bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Host Factor Polyhedrin Promoter Binding Protein (PPBP) Is Involved in Transcription from the Baculovirus Polyhedrin Gene Promoter
Authors: Ghosh Sudip , Jain Anjali , Mukherjee Bipasha , Habib Saman , Hasnain Seyed E. 
Published: December 2019

ABSTRACT

Hypertranscription and temporal expression from the
Autographa californica
nuclear polyhedrosis (AcNPV) baculovirus polyhedrin promoter involves an α-amanitin-resistant RNA polymerase and requires a
trans
-acting viral factor(s). We previously reported that a 30-kDa host factor, polyhedrin promoter binding protein (PPBP), binds with unusual affinity, specificity, and stability to the transcriptionally important motif AATAAATAAGTATT within the polyhedrin (
polh
) initiator promoter and also displays coding strand-specific single-stranded DNA (ssDNA)-binding activity (S. Burma, B. Mukherjee, A. Jain, S. Habib, and S. E. Hasnain, J. Biol. Chem. 269:2750–2757, 1994; B. Mukherjee, S. Burma, and S. E. Hasnain, J. Biol. Chem. 270:4405–4411, 1995). We now present evidence which indicates that an additional factor(s) is involved in stabilizing PPBP-duplex promoter and PPBP-ssDNA interactions. TBP (TATA box binding protein) present in
Spodoptera frugiperda
(
Sf
9) cells is characteristically distinct from PPBP and does not interact directly with the
polh
promoter. Replacement of PPBP cognate sequences within the
polh
promoter with random nucleotides abolished PPBP binding in vitro and also failed to express the luciferase reporter gene in vivo. Phosphocellulose fractions of total nuclear extract from virus-infected cells which support in vitro transcription from the
polh
promoter contain PPBP activity. When PPBP was sequestered by the presence of oligonucleotides containing PPBP cognate sequence motifs, in vitro transcription of a C-free reporter cassette was affected but was restored by the exogenous addition of nuclear extract containing PPBP. When PPBP was mopped out in vivo by a plasmid carrying PPBP cognate sequence present in
trans
,
polh
promoter-driven expression of the luciferase reporter was abolished, demonstrating that binding of PPBP to the
polh
promoter is essential for transcription.
Human Trophoblast Cells Are Permissive to the Complete Replicative Cycle of Human Cytomegalovirus
Authors: Halwachs-Baumann G. , Wilders-Truschnig M. , Desoye G. , Hahn T. , Kiesel L. , Klingel K. , Rieger P. , Jahn G. , Sinzger C. 
Published: December 2019

ABSTRACT
Human trophoblast cells were permissively infected by human cytomegalovirus. The kinetics of viral immediate-early, early, and late gene expression was clearly delayed compared to that in fibroblasts. Productive infection was unequivocally proven by the detection of virion particles, infectious virus in trophoblast culture supernatant, and cell-to-cell spread of cytomegalovirus from infected trophoblasts to uninfected fibroblasts. These observations indicate that infected trophoblasts may be involved in maternofetal transmission of human cytomegalovirus.
The Nucleolus Is the Site of Borna Disease Virus RNA Transcription and Replication
Authors: Pyper J. M. , Clements J. E. , Zink M. C. 
Published: December 2019

ABSTRACT
Borna disease virus (BDV) is a neurotropic nonsegmented negative-strand RNA virus with limited homology to rhabdoviruses and paramyxoviruses. A distinguishing feature of BDV is that it replicates in the nucleus of infected cells. Strand-specific probes used for in situ hybridization of infected rat brain showed that there was differential localization of positive- and negative-strand RNAs within the nucleus of neurons. Within nuclei, sense-strand RNAs were preferentially localized within nucleolar regions while genomic-sense RNAs were found in both nucleolar and nonnucleolar regions. These results suggested a role for the nucleolus in BDV replication. Nucleoli isolated from persistently infected neuroblastoma cells contained both genomic and antigenomic BDV RNA species as well as an enrichment of the 39/38-kDa and gp18 BDV proteins. Since the nucleolus is the site of rRNA transcription, we examined BDV transcription in the presence of inhibitors of RNA polymerase I. Inhibition of RNA polymerase I did not affect levels of BDV transcription.
Colocalization of Baculovirus IE-1 and Two DNA-Binding Proteins, DBP and LEF-3, to Viral Replication Factories
Authors: Okano Keiju , Mikhailov Victor S. , Maeda Susumu 
Published: December 2019

ABSTRACT

We have recently identified a DNA-binding protein (DBP) from the baculovirus
Bombyx mori
nucleopolyhedrovirus (BmNPV) which can destabilize double-stranded DNA (V. S. Mikhailov, A. L. Mikhailova, M. Iwanaga, S. Gomi, and S. Maeda, J. Virol. 72:3107–3116, 1998). DBP was found to be an early gene product that was not present in budded or occlusion-derived virions. In order to characterize the localization of DBP during viral replication, BmNPV-infected BmN cells were examined by immunostaining and confocal microscopy with DBP antibodies. DBP first appeared as diffuse nuclear staining at 4 to 6 h postinfection (p.i.) and then localized to several specific foci within the nucleus at 6 to 8 h p.i. After the onset of viral DNA replication at around 8 h p.i., these foci began to enlarge and eventually occupied more than half of the nucleus by 14 h p.i. After the termination of viral DNA replication at about 20 h p.i., the DBP-stained regions appeared to break down into approximately 100 small foci within the nucleus. At 8 h p.i., the distribution of DBP as well as that of IE-1 or LEF-3 (two proteins involved in baculovirus DNA replication) overlapped well with that of DNA replication sites labeled with bromodeoxyuridine incorporation. Double-staining experiments with IE-1 and DBP or IE-1 and LEF-3 further confirmed that, between 8 and 14 h p.i., the distribution of IE-1 and LEF-3 overlapped with that of DBP. However, IE-1 localized to the specific foci prior to DBP or LEF-3 at 4 h p.i. In the presence of aphidicolin, an inhibitor of DNA synthesis, immature foci containing IE-1, LEF-3, and DBP were observed by 8 h p.i. However, the subsequent enlargement of these foci was completely suppressed, suggesting that the enlargement depended upon viral DNA replication. At 4 h p.i., the number of IE-1 foci correlated with the multiplicity of infection (MOI) between 0.4 and 10. At higher MOIs (e.g., 50), the number of foci plateaued at around 15. These results suggested that there are about 15 preexisting sites per nucleus which are associated with the initiation of viral DNA replication and assembly of viral DNA replication factories.
Nucleoporins Nup98 and Nup214 Participate in Nuclear Export of Human Immunodeficiency Virus Type 1 Rev
Authors: Zolotukhin Andrei S. , Felber Barbara K. 
Published: December 2019

ABSTRACT
Human immunodeficiency virus type 1 (HIV-1) Rev contains a leucine-rich nuclear export signal that is essential for its nucleocytoplasmic export mediated by hCRM1. We examined the role of selected nucleoporins, which are located in peripheral structures of the nuclear pore complex and are thought to be involved in export, in Rev function in human cells. First, we found that upon actinomycin D treatment, Nup98, but not Nup214 or Nup153, is able to translocate to the cytoplasm of HeLa cells, demonstrating that Nup98 may act as a soluble factor. We further showed that Rev can recruit Nup98 and Nup214, but not Nup153, to the nucleolus. We also found that the isolated FG-containing repeat domains of Nup98 and Nup214, but not those of Nup153, competitively inhibit the Rev/RRE-mediated expression of HIV. Taken together, the recruitment of Nup98 and Nup214 by Rev and the competitive inhibition exhibited by their NP domains demonstrate direct participation of Nup98 and Nup214 in the Rev-hCRM1-mediated export.
Inhibition of Influenza A Virus Replication by Compounds Interfering with the Fusogenic Function of the Viral Hemagglutinin
Authors: Plotch Stephen J. , O’Hara Bryan , Morin John , Palant Olga , LaRocque James , Bloom Jonathan D. , Lang Stanley A. , DiGrandi Martin J. , Bradley Mary , Nilakantan Ramaswamy , Gluzman Yakov 
Published: December 2019

ABSTRACT
Several compounds that specifically inhibited replication of the H1 and H2 subtypes of influenza virus type A were identified by screening a chemical library for antiviral activity. In single-cycle infections, the compounds inhibited virus-specific protein synthesis when added before or immediately after infection but were ineffective when added 30 min later, suggesting that an uncoating step was blocked. Sequencing of hemagglutinin (HA) genes of several independent mutant viruses resistant to the compounds revealed single amino acid changes that clustered in the stem region of the HA trimer in and near the HA2 fusion peptide. One of the compounds, an N-substituted piperidine, could be docked in a pocket in this region by computer-assisted molecular modeling. This compound blocked the fusogenic activity of HA, as evidenced by its inhibition of low-pH-induced cell-cell fusion in infected cell monolayers. An analog which was more effective than the parent compound in inhibiting virus replication was synthesized. It was also more effective in blocking other manifestations of the low-pH-induced conformational change in HA, including virus inactivation, virus-induced hemolysis of erythrocytes, and susceptibility of the HA to proteolytic degradation. Both compounds inhibited viral protein synthesis and replication more effectively in cells infected with a virus mutated in its M2 protein than with wild-type virus. The possible functional relationship between M2 and HA suggested by these results is discussed.
Diverse Gene Junctions of Respiratory Syncytial Virus Modulate the Efficiency of Transcription Termination and Respond Differently to M2-Mediated Antitermination
Authors: Hardy Richard W. , Harmon Shawn B. , Wertz Gail W. 
Published: December 2019

ABSTRACT
The ability of the diverse gene junctions of respiratory syncytial (RS) virus to signal the termination of transcription was analyzed. Nine dicistronic subgenomic replicons of RS virus were constructed; each contained one of the RS virus gene junctions in its natural upstream and downstream sequence context. The RNA synthesis activities of these subgenomic replicons were analyzed in the absence and presence of the M2 protein, which we showed previously to function as a transcription antiterminator. Our data showed that the efficiency with which the polymerase terminated transcription was affected by the gene junction that it encountered. The M2 protein significantly decreased the efficiency of the termination of transcription, resulting in increased levels of readthrough transcription at all the gene junctions. The diverse gene junctions fell into three broad groups with respect to their ability to signal transcription termination. One group of gene junctions (NS1/NS2, NS2/N, M2/L, and L/trailer) showed inefficient termination in the absence or the presence of the M2 protein. A second group of gene junctions (N/P, P/M, M/SH, SH/G, and G/F) terminated transcription efficiently. The SH/G gene junction terminated transcription with the greatest efficiency and produced low levels of readthrough transcripts in the absence or the presence of the M2 protein, correlating with the absence of SH/G polycistronic transcripts in RS virus-infected cells. The F/M2 gene junction was particularly sensitive to the M2 protein: it efficiently signaled termination in the absence of the M2 protein but produced high levels of readthrough transcripts in the presence of the M2 protein. This result suggests that the M2 protein may regulate its own production by negative feedback. The data presented here show that the different gene junctions of RS virus do modulate RS virus transcription termination. The M2 protein reduced termination at all gene junctions. The magnitude of antitermination due to the M2 protein, however, varied at the different gene junctions. The data presented here indicate that the mechanism for the regulation of RS virus gene expression is more complex than was previously appreciated.
Mutational Analysis of the Hydrophobic Tail of the Human Immunodeficiency Virus Type 1 p6
Gag
Protein Produces a Mutant That Fails To Package Its Envelope Protein
Authors: Ott David E. , Chertova Elena N. , Busch Laura K. , Coren Lori V. , Gagliardi Tracy D. , Johnson Donald G. 
Published: December 2019

ABSTRACT

The p6
Gag
protein of human immunodeficiency virus type 1 (HIV-1) is produced as the carboxyl-terminal sequence within the Gag polyprotein. The amino acid composition of this protein is high in hydrophilic and polar residues except for a patch of relatively hydrophobic amino acids found in the carboxyl-terminal 16 amino acids. Internal cleavage of p6
Gag
between Y
36
and P
37
, apparently by the HIV-1 protease, removes this hydrophobic tail region from approximately 30% of the mature p6
Gag
proteins in HIV-1
MN
. To investigate the importance of this cleavage and the hydrophobic nature of this portion of p6
Gag
, site-directed mutations were made at the minor protease cleavage site and within the hydrophobic tail. The results showed that all of the single-amino-acid-replacement mutants exhibited either reduced or undetectable cleavage at the site yet almost all were nearly as infectious as wild-type virus, demonstrating that processing at this site is not important for viral replication. However, one exception, Y36F, was 300-fold as infectious the wild type. In contrast to the single-substitution mutants, a virus with two substitutions in this region of p6
Gag
, Y36S-L41P, could not infect susceptible cells. Protein analysis showed that while the processing of the Gag precursor was normal, the double mutant did not incorporate Env into virus particles. This mutant could be complemented with surface glycoproteins from vesicular stomatitis virus and murine leukemia virus, showing that the inability to incorporate Env was the lethal defect for the Y36S-L41P virus. However, this mutant was not rescued by an HIV-1 Env with a truncated gp41
TM
cytoplasmic domain, showing that it is phenotypically different from the previously described MA mutants that do not incorporate their full-length Env proteins. Cotransfection experiments with Y36S-L41P and wild-type proviral DNAs revealed that the mutant Gag dominantly blocked the incorporation of Env by wild-type Gag. These results show that the Y36S-L41P p6
Gag
mutation dramatically blocks the incorporation of HIV-1 Env, presumably acting late in assembly and early during budding.
Porcine Reproductive and Respiratory Syndrome Virus Comparison: Divergent Evolution on Two Continents
Authors: Nelsen Chris J. , Murtaugh Michael P. , Faaberg Kay S. 
Published: December 2019

ABSTRACT
Porcine reproductive and respiratory syndrome virus (PRRSV) is a recently described arterivirus responsible for disease in swine worldwide. Comparative sequence analysis of 3′-terminal structural genes of the single-stranded RNA viral genome revealed the presence of two genotypic classes of PRRSV, represented by the prototype North American and European strains, VR-2332 and Lelystad virus (LV), respectively. To better understand the evolution and pathogenicity of PRRSV, we obtained the 12,066-base 5′-terminal nucleotide sequence of VR-2332, encoding the viral replication activities, and compared it to those of LV and other arteriviruses. VR-2332 and LV differ markedly in the 5′ leader and sections of the open reading frame (ORF) 1a region. The ORF 1b sequence was nearly colinear but varied in similarity of proteins encoded in identified regions. Furthermore, molecular and biochemical analysis of subgenomic mRNA (sgmRNA) processing revealed extensive variation in the number of sgmRNAs which may be generated during infection and in the lengths of noncoding sequence between leader-body junctions and the translation-initiating codon AUG. In addition, VR-2332 and LV select different leader-body junction sites from a pool of similar candidate sites to produce sgmRNA 7, encoding the viral nucleocapsid protein. The presence of substantial variations across the entire genome and in sgmRNA processing indicates that PRRSV has evolved independently on separate continents. The near-simultaneous global emergence of a new swine disease caused by divergently evolved viruses suggests that changes in swine husbandry and management may have contributed to the emergence of PRRS.
Multiple Nucleocapsid Packaging of
Autographa californica
Nucleopolyhedrovirus Accelerates the Onset of Systemic Infection in
Trichoplusia ni
Authors: Washburn Jan O. , Lyons Eric H. , Haas-Stapleton Eric J. , Volkman Loy E. 
Published: December 2019

ABSTRACT

Among the nucleopolyhedroviruses (
Baculoviridae
), the occlusion-derived virus (ODV), which initiates infection in host insects, may contain only a single nucleocapsid per virion (the SNPVs) or one to many nucleocapsids per virion (the MNPVs), but the significance of this difference is unclear. To gain insight into the biological relevance of these different packaging strategies, we compared pathogenesis induced by ODV fractions enriched for multiple nucleocapsids (ODV-M) or single nucleocapsids (ODV-S) of
Autographa californica
multicapsid nucleopolyhedrovirus (Ac
M
NPV) containing a β-galactosidase reporter gene. In time course experiments wherein newly molted fourth-instar
Trichoplusia ni
were challenged with doses of ODV-S or ODV-M that yielded the same final mortality (∼70%), we characterized viral foci as either being restricted to the midgut or involving tracheal cells (the secondary target tissue, indicative of systemic infection). We found that while the timing of primary infection by ODV-S and ODV-M was similar, ODV-S established significantly more primary midgut cell foci than ODV-M, but ODV-M infected tracheal cells at twice the rate of ODV-S. The more efficient establishment of tracheal infections by ODV-M decreased the probability that infections were lost by midgut cell sloughing, explaining why higher numbers of primary infections established by ODV-S within larvae were needed to achieve the same final mortality. These results showed that the multiple nucleocapsid packaging strategy of Ac
M
NPV accelerates the onset of irreversible systemic infections and may indicate why MNPVs have wider individual host ranges than SNPVs.
Mutations in Nonconserved Domains of Ty3 Integrase Affect Multiple Stages of the Ty3 Life Cycle
Authors: Nymark-McMahon M. Henrietta , Sandmeyer Suzanne B. 
Published: December 2019

ABSTRACT

Ty3, a retroviruslike element of
Saccharomyces cerevisiae
, transposes into positions immediately upstream of RNA polymerase III-transcribed genes. The Ty3 integrase (IN) protein is required for integration of the replicated, extrachromosomal Ty3 DNA. In retroviral IN, a conserved core region is sufficient for strand transfer activity. In this study, charged-to-alanine scanning mutagenesis was used to investigate the roles of the nonconserved amino- and carboxyl-terminal regions of Ty3 IN. Each of the 20 IN mutants was defective for transposition, but no mutant was grossly defective for capsid maturation. All mutations affecting steady-state levels of mature IN protein resulted in reduced levels of replicated DNA, even when polymerase activity was not grossly defective as measured by exogenous reverse transcriptase activity assay. Thus, IN could contribute to nonpolymerase functions required for DNA production in vivo or to the stability of the DNA product. Several mutations in the carboxyl-terminal domain resulted in relatively low levels of processed 3′ ends of the replicated DNA, suggesting that this domain may be important for binding of IN to the long terminal repeat. Another class of mutants produced wild-type amounts of DNA with correctly processed 3′ ends. This class could include mutants affected in nuclear entry and target association. Collectively, these mutations demonstrate that in vivo, within the preintegration complex, IN performs a central role in coordinating multiple late stages of the retrotransposition life cycle.
Influenza C Virus CM2 Protein Is Produced from a 374-Amino-Acid Protein (P42) by Signal Peptidase Cleavage
Authors: Hongo Seiji , Sugawara Kanetsu , Muraki Yasushi , Matsuzaki Yoko , Takashita Emi , Kitame Fumio , Nakamura Kiyoto 
Published: December 2019

ABSTRACT

Although unspliced mRNA from influenza C virus RNA segment 6 (M gene) has a single open reading frame capable of encoding a 374-amino-acid protein (M
r
, 42,000), the major polypeptide synthesized from this mRNA species is the CM2 protein, with an M
r
of 18,000. The present study was performed to investigate the mechanism by which CM2 is generated from the unspliced mRNA. It was reported previously that the 374-amino-acid protein (P42) is an integral membrane protein having two internal hydrophobic domains, one of which (residues 241 to 252) is followed by two sequences (252 Ile-Thr-Ser and 257 Ala-Ser-Ala) favorable for cleavage by signal peptidase. To examine the possibility that P42 is cleaved by signal peptidase after Ser residue 254 or Ala residue 259 to yield CM2, we constructed three mutated M gene cDNAs in which either or both of the two sequences were eliminated and tested their ability to synthesize CM2 in the transfected COS cells. The results showed that CM2 synthesis was blocked completely when the second recognition motif for signal peptidase was removed. It was also found that when the mRNA transcript of the wild-type M gene was translated in vitro, P42, but not CM2, was synthesized in the absence of dog pancreas microsomal membranes, whereas CM2, in addition to a polypeptide (designated M1′) slightly larger than matrix protein (M1), was synthesized in the presence of microsomes. When the same experiment was done with the transcript of the mutated M gene in which the second recognition motif was removed, synthesis of CM2 could not be seen, even in the presence of microsomes. From these results, we conclude that cleavage of P42 by signal peptidase after Ala residue 259 produces CM2, composed of the C-terminal 115 amino acids, in addition to M1′, composed of the N-terminal 259 amino acids.
Spontaneous Mutation Rate of Measles Virus: Direct Estimation Based on Mutations Conferring Monoclonal Antibody Resistance
Authors: Schrag Stephanie J. , Rota Paul A. , Bellini William J. 
Published: December 2019

ABSTRACT

High mutation rates typical of RNA viruses often generate a unique viral population structure consisting of a large number of genetic microvariants. In the case of viral pathogens, this can result in rapid evolution of antiviral resistance or vaccine-escape mutants. We determined a direct estimate of the mutation rate of measles virus, the next likely target for global elimination following poliovirus. In a laboratory tissue culture system, we used the fluctuation test method of estimating mutation rate, which involves screening a large number of independent populations initiated by a small number of viruses each for the presence or absence of a particular single point mutation. The mutation we focused on, which can be screened for phenotypically, confers resistance to a monoclonal antibody (MAb 80-III-B2). The entire H gene of a subset of mutants was sequenced to verify that the resistance phenotype was associated with single point mutations. The epitope conferring MAb resistance was further characterized by Western blot analysis. Based on this approach, measles virus was estimated to have a mutation rate of 9 × 10
−5
per base per replication and a genomic mutation rate of 1.43 per replication. The mutation rates we estimated for measles virus are comparable to recent in vitro estimates for both poliovirus and vesicular stomatitis virus. In the field, however, measles virus shows marked genetic stability. We briefly discuss the evolutionary implications of these results.