bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of the Single-Stranded DNA Binding Protein Encoded by the Vaccinia Virus I3 Gene
Authors: Rochester S. Craig , Traktman Paula 
Published: December 2019

ABSTRACT

The 34-kDa protein encoded by the I3 gene of vaccinia virus is expressed at early and intermediate times postinfection and is phosphorylated on serine residues. Recombinant I3 has been expressed in
Escherichia coli
and purified to near homogeneity, as has the protein from infected cells. Both recombinant and endogenous I3 protein demonstrate a striking affinity for single-stranded, but not for double-stranded, DNA. The interaction with DNA is resistant to salt, exhibits low cooperativity, and appears to involve a binding site of approximately 10 nucleotides. Electrophoretic mobility shift assays indicate that numerous I3 molecules can bind to a template, reflecting the stoichiometric interaction of I3 with DNA. Sequence analysis reveals that a pattern of aromatic and charged amino acids common to many replicative single-stranded DNA binding proteins (SSBs) is conserved in I3. The inability to isolate viable virus containing an interrupted I3 allele provides strong evidence that the I3 protein plays an essential role in the viral life cycle. A likely role for I3 as an SSB involved in DNA replication and/or repair is discussed.
Specific Methylation Patterns in Two Control Regions of Epstein-Barr Virus Latency: the LMP-1-Coding Upstream Regulatory Region and an Origin of DNA Replication (oriP)
Authors: Falk Kerstin I. , Szekely Laszlo , Aleman Anna , Ernberg Ingemar 
Published: December 2019

ABSTRACT
The Epstein-Barr virus (EBV) can establish at least four different forms of latent infection. Previously, we have shown that the level of methylation of the EBV genome varies, depending on the form of latency. The methylation status of CpGs was analyzed by the bisulfite genomic sequencing technique in four different cell types representing different forms of latency. The dyad symmetry element of the origin of replication (oriP) region and the latent membrane protein 1 (LMP-1) regulatory sequence (LRS) were studied. The dyad symmetry element has four binding sites for EBNA-1. In a cell with type I latency, a region upstream of the dyad symmetry element was highly methylated, whereas the dyad symmetry element was unmethylated in the EBNA-1-binding region. The LRS was extensively methylated in the LMP-1-negative cell line Rael, in contrast to a LMP-1-expressing nasopharyngeal carcinoma tumor (NPC C15), which was almost completely unmethylated. The methylation pattern of LRS in type I and type III Burkitt lymphoma cells of similar parental origins confirmed that demethylation of some regions takes place upon phenotypic drift.
In Vitro Transcription of pe38/Polyhedrin Hybrid Promoters Reveals Sequences Essential for Recognition by the Baculovirus-Induced RNA Polymerase and for the Strength of Very Late Viral Promoters
Authors: Mans Ruud M. W. , Knebel-Mörsdorf Dagmar 
Published: December 2019

ABSTRACT

In vitro transcription was used to analyze the promoter specificity of the α-amanitin-resistant RNA polymerase that is induced late during infection of
Autographa californica
multicapsid nuclear polyhedrosis virus. By modifying the preparation of crude nuclear extracts, we have established an assay that permits differentiation between weak late and strong very late viral promoters. The virus-induced RNA polymerase initiates at a TAAG sequence motif in both late and very late promoters. Based on the sensitivity of our in vitro transcription system, we have investigated the sequences responsible for a functional TAAG motif and their putative role with respect to the strength of very late promoters. By constructing hybrid promoters between the early pe38 and the very late polyhedrin promoters, we demonstrated that the replacement of 7 nucleotides upstream of the nonfunctional TAAG sequences in the pe38 promoter with the corresponding sequences of the polyhedrin promoter was sufficient for recognition by the virus-induced RNA polymerase. The strength of the very late polyhedrin promoter was established after replacing the 5′ untranslated sequences of the pe38 promoter by those of the polyhedrin promoter in addition to the 7 nucleotides upstream of the TAAG motif.
Bovine Herpesvirus 1 Glycoprotein M Forms a Disulfide-Linked Heterodimer with the U
L
49.5 Protein
Authors: Wu S. X. , Zhu X. P. , Letchworth G. J. 
Published: December 2019

ABSTRACT

Nine glycoproteins (gB, gC, gD, gE, gG, gH, gI, gK, and gL) have been identified in bovine herpesvirus 1 (BHV-1). gM has been identified in many other alpha-, beta-, and gammaherpesviruses, in which it appears to play a role in membrane penetration and cell-to-cell fusion. We sought to express BHV-1 open reading frame U
L
10, which encodes gM, and specifically identify the glycoprotein. We corrected a frameshift error in the published sequence and used the corrected sequence to design coterminal peptides from the C terminus. These were expressed as glutathione
S
-transferase fusion proteins in
Escherichia coli
. The fusion protein containing the 63 C-terminal amino acids from the corrected gM sequence engendered antibodies that immunoprecipitated a 30-kDa protein from in vitro translation reactions programmed with the U
L
10 gene. Proteins immunoprecipitated by this antibody from virus-infected cells ran at 36 and 43 kDa in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 43 and 48 kDa in nonreducing SDS-PAGE. Only the larger of the pair was present in virions. A 7-kDa protein was released from gM by reducing agents. The 7-kDa protein was not recognized in Western blots probed with the anti-gM antibody but reacted specifically with antibodies prepared against BHV-1 U
L
49.5, previously reported to be a 9-kDa protein associated with an unidentified 39-kDa protein (X. Liang, B. Chow, C. Raggo, and L. A. Babiuk, J. Virol. 70:1448–1454, 1996). This is the first report of a small protein covalently bound to any herpesvirus gM. Similar patterns of hydrophobic domains and cysteines in all known gM and U
L
49.5 homologs suggest that these two proteins may be linked by disulfide bonds in all herpesviruses.
Herpes Simplex Virus DNA Cleavage and Packaging: Association of Multiple Forms of U
L
15-Encoded Proteins with B Capsids Requires at Least the U
L
6, U
L
17, and U
L
28 Genes
Authors: Salmon Brandy , Baines Joel D. 
Published: December 2019

ABSTRACT

The U
L
15 gene of herpes simplex virus (HSV) is one of several genes required for the packaging of viral DNA into intranuclear B capsids to produce C capsids that become enveloped at the inner nuclear membrane. A rabbit antiserum directed against U
L
15-encoded protein recognized three proteins with apparent
M
r
s of 79,000, 80,000, and 83,000 in highly purified B capsids. The 83,000-
M
r
protein was detected in type C capsids and comigrated with the product of a U
L
15 cDNA transcribed and translated in vitro. The 83,000- and 80,000-
M
r
proteins were readily detected in purified virions. Inasmuch as (i) none of these proteins were detectable in capsids purified from cells infected with HSV-1(ΔU
L
15), a virus lacking an intact U
L
15 gene, and (ii) corresponding proteins in capsids purified from cells infected with a recombinant virus [HSV-1(R7244), containing a 20-codon tag at the 3′ end of U
L
15] were decreased in electrophoretic mobility relative to the wild-type proteins, we conclude that the proteins with apparent
M
r
s of 83,000, 80,000, and 79,000 are products of U
L
15 with identical C termini. The 79,000-, 80,000-, and 83,000-
M
r
proteins remained associated with B capsids in the presence of 0.5 M guanidine HCl and remained detectable in capsids treated with 2.0 M guanidine HCl and lacking proteins associated with the capsid core. These data, therefore, indicate that U
L
15-encoded proteins are integral components of B capsids. Only the 83,000-
M
r
protein was detected in B capsids purified from cells infected with viruses lacking the U
L
6, U
L
17, or U
L
28 genes, which are required for DNA cleavage and packaging, suggesting that capsid association of the 80,000- and 79,000-
M
r
proteins requires intact cleavage and packaging machinery. These data, therefore, indicate that capsid association of the 80,000- and 79,000-
M
r
U
L
15-encoded proteins reflects a previously unrecognized step in the DNA cleavage and packaging reaction.
Cleavage of the Feline Calicivirus Capsid Precursor Is Mediated by a Virus-Encoded Proteinase
Authors: Sosnovtsev Stanislav V. , Sosnovtseva Svetlana A. , Green Kim Y. 
Published: December 2019

ABSTRACT

Feline calicivirus (FCV), a member of the
Caliciviridae
, produces its major structural protein as a precursor polyprotein from a subgenomic-sized mRNA. In this study, we show that the proteinase responsible for processing this precursor into the mature capsid protein is encoded by the viral genome at the 3′-terminal portion of open reading frame 1 (ORF1). Protein expression studies of either the entire or partial ORF1 indicate that the proteinase is active when expressed either in in vitro translation or in bacterial cells. Site-directed mutagenesis was used to characterize the proteinase Glu-Ala cleavage site in the capsid precursor, utilizing an in vitro cleavage assay in which mutant precursor proteins translated from cDNA clones were used as substrates for
trans
cleavage by the proteinase. In general, amino acid substitutions in the P1 position (Glu) of the cleavage site were less well tolerated by the proteinase than those in the P1′ position (Ala). The precursor cleavage site mutations were introduced into an infectious cDNA clone of the FCV genome, and transfection of RNA derived from these clones into feline kidney cells showed that efficient cleavage of the capsid precursor by the virus-encoded proteinase is a critical determinant in the growth of the virus.
Bombyx mori
Nucleopolyhedrovirus Encodes a DNA-Binding Protein Capable of Destabilizing Duplex DNA
Authors: Mikhailov Victor S. , Mikhailova Alla L. , Iwanaga Masashi , Gomi Sumiko , Maeda Susumu 
Published: December 2019

ABSTRACT

A DNA-binding protein (designated DBP) with an apparent molecular mass of 38 kDa was purified to homogeneity from BmN cells (derived from
Bombyx mori
) infected with the
B. mori
nucleopolyhedrovirus (BmNPV). Six peptides obtained after digestion of the isolated protein with
Achromobacter
protease I were partially or completely sequenced. The determined amino acid sequences indicated that DBP was encoded by an open reading frame (ORF16) located at nucleotides (nt) 16189 to 17139 in the BmNPV genome (GenBank accession no.
L33180
). This ORF (designated
dbp
) is a homolog of
Autographa californica
multicapsid NPV ORF25, whose product has not been identified. BmNPV DBP is predicted to contain 317 amino acids (calculated molecular mass of 36.7 kDa) and to have an isoelectric point of 7.8. DBP showed a tendency to multimerization in the course of purification and was found to bind preferentially to single-stranded DNA. When bound to oligonucleotides, DBP protected them from hydrolysis by phage T4 DNA polymerase-associated 3′→5′ exonuclease. The sizes of the protected fragments indicated that a binding site size for DBP is about 30 nt per protein monomer. DBP, but not BmNPV LEF-3, was capable of unwinding partial DNA duplexes in an in vitro system. This helix-destabilizing ability is consistent with the prediction that DBP functions as a single-stranded DNA binding protein in virus replication.
The Activity of Sendai Virus Genomic and Antigenomic Promoters Requires a Second Element Past the Leader Template Regions: a Motif (GNNNNN)
3
Is Essential for Replication
Authors: Tapparel Caroline , Maurice Diane , Roux Laurent 
Published: December 2019

ABSTRACT

The paramyxovirus genome, a nonsegmented, negative-polarity, single-stranded RNA of ∼15 kb, contains six transcription units flanked at the 3′ and 5′ ends by a short (∼ 50- to 60-nucleotide) extracistronic sequence, dubbed the positive and negative leader regions. These leader template regions, present at the 3′ end of the genome and the antigenome, have been shown to contain essential signals governing RNA replication activity. Whether they are sufficient to promote replication is still open to question. By using a series of Sendai virus defective interfering RNAs carrying a nested set of deletions in the promoter regions, it is shown here that for both the genomic and antigenomic promoters, a 3′-end RNA sequence of 96 nucleotides is required to allow replication. Sequence comparison of active and inactive promoters led to the identification of a set of three nucleotide hexamers (nucleotides 79 to 84, 85 to 90, and 91 to 96) containing a repeated motif RXXYXX [shown as 5′-3′ positive-strand]. Sequential mutation of each hexamer into its complementary sequence confirmed their essential role. The three hexamers are required, and their relative positioning is important, since displacing them by 6 nucleotides destroyed promoter function. RNAs carrying degenerate nucleotides in the three hexamers were used as replication templates. They led to the selection of actively replicating RNA species exclusively carrying the basic motif (GNNNNN)
3
from nucleotides 79 to 96. These results clearly show that, apart from the region from nucleotides 1 to 31, previously identified as governing Sendai virus replication activity, a second element, spanning at the most nucleotides 79 to 96, appears essential. Thus, the paramyxovirus replication promoters are not confined to the leader template regions, as seems to be the case for the rhabdoviruses.
The Proteolytic Cleavage of Human Immunodeficiency Virus Type 1 Nef Does Not Correlate with Its Ability To Stimulate Virion Infectivity
Authors: Chen Yen-Liang , Trono Didier , Camaur Diana 
Published: December 2019

ABSTRACT

The Nef protein of human immunodeficiency virus type 1 (HIV-1) promotes virion infectivity through mechanisms that are yet ill defined. Some Nef is incorporated into particles, where it is cleaved by the viral protease between amino acids 57 and 58. The functional significance of this event, which liberates the C-terminal core domain of the protein from its membrane-associated N terminus, is unknown. To address this question, we examined the modalities of Nef virion association and processing. We found that although significant levels of Nef were detected in HIV-1 virions partly in a cleaved form, cell-specific variations existed in the efficiency of Nef proteolytic processing. The virion association of Nef was strongly enhanced by myristoylation but did not require other HIV-1-specific proteins, since Nef was efficiently incorporated into and cleaved inside murine leukemia virus particles. Substituting alanine for tryptophan
57
decreased the efficiency of Nef processing, while mutating leucine
58
had little effect. In contrast, replacing both of these residues simultaneously almost completely prevented this process. However, when the resulting mutants were compared with a wild-type control in viral infectivity assays, no correlation was found between the levels of cleavage and the ability to stimulate virion infectivity. Furthermore, simian immunodeficiency virus Nef, which lacks the sequence recognized by the protease and as a consequence is not cleaved despite its incorporation into virions, could stimulate the infectivity of a
nef
-defective HIV-1 variant as efficiently as HIV-1 Nef. On these bases, we conclude that the proteolytic processing of Nef is not required for the ability of this protein to enhance virion infectivity.
In Vivo Replication Capacity Rather Than In Vitro Macrophage Tropism Predicts Efficiency of Vaginal Transmission of Simian Immunodeficiency Virus or Simian/Human Immunodeficiency Virus in Rhesus Macaques
Authors: Miller Christopher J. , Marthas Marta , Greenier Jennifer , Lu Ding , Dailey Peter J. , Lu Yichen 
Published: December 2019

ABSTRACTWe used the rhesus macaque model of heterosexual human immunodeficiency virus (HIV) transmission to test the hypothesis that in vitro measures of macrophage tropism predict the ability of a primate lentivirus to initiate a systemic infection after intravaginal inoculation. A single atraumatic intravaginal inoculation with a T-cell-tropic molecular clone of simian immunodeficiency virus (SIV), SIVmac239, or a dualtropic recombinant molecular clone of SIV, SIVmac239/1A11/239, or uncloned dualtropic SIVmac251 or uncloned dualtropic simian/human immunodeficiency virus (SHIV) 89.6-PD produced systemic infection in all rhesus macaques tested. However, vaginal inoculation with a dualtropic molecular clone of SIV, SIVmac1A11, resulted in transient viremia in one of two rhesus macaques. It has previously been shown that 12 intravaginal inoculations with SIVmac1A11 resulted in infection of one of five rhesus macaques (M. L. Marthas, C. J. Miller, S. Sutjipto, J. Higgins, J. Torten, B. L. Lohman, R. E. Unger, H. Kiyono, J. R. McGhee, P. A. Marx, and N. C. Pedersen, J. Med. Primatol. 21:99–107, 1992). In addition, SHIV HXBc2, which replicates in monkey macrophages, does not infect rhesus macaques following multiple vaginal inoculations, while T-cell-tropic SHIV 89.6 does (Y. Lu, P. B. Brosio, M. Lafaile, J. Li, R. G. Collman, J. Sodroski, and C. J. Miller, J. Virol. 70:3045–3050, 1996). These results demonstrate that in vitro measures of macrophage tropism do not predict if a SIV or SHIV will produce systemic infection after intravaginal inoculation of rhesus macaques. However, we did find that the level to which these viruses replicate in vivo after intravenous inoculation predicts the outcome of intravaginal inoculation with each virus.
Enhancement of Aquareovirus Infectivity by Treatment with Proteases: Mechanism of Action
Authors: McPhillips Thomas H. , Dinan David , Subramanian Kothandaraman , Samal Siba K. 
Published: December 2019

ABSTRACT
The effects of protease digestion on the polypeptide composition and on the infectivity of striped bass virus, an aquareovirus, were examined. Both trypsin and chymotrypsin enhanced the infectivity of the virus. Enhancement of infectivity was correlated with the digestion of the outer capsid protein, VP7. These studies support the assertion that VP7 is the outermost capsid protein and suggest that VP4 and VP5 are exposed on the outer surface of infectious particles. The possible role of VP7 in the variation in virulence observed among aquareovirus isolates is discussed.
T-Cell Lymphoma Caused by Herpesvirus Saimiri C488 Independently ofie14/vsag, a Viral Gene with Superantigen Homology
Authors: Knappe Andrea , Thurau Mathias , Niphuis Henk , Hiller Christian , Wittmann Sabine , Kuhn Eva-Maria , Rosenwirth Brigitte , Fleckenstein Bernhard , Heeney Jonathan , Fickenscher Helmut 
Published: December 2019

ABSTRACTThe immediate-early geneie14/vsagof herpesvirus saimiri has homology with murine superantigens. We compared the pathogenesis of infection with eitherie14/vsagdeletion mutants or wild-type virus C488 in cottontop tamarin monkeys (Saguinus oedipus). Two weeks after infection, all animals developed acute T-cell lymphomas independently of the presence of the viralie14/vsaggene.
Induction of Mucosal B-Cell Memory by Intramuscular Inoculation of Mice with Rotavirus
Authors: Coffin Susan E. , Offit Paul A. 
Published: December 2019

ABSTRACTWe investigated the capacity of intramuscular (i.m.) immunization with heterologous-host rotavirus (simian strain RRV) to induce mucosal virus-specific memory B cells in mice. We found that prior i.m. immunization enhanced the magnitude of mucosal virus-specific immunoglobulin A (IgA) production but did not alter the site and timing of induction of virus-specific IgA responses after challenge.
Identification and Characterization of a Porcine Torovirus
Authors: Kroneman A. , Cornelissen L. A. H. M. , Horzinek M. C. , de Groot R. J. , Egberink H. F. 
Published: December 2019

ABSTRACTA porcine torovirus (PoTV) was identified and characterized; it is a novel member of the genusTorovirus(familyCoronaviridae, orderNidovirales), closely related to but clearly distinct from the already recognized equine torovirus (ETV) and bovine torovirus (BoTV) representatives. Immunoelectron microscopy of feces from piglets revealed elongated, 120- by 55-nm particles which were recognized by a torovirus-specific antiserum. Amplification by reverse transcriptase (RT) PCR with primers designed to detect conserved regions (on the basis of the genomes of BoTV strain Breda and ETV strain Berne) resulted in the identification of the 489-bp nucleocapsid gene, encoding a 18.7-kDa protein. The sequence identity in this region between PoTV and both ETV and BoTV was only about 68%, whereas the latter two show 81% identity. Neutralizing antibodies directed against ETV were found in sera of adult and young pigs. In all 10 herds sampled, seropositive animals were present, and 81% of randomly selected adult sows possessed antibodies. A longitudinal study with RT PCR showed that piglets shed virus in the feces for 1 or more days, starting 4 to 14 days after weaning.
Neutralization of Human Immunodeficiency Virus Type 1 by Antibody to gp120 Is Determined Primarily by Occupancy of Sites on the Virion Irrespective of Epitope Specificity
Authors: Parren Paul W. H. I. , Mondor Isabelle , Naniche Denise , Ditzel Henrik J. , Klasse P. J. , Burton Dennis R. , Sattentau Quentin J. 
Published: December 2019

ABSTRACTWe investigated the relative importance of binding site occupancy and epitope specificity in antibody neutralization of human immunodeficiency virus (HIV) type 1 (HIV-1). The neutralization of a T-cell-line-adapted HIV-1 isolate (MN) was analyzed with a number of monovalent recombinant Fab fragments (Fabs) and monoclonal antibodies with a range of specificities covering all confirmed gp120-specific neutralization epitopes. Binding of Fabs to recombinant monomeric gp120 was determined by surface plasmon resonance, and binding of Fabs and whole antibodies to functional oligomeric gp120 was determined by indirect immunofluorescence and flow cytometry on HIV-infected cells. An excellent correlation between neutralization and oligomeric gp120 binding was observed, and a lack of correlation with monomeric gp120 binding was confirmed. A similar degree of correlation was observed between oligomeric gp120 binding and neutralization with a T-cell-line-adapted HIV-1 molecular clone (Hx10). The ratios of oligomer binding/neutralization titer fell, in general, within a relatively narrow range for antibodies to different neutralization epitopes. These results suggest that the occupancy of binding sites on HIV-1 virions is the major factor in determining neutralization, irrespective of epitope specificity. Models to account for these observations are proposed.