter remained unmethylated (examined in 10 cell lines), and the cat gene was expressed in 18 cell lines. On the other hand, among 14 cell lines which were generated by transfection with the methylated construct, 7 cell lines did not express the cat gene, and the three 5'-CCGG-3' sequences in the late E2A promoter remained almost completely methylated. In five cell lines, the E2A promoter sequences were partly demethylated and the cat gene was expressed at low levels. Last, in two cell lines, demethylations were found to be extensive and strong cat expression was observed. It remained a question of considerable interest what factors determined the stability of methylation patterns that had been preimposed by in vitro methylation on specific sequences in a promoter, after this promoter was fixed by integration in the mammalian genome.(ABSTRACT TRUNCATED AT 400 WORDS)
Detection of herpes simplex virus type 1 transcripts during latent infection in mice
Authors: Spivack J G , Fraser N W
Published: January 2020

A latent infection can be established in the trigeminal ganglia of mice after corneal inoculation of herpes simplex virus type 1 (HSV-1). With a virion DNA probe, three transcripts (2.0, 1.5, and 1.45 kilobases [kb]) were detected by Northern blot (RNA blot) analysis of RNAs isolated from the ganglia of latently infected mice. All three transcripts hybridized to a nick-translated HSV-1 DNA probe from BamHI restriction fragment B (strain F). These RNAs were mapped with subfragments of BamHI-B and with strand-specific probes. They are at least partially colinear with each other, map to a 3.0-kb PstI-MluI subfragment of BamHI-B, and are transcribed from left to right. The latent HSV-1 RNAs partially overlap the 3' end of ICP0 mRNA but are transcribed in the opposite direction. The latent RNAs were not as extensively poly(A)+ as actin mRNA. The HSV-1 transcripts detected in latently infected trigeminal ganglia did not correspond with any that have been previously identified in permissively infected cells in tissue culture. However, the 2.0-kb HSV-1 RNA present during latency was detectable at reduced levels in the trigeminal ganglia of acutely infected mice and in infected tissue culture cells. The data indicate that the pattern of viral gene expression during HSV-1 latency in the trigeminal ganglia of mice does not represent restriction of the genes actively transcribed during the lytic replication cycle in tissue culture.
Functional and antigenic domains of the matrix (M1) protein of influenza A virus
Authors: Ye Z P , Pal R , Fox J W , Wagner R R
Published: January 2020

The membrane- and ribonucleocapsid (RNP)-binding domains of the matrix (M1) protein of influenza A virus (WSN strain) were partially mapped and characterized by reactivity with monoclonal antibodies (MAb) as well as by proteolytic cleavages and amino acid sequencing of the resulting peptides. Of two peptides formed by formic acid hydrolysis, a 9-kilodalton fragment at the amino-terminal third of the M1 protein was recognized by MAb M2-1C6 (to epitope 1), and a 15-kilodalton fragment at the carboxy-terminal two-thirds was recognized by MAb 289/4 (to epitope 2). Partial cleavage by staphylococcal V8 protease gave rise to a 16-kilodalton peptide, mapping to amino acid 8, which was recognized by MAbs to all three epitopes but rather weakly by MAb 904/6 to epitope 3. These studies suggest that epitope 1 of the M1 protein resides between amino acids 8 and 89, whereas epitopes 2 and possibly 3 are located between amino acids 89 and 141 or somewhat more carboxy distal. The intact M1 protein and its N-terminal 9- and 10-kilodalton peptides generated by formic acid or V8 protease cleavage, respectively, reconstituted with dipalmitoylphosphatidylcholine vesicles, but these N-terminal peptides had little effect on in vitro transcription of the RNP core. In sharp contrast, both intact M1 protein and the C-terminal 15-kilodalton formic acid fragment were able to inhibit viral transcription markedly. Moreover, MAb 289/4 (to epitope 2) reversed this inhibited transcription significantly. These studies suggest that the lipid-binding domain of the M1 protein is located within the amino-terminal third, whereas the site involved in the interaction of the M1 protein with RNP cores is located within the carboxy-terminal two-thirds.
Molecular characterization of the Akvr-1 restriction gene: a defective endogenous retrovirus-borne gene identical to Fv-4r
Authors: Dandekar S , Rossitto P , Pickett S , Mockli G , Bradshaw H , Cardiff R , Gardner M
Published: January 2020

A dominant restriction allele, Akvr-1r, from California wild mice (Mus musculus domesticus) confers resistance to exogenous ecotropic murine leukemia virus (MuLV) infection. The presence of an ecotropic MuLV envelope-related glycoprotein in uninfected virus-resistant cells suggests that viral interference is a possible mechanism for this resistance. We molecularly cloned the ecotropic MuLV envelope-related sequence from the genomic DNA of a wild mouse homozygous for the Akvr-1r locus. The cloned provirus was defective and contained a C-terminal end of the pol gene, a complete envelope gene, and a 3' long terminal repeat. The presence of this provirus was directly correlated with Akvr-1r-mediated virus resistance in cell cultures and hybrid mice. The Akvr-1r provirus restriction map and partial DNA sequence were identical to those of the Fv-4r allele, an ecotropic MuLV resistance locus from Japanese feral mice (M. musculus molossinus), which was previously shown to be allelic with the Akvr-1r gene. The 3' host flanking sequences of Fv-4r and Akvr-1r also had identical restriction maps. These findings indicate that Akvr-1r and Fv-4r are the same gene. It was probably acquired by interbreeding of these feral species in recent times. Conservation of this locus might be favored by the useful function that it performs in protection against ecotropic MuLV infection endemic in both populations of wild mice.
A 14,000-Mr envelope protein of vaccinia virus is involved in cell fusion and forms covalently linked trimers
Authors: Rodriguez J F , Paez E , Esteban M
Published: January 2020

A monoclonal antibody, MAbC3, that reacts with a 14,000-molecular-weight envelope protein (14K protein) of vaccinia virus completely inhibited virus-induced cell fusion during infection. Immunoblot and immunofluorescence studies revealed that the 14K protein was synthesized at about 6 to 7 h postinfection and transported from the cytoplasm to the cell surface. Synthesis and transport of the 14K protein during infection occurred in the presence of rifampin, an inhibitor of virus maturation. One- and two-dimensional gel electrophoretic analyses demonstrated that the 14K protein forms largely trimers (42K) that are covalently linked by disulfide bonds. The facts that MAbC3 prevents virus uncoating and blocks virus-induced cell fusion but does not prevent virus attachment to cells and the 14K envelope protein forms trimers all suggest that this protein plays major role in virus penetration.
Monoclonal antibodies as probes for a function of large T antigen during the elongation process of simian virus 40 DNA replication
Authors: Wiekowski M , Dröge P , Stahl H
Published: January 2020

Various monoclonal antibodies specific for simian virus 40 large tumor antigen (T antigen) inhibit the elongation process of viral DNA replication in an in vitro system. The results provide strong evidence for a function intrinsic to T antigen during ongoing replicative-chain elongation. The antibody inhibition studies were further used to establish a correlation between the known biochemical activities of T antigen and its function during the elongation phase. The data demonstrate that, in addition to DNA binding and ATPase, a third function of T antigen is required for replicative chain elongation. This function is most probably related to the recently described DNA helicase activity of T antigen. This conclusion is based on the following results: aphidicolin treatment of actively replicating simian virus 40 minichromosomes causes a partial uncoupling of parental DNA strand separation and DNA synthesis; the strand separation reaction is blocked by the same monoclonal antibodies which strongly inhibit the elongation process. DNA helicase activity of isolated T antigen is equally well inhibited by the same set of monoclonal antibodies that affect minichromosome replication in vitro.
Selective inhibition of translation of the mRNA coding for measles virus membrane protein at elevated temperatures
Authors: Ogura H , Baczko K , Rima B K , ter Meulen V
Published: January 2020

The elevation of culture temperatures of C6 cells that were persistently infected with the Lec strain of the subacute sclerosing panencephalitis (SSPE) virus (C6/SSPE) resulted in immediate selective inhibition of membrane (M) protein synthesis. This phenomenon was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cytoplasmic lysates and immunoprecipitation with monoclonal antibody against the M protein in short-time labeling experiments. The synthesis of various viral mRNAs in the presence of actinomycin D decreased gradually at similar rates after a shift to 39 degrees C. No specific disappearance of the mRNA coding for the M protein was observed when viral RNAs isolated from the infected cells were compared before and after a shift up by Northern blot analysis. Results of pulse-chase experiments did not show any significant difference in M protein stability between 35 and 39 degrees C. This rapid block of M protein synthesis was observed not only in Vero cells that were lytically infected with plaque-purified clones from the Lec strain, clones isolated from C6/SSPE cells and the standard Edmonston strain of measles virus but also in CV1, MA160, and HeLa cells that were lytically infected with the Edmonston strain. Poly(A)+ RNAs that were extracted from C6/SSPE cells before and after a shift to 39 degrees C produced detectable phospho, nucleocapsid, and M proteins in cell-free translation systems at 32 degrees C. Even higher incubation temperatures did not demonstrate the selective depression of M protein synthesis described above in vitro. All these data indicate that M protein synthesis of measles virus is selectively suppressed at elevated temperatures because of an inability of the translation apparatus to interact with the M protein-encoded mRNA.
Neutralization escape mutants define a dominant immunogenic neutralization site on hepatitis A virus
Authors: Stapleton J T , Lemon S M
Published: January 2020

Hepatitis A virus is an hepatotrophic human picornavirus which demonstrates little antigenic variability. To topologically map immunogenic sites on hepatitis A virus which elicit neutralizing antibodies, eight neutralizing monoclonal antibodies were evaluated in competition immunoassays employing radiolabeled monoclonal antibodies and HM-175 virus. Whereas two antibodies (K3-4C8 and K3-2F2) bound to intimately overlapping epitopes, the epitope bound by a third antibody (B5-B3) was distinctly different as evidenced by a lack of competition between antibodies for binding to the virus. The other five antibodies variably blocked the binding of both K3-4C8-K3-2F2 and B5-B3, suggesting that these epitopes are closely spaced and perhaps part of a single neutralization immunogenic site. Several combinations of monoclonal antibodies blocked the binding of polyclonal human convalescent antibody by greater than 96%, indicating that the neutralization epitopes bound by these antibodies are immunodominant in humans. Spontaneously arising HM-175 mutants were selected for resistance to monoclonal antibody-mediated neutralization. Fourteen clonally isolated mutants demonstrated substantial resistance to multiple monoclonal antibodies, including K3-4C8-K3-2F2 and B5-B3. In addition, 13 mutants demonstrated a 10-fold or greater reduction in neutraliztion mediated by polyclonal human antibody. Neutralization resistance was associated with reduced antibody binding. These results suggest that hepatitis A virus may differ from poliovirus in possessing a single, dominant neutralization immunogenic site and therefore may be a better candidate for synthetic peptide or antiidiotype vaccine development.
Expression of polyomavirus virion proteins by a vaccinia virus vector: association of VP1 and VP2 with the nuclear framework
Authors: Stamatos N M , Chakrabarti S , Moss B , Hare J D
Published: January 2020

The polyomavirus proteins VP1, VP2, and VP3 move from their cytoplasmic site of synthesis into the nucleus, where virus assembly occurs. To identify cellular or viral components which might control this process, we determined the distribution of VP1, VP2, and VP3 in a soluble fraction, a cytoplasmic cytoskeleton fraction, and a nuclear framework fraction of infected cells. All three proteins were detected in a detergent-extractable form immediately after their synthesis in polyomavirus-infected cells. Approximately 50, 25, and 40% of pulse-labeled VP1, VP2, and VP3, respectively, associated with the skeletal framework of the nucleus within 10 min after their synthesis. The remaining portion of each labeled protein failed to accumulate on the nuclear framework during a 40-min chase and was degraded. When expressed separately by recombinant vaccinia viruses, VP1 and VP2, but not VP3, accumulated on the nuclear framework. This association was not dependent on other polyomavirus proteins or viral DNA. The amount of total VP1 and VP2 which was bound to the nuclear framework approximated 45 and 20%, respectively. Indirect immunofluorescence demonstrated an exclusive nuclear localization of VP1 in situ. In coinfection experiments, a greater percentage of total VP2 and VP3 was bound to the nuclear framework of cells which cosynthesized VP1. These results indicate that although VP1 and VP2 can bind independently to the insoluble nuclear framework, the association of VP3 with this nuclear structure is promoted by the presence of VP1.
Identification of the gene encoding an RNA polymerase-binding protein of bacteriophage T4
Authors: Williams K P , Kassavetis G A , Esch F S , Geiduschek E P
Published: January 2020

One of five bacteriophage T4-specified proteins that bind to host RNA polymerase core has been purified and partially sequenced. A mixed oligonucleotide, based on the amino acid sequence, was used to probe genomic restriction fragments. The gene for this protein, previously designated the 15K protein, has been located between T4 genes 45 and 46 and designated rpbA.
Direct identification of class II histocompatibility DR proteins in preparations of human T-cell lymphotropic virus type III
Authors: Henderson L E , Sowder R , Copeland T D , Oroszlan S , Arthur L O , Robey W G , Fischinger P J
Published: January 2020

Class II histocompatibility DR antigen alpha and beta chains were isolated from preparations of human T-cell lymphotropic virus type III grown in human H-9 cells. The proteins were purified by reversed-phase high-pressure liquid chromatography and identified by direct N-terminal amino acid sequence analysis of each chain. The purified DR alpha chain had an N-terminal amino acid sequence identical to the known sequence of human DR alpha chain through the first 37 residues. The N-terminal amino acid sequence of the purified DR beta chain was identical to that of human DR4 beta chain. The DR alpha and beta chains appeared to be identical to the p34-36K and p30-32K proteins, respectively, concentrated in immunostimulatory complexes prepared from unfractionated virus and were the major immunogens in these complexes. These proteins represent a ready source of antigens which can cause false-positive enzyme-linked immunosorbent assay reactions in individuals previously exposed to allogenic histocompatibility antigens. The removal of the DR chains from virus preparations by use of available monoclonal antibodies or other means should result in a lower rate of initial false-positive enzyme-linked immunosorbent assay reactions.
Linker-insertion nonsense and restriction-site deletion mutations of the gB glycoprotein gene of herpes simplex virus type 1
Authors: Cai W Z , Person S , Warner S C , Zhou J H , DeLuca N A
Published: January 2020

To study the effects of missense, nonsense, and deletion mutations of the gB glycoprotein gene of herpes simplex virus type 1, a gB-transformed cell line was isolated that, after virus infection, would express sufficient quantities of gB from the cellular chromosome to complement temperature-sensitive gB mutants. The transformed cell line was then used as a permissive cell to transfer two gB mutations from plasmid to viral DNA. One of the mutants, K082, harbored an HpaI linker insertion that introduced one new amino acid and a chain terminator codon within amino acid residue 43. The other mutant contained a 969-base-pair deletion in a part of the gene that includes the membrane-spanning region; a correspondingly shorter gB polypeptide was detected by sodium dodecyl sulfate-gel electrophoresis after immunoprecipitation of infected-cell extracts with four pooled monoclonal antibodies. No polypeptide was observed from K082-infected cells. The shortened gB polypeptide was efficiently processed and secreted into the growth medium. Each of the four monoclonal antibodies precipitated full-length gB, and three of the four precipitated the shortened polypeptide. Enveloped virus particles could be purified after infection of nonpermissive cells with either mutant virus. Virus particles appeared to possess normal polypeptide and glycopeptide profiles except for the absence of gB. Therefore, the presence of gB is not essential for viral assembly, including envelopment. Recombinants in virus stocks grown on the gB-transformed cells occurred at frequencies on the order of 10(-7) to 10(-5), compared with a frequency of approximately 10(-2) in mixed infections with the two mutants.
Mechanisms of antiviral immunity induced by a vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D: cytotoxic T cells
Authors: Martin S , Moss B , Berman P W , Laskey L A , Rouse B T
Published: January 2020

We used a transfected L cell and a vaccinia vector carrying the herpes simplex virus type 1 (HSV-1) gene coding for glycoprotein D (gD) to characterize HSV-specific T-cell responses. Various studies with mice revealed that the vectors could stimulate some HSV-specific T-cell responses. Although the majority of the T cells contributing to the HSV-1 gD-specific proliferative response were of the Lyt-2.1+ phenotype, cytotoxic T cells (Tc), surprisingly, were not induced by these gD vectors. Even though gD appeared to be a target for a class II major histocompatibility complex (MHC)-restricted killer cell, neither gD vector was capable of forming a target cell complex which could be recognized by class I MHC-restricted HSV-specific Tc. Further investigation of the gD-specific responses revealed the presence of potent suppressor cells and factors capable of inhibiting HSV-specific Tc induction in in vitro assays. One interpretation of these data is that class I MHC-restricted HSV- and gD-specific Tc do not develop during HSV infection because of active suppression.
Influenza virus gene expression: control mechanisms at early and late times of infection and nuclear-cytoplasmic transport of virus-specific RNAs
Authors: Shapiro G I , Gurney T , Krug R M
Published: January 2020

Single-stranded M13 DNAs specific for various influenza virus genomic segments were used to analyze the synthesis of virus-specific RNAs in infected cells. The results show that influenza virus infection is divided into two distinct phases. During the early phase, the syntheses of specific virion RNAs, viral mRNAs, and viral proteins were coupled. Thus, the NS (nonstructural) virion RNA was preferentially synthesized early, leading to the preferential synthesis of NS1 viral mRNA and NS1 protein; in contrast, M (matrix) RNA synthesis was delayed, leading to the delayed synthesis of M1 viral mRNA and M1 protein. This phase lasted for 2.5 h in BHK-21 cells, the time at which the rate of synthesis of all the viral mRNAs was maximal. During the second phase, the synthesis of all the virion RNAs remained at or near maximum until at least 5.5 h postinfection, whereas the rate of synthesis of all the viral mRNAs declined dramatically. By 4.5 h, the rate of synthesis of all the viral mRNAs was 5% of the maximum rate. Viral mRNA and protein syntheses were also not coupled, as the synthesis of all the viral proteins continued at maximum levels, indicating that protein synthesis during this phase was directed principally by previously synthesized viral mRNAs. Short pulses (3 min) with [3H]uridine and nonaqueous fractionation of cells were used to show that influenza virion RNA synthesis occurred in the nucleus, demonstrating that all virus-specific RNA synthesis was nuclear. Virion RNAs, like viral mRNAs, were efficiently transported to the cytoplasm at both early and late times of infection. In contrast, the full-length transcripts of the virion RNAs, which are the templates for virion RNA synthesis, were sequestered in the nucleus. Thus, the template RNAs, which were synthesized only at early times, remained in the nucleus to direct virion RNA synthesis throughout infection. These results enabled us to present an overall scheme for the control of influenza virus gene expression.
Adenovirus early region 1A modulation of interferon antiviral activity
Authors: Anderson K P , Fennie E H
Published: January 2020

Human adenovirus type 5 (Ad5) is a DNA virus which replicates as efficiently in human A549 cells treated with human interferon-alpha 2 (IFN) as in untreated cells. Vesicular stomatitis virus (VSV), on the other hand, is a negative-strand RNA virus which is very sensitive to the effects of IFN treatment in A549 cells. The IFN-mediated inhibition of VSV replication was not observed in cells coinfected with Ad5. Abrogation of IFN-mediated antiviral activity was maximal when Ad5 infection preceded VSV infection by at least 36 h, but did not require adenovirus DNA synthesis for manifestation. Coinfection experiments with VSV and deletion variants of adenovirus demonstrated that neither virus-associated RNA synthesis nor expression of adenovirus early regions E1B, E2A, E3, or E4 are required for abrogation of IFN-mediated inhibition of VSV replication. However, expression of early region E1A was essential, suggesting that E1A products can modulate, either directly or indirectly, IFN activity in adenovirus-infected cells.