bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of Human Papillomavirus Type 31 Polyadenylation during the Differentiation-Dependent Life Cycle
Authors: Terhune Scott S. , Milcarek Christine , Laimins Laimonis A. 
Published: December 2019

ABSTRACT
The L1 and L2 capsid genes of human papillomavirus type 31 (HPV-31) are expressed late in the differentiation-dependent life cycle from a promoter located in the E7 open reading frame (ORF) of the early region. These late HPV genes are transcribed by RNA polymerase II which reads through the region containing early polyadenylation signals and proceeds to a poly(A) site downstream of L1. In this study, we have investigated the mechanisms regulating differentiation-dependent polyadenylation and read-through in HPV-31. HPV-31 early transcripts were found to utilize a heterogeneous series of polyadenylation sites in undifferentiated cells. The sites for polyadenylation extended over a range of 100 nucleotides from within the E5 ORF to upstream of L2. Upon differentiation, the transcription of early genes increased, but no change in the heterogeneous distribution of 3′ ends was detected. The early polyadenylation region was found to contain a single consensus hexanucleotide sequence, AAUAAA, as well as three weak binding sites for the cleavage stimulatory factor, CstF. In contrast to the heterogeneity at the early site, the 3′ ends of late transcripts encoding L1 and L2 were localized to a narrow region downstream of the late AAUAAA element. The late polyadenylation signal was found to contain a single high-affinity site for CstF, as well as one consensus hexanucleotide sequence. By using a reporter assay, it was determined that the HPV-31 early polyadenylation sequences allowed significant levels of read-through into the late region in undifferentiated cells. Upon differentiation, this read-through was increased by approximately 50%, indicating that use of the early site decreased. Differentiation was also found to induce a 40% reduction in the levels of CstF subunits, which may contribute to the increased read-through of the early sequence. The insertion of the late high-affinity binding site for CstF into the early polyadenylation region significantly reduced the level of read-through, suggesting that these factors modulate read-through activity. Our studies demonstrate that HPV-31 late gene expression is regulated in a large part by posttranscriptional mechanisms, including the polyadenylation of early transcripts.
Moloney Murine Sarcoma Virus Genomic RNAs Dimerize via a Two-Step Process: a Concentration-Dependent Kissing-Loop Interaction Is Driven by Initial Contact between Consecutive Guanines
Authors: Ly Hinh , Nierlich Donald P. , Olsen John C. , Kaplan Andrew H. 
Published: December 2019

ABSTRACT
Retroviruses contain two plus-strand genomic RNAs, which are stably but noncovalently joined in their 5′ regions by a dimer linkage structure (DLS). Two models have been put forward to explain the mechanisms by which the RNAs dimerize; each model emphasizes the role of specific molecular determinants. The kissing-loop model implicates interactions between palindromic sequences in the DLS region. The second model proposes that purine-rich stretches in the region form purine quartet structures. Here, we present an examination of the in vitro dimerization of Moloney murine sarcoma virus (MuSV) RNA in the context of these two models. Dimers were found to form spontaneously in a temperature-, time-, concentration-, and salt-dependent manner. In contrast to earlier reports, we found that deletion of neither the palindrome nor the consensus purine motifs (PuGGAPuA) affected the level of dimer formation at low concentrations of RNA. Rather, different purine-rich sequences, i.e., consecutive stretches of guanines, were found to enhance both in vitro RNA dimerization and in vivo viral replication. Biochemical evidence further suggests that these guanine-rich (G-rich) stretches form guanine quartet structures. We also found that the palindromic sequences could support dimerization at significantly higher RNA concentrations. In addition, the G-rich stretches were as important as the palindromic sequence for maintaining efficient viral replication. Overall, our data support a model that entails contributions from both of the previously proposed mechanisms of retroviral RNA dimerization.
Inhibition of Antigen Presentation by the Glycine/Alanine Repeat Domain Is Not Conserved in Simian Homologues of Epstein-Barr Virus Nuclear Antigen 1
Authors: Blake Neil W. , Moghaddam Amir , Rao Pasupuleti , Kaur Amitinder , Glickman Rhona , Cho Young-gyu , Marchini Andrew , Haigh Tracey , Johnson R. Paul , Rickinson Alan B. , Wang Fred 
Published: December 2019

ABSTRACTMost humans and Old World nonhuman primates are infected for life with Epstein-Barr virus (EBV) or closely related gammaherpesviruses in the same lymphocryptovirus (LCV) subgroup. Several potential strategies for immune evasion and persistence have been proposed based on studies of EBV infection in humans, but it has been difficult to test their actual contribution experimentally. Interest has focused on the EBV nuclear antigen 1 (EBNA1) because of its essential role in the maintenance and replication of the episomal viral genome in latently infected cells and because EBNA1 endogenously expressed in these cells is protected from presentation to the major histocompatibility complex class-I restricted cytotoxic T-lymphocyte (CTL) response through the action of an internal glycine-alanine repeat (GAR). Given the high degree of biologic conservation among LCVs which infect humans and Old World primates, we hypothesized that strategies essential for viral persistence would be well conserved among viruses of this subgroup. We show that the rhesus LCV EBNA1 shares sequence homology with the EBV and baboon LCV EBNA1 and that the rhesus LCV EBNA1 is a functional homologue for EBV EBNA1-dependent plasmid maintenance and replication. Interestingly, all three LCVs possess a GAR domain, but the baboon and rhesus LCV EBNA1 GARs fail to inhibit antigen processing and presentation as determined by using three different in vitro CTL assays. These studies suggest that inhibition of antigen processing and presentation by the EBNA1 GAR may not be an essential mechanism for persistent infection by all LCV and that other mechanisms may be important for immune evasion during LCV infection.
Catalytically Inactive Protein Phosphatase 2A Can Bind to Polyomavirus Middle Tumor Antigen and Support Complex Formation with pp60

c-
src
Authors: Ogris Egon , Mudrak Ingrid , Mak Elsa , Gibson Daryl , Pallas David C. 
Published: December 2019

ABSTRACT

Interaction between the heterodimeric form of protein phosphatase 2A (PP2A) and polyomavirus middle T antigen (MT) is required for the subsequent assembly of a transformation-competent MT complex. To investigate the role of PP2A catalytic activity in MT complex formation, we undertook a mutational analysis of the PP2A 36-kDa catalytic C subunit. Several residues likely to be involved in the dephosphorylation mechanism were identified and mutated. The resultant catalytically inactive C subunit mutants were then analyzed for their ability to associate with a cellular (B subunit) or a viral (MT) B-type subunit. Strikingly, while all of the inactive mutants were severely impaired in their interaction with B subunit, most of these mutants formed complexes with polyomavirus MT. These findings indicate a potential role for these catalytically important residues in complex formation with cellular B subunit, but not in complex formation with MT. Transformation-competent MT is known to associate with, and modulate the activity of, several cellular proteins, including pp60

c-
src

family kinases. To determine whether association of MT with an active PP2A A-C heterodimer is necessary for subsequent association with pp60

c-
src

, catalytically inactive C subunits were examined for their ability to form complexes containing pp60

c-
src

in MT-expressing cells. Two catalytically inactive C subunit mutants that efficiently formed complexes with MT also formed complexes that included an active pp60

c-
src

kinase, demonstrating that PP2A activity is not essential in
cis
in MT complexes for subsequent pp60

c-
src

association.
Factors Associated with Slow Disease Progression in Macaques Immunized with an Adenovirus-Simian Immunodeficiency Virus (SIV) Envelope Priming-gp120 Boosting Regimen and Challenged Vaginally with SIVmac251
Authors: Buge Suzan L. , Murty Lalita , Arora Kamalpreet , Kalyanaraman V. S. , Markham Phillip D. , Richardson Ersell S. , Aldrich Kristine , Patterson L. Jean , Miller Christopher J. , Cheng Sheau-Mei , Robert-Guroff Marjorie 
Published: December 2019

ABSTRACTRhesus macaques were immunized with a combination vaccine regimen consisting of adenovirus type 5 host range mutant-simian immunodeficiency virus envelope (Ad5hr-SIVenv) recombinant priming and boosting with native SIV gp120. Upon intravaginal challenge with SIVmac251, both persistently and transiently viremic animals were observed (S. L. Buge, E. Richardson, S. Alipanah, P. Markham, S. Cheng, N. Kalyan, C. J. Miller, M. Lubeck, S. Udem, J. Eldridge, and M. Robert-Guroff, J. Virol. 71:8531–8541, 1997). Long-term follow-up of the persistently viremic immunized macaques, which displayed significantly reduced viral burdens during the first 18 weeks postchallenge compared to controls, has now shown that one of four became a slow progressor, clearing virus from plasma and remaining asymptomatic with stable CD4 counts for 134 weeks postchallenge. Reboosting of the transiently viremic macaques did not reactivate latent virus. Rechallenge with two sequential SIVmac251 intravaginal exposures again resulted in partial protection of one of two immunized macaques, manifested by viral clearance and stable CD4 counts. No single immune parameter was associated with partial protection. Development of a strong antibody response capable of neutralizing a primary SIVmac251 isolate together with SIV-specific cytotoxic T lymphocytes were implicated, while CD8+T-cell antiviral activity and mucosal immune responses were not associated with delayed disease progression. Our data show that even a third immunization with the same Ad5hr-SIVenv recombinant can elicit significant immune responses to the inserted gene product, suggesting that preexisting Ad antibodies may not preclude effective immunization. Further, the partial protection against a virulent, pathogenic SIV challenge observed in two of six macaques immunized with a vaccine regimen based solely on the viral envelope indicates that this vectored-vaccine approach has promise and that multicomponent vaccines based in the same system merit further investigation.
Mapping of the Coronavirus Membrane Protein Domains Involved in Interaction with the Spike Protein
Authors: de Haan Cornelis A. M. , Smeets M. , Vernooij F. , Vennema H. , Rottier P. J. M. 
Published: December 2019

ABSTRACT
The coronavirus membrane (M) protein is the key player in virion assembly. One of its functions is to mediate the incorporation of the spikes into the viral envelope. Heterotypic interactions between M and the spike (S) protein can be demonstrated by coimmunoprecipitation and by immunofluorescence colocalization, after coexpression of their genes in eukaryotic cells. Using these assays in a mutagenetic approach, we have mapped the domains in the M protein that are involved in complex formation between M and S. It appeared that the 25-residue luminally exposed amino-terminal domain of the M protein is not important for M-S interaction. A 15-residue deletion, the insertion of a His tag, and replacement of the ectodomain by that of another coronavirus M protein did not affect the ability of the M protein to associate with the S protein. However, complex formation was sensitive to changes in the transmembrane domains of this triple-spanning protein. Deletion of either the first two or the last two transmembrane domains, known not to affect the topology of the protein, led to a considerable decrease in complex formation, but association was not completely abrogated. Various effects of changes in the part of the M protein that is located at the cytoplasmic face of the membrane were observed. Deletions of the extreme carboxy-terminal tail appeared not to interfere with M-S complex formation. However, deletions in the amphipathic domain severely affected M-S interaction. Interestingly, changes in the amino-terminal and extreme carboxy-terminal domains of M, which did not disrupt the interaction with S, are known to be fatal to the ability of the protein to engage in virus particle formation (C. A. M. de Haan, L. Kuo, P. S. Masters, H. Vennema, and P. J. M. Rottier, J. Virol. 72:6838–6850, 1998). Apparently, the structural requirements of the M protein for virus particle assembly differ from the requirements for the formation of M-S complexes.
B-
myb
Promoter Retargeting of Herpes Simplex Virus γ34.5 Gene-Mediated Virulence toward Tumor and Cycling Cells
Authors: Chung Richard Y. , Saeki Yoshinaga , Chiocca E. Antonio 
Published: December 2019

ABSTRACT

Deletion of the γ34.5 gene coding for virulence markedly reduces cytotoxicity mediated by herpes simplex virus type 1 (HSV-1) (J. M. Markert et al., Neurosurgery 32:597–603, 1993; N. S. Markovitz et al., J. Virol. 71:5560–5569, 1997). To target lytic virulence to tumors, we have created a novel HSV-1 mutant, designated Myb34.5. This viral mutant is characterized by a deletion of the gene for infected cell polypeptide 6 (ICP6; also known as UL39 or ribonucleotide reductase) and of the two endogenous copies of the γ34.5 gene (RL1) and by reintroduction of one copy of γ34.5 under control of the E2F-responsive, cellular B-
myb
promoter. On direct intracerebral inoculation in BALB/c mice, the 50% lethal dose (LD
50
) for Myb34.5 was 2.7 × 10
7
PFU while that for HSVs with mutations in the γ34.5 gene could not be technically achieved with available viral stocks and it was estimated as >1 × 10
7
PFU. The LD
50
for an HSV with a single defect in ICP6 function was 1.3 × 10
6
PFU. Conversely, Myb34.5’s oncolytic efficacy against a variety of human glioma cells in culture and in vivo was enhanced compared to that of HSVs with γ34.5 mutations, and in fact, it was comparable to that of the wild-type F strain and of viral mutants that possess a wild-type γ34.5 gene. The characteristic shutoff of host protein synthesis, occurring after infection of human SK-N-SH neuroblastoma cells by γ34.5 mutant viruses (J. Chou and B. Roizman, Proc. Natl. Acad. Sci. USA 89:3266–3270, 1992), was not present after infection with Myb34.5. There was an increase of almost 3 logarithmic units in the production of progeny virus in arrested fibroblasts compared to that in cycling fibroblasts infected with Myb34.5. These results suggest that transcriptional regulation of γ34.5 by cell cycle-regulated promoters can be used to target HSV-1 virulence toward tumors while maintaining the desirable neuroattenuated phenotype of a γ34.5 mutant.
Antibody-Independent Protection against Rotavirus Infection of Mice Stimulated by Intranasal Immunization with Chimeric VP4 or VP6 Protein
Authors: Choi Anthony H.-C. , Basu Mitali , McNeal Monica M. , Clements John D. , Ward Richard L. 
Published: December 2019

ABSTRACT

This study was to determine whether individual rotavirus capsid proteins could stimulate protection against rotavirus shedding in an adult mouse model. BALB/c mice were intranasally or intramuscularly administered purified
Escherichia coli
-expressed murine rotavirus strain EDIM VP4, VP6, or truncated VP7 (TrVP7) protein fused to the 42.7-kDa maltose-binding protein (MBP). One month after the last immunization, mice were challenged with EDIM and shedding of rotavirus antigen was measured. When three 9-μg doses of one of the three rotavirus proteins fused to MBP were administered intramuscularly with the saponin adjuvant QS-21, serum rotavirus immunoglobulin G (IgG) was induced by each protein. Following EDIM challenge, shedding was significantly (
P
= 0.02) reduced (i.e., 38%) in MBP::VP6-immunized mice only. Three 9-μg doses of chimeric MBP::VP6 or MBP::TrVP7 administered intranasally with attenuated
E. coli
heat-labile toxin LT(R192G) also induced serum rotavirus IgG, but MBP::VP4 immunization stimulated no detectable rotavirus antibody. No protection against EDIM shedding was observed in the MBP::TrVP7-immunized mice. However, shedding was reduced 93 to 100% following MBP::VP6 inoculation and 56% following MBP::VP4 immunization relative to that of controls (
P
= <0.001). Substitution of cholera toxin for LT(R192G) as the adjuvant, reduction of the number of doses to 1, and challenge of the mice 3 months after the last immunization did not reduce the level of protection stimulated by intranasal administration of MBP::VP6. When MBP::VP6 was administered intranasally to B-cell-deficient μMt mice that made no rotavirus antibody, shedding was still reduced to <1% of that of controls. These results show that mice can be protected against rotavirus shedding by intranasal administration of individual rotavirus proteins and that this protection can occur independently of rotavirus antibody.
Epstein-Barr Virus BARF1 Protein Is Dispensable for B-Cell Transformation and Inhibits Alpha Interferon Secretion from Mononuclear Cells
Authors: Cohen Jeffrey I. , Lekstrom Kristen 
Published: December 2019

ABSTRACTThe Epstein-Barr virus (EBV) BARF1 gene encodes a soluble colony-stimulating factor 1 (CSF-1) receptor that neutralizes the effects of CSF-1 in vitro. To study the effect of BARF1 on EBV-induced transformation, we added recombinant BARF1 to B cells in the presence of EBV. BARF1 did not enhance transformation of B cells by EBV in vitro. To study the role of BARF1 in the context of EBV infection, we constructed a recombinant EBV mutant with a large deletion followed by stop codons in the BARF1 gene as well as a recombinant virus with a wild-type BARF1 gene. While BARF1 has previously been shown to act as an oncogene in several cell lines, the EBV BARF1 deletion mutant transformed B cells and initiated latent infection, and the B cells transformed with the BARF1 mutant virus induced tumors in SCID mice with an efficiency similar to that of the wild-type recombinant virus. Since human CSF-1 stimulates secretion of alpha interferon from mononuclear cells and BARF1 encodes a soluble CSF-1 receptor, we examined whether recombinant BARF1 or BARF1 derived from EBV-infected B cells could inhibit alpha interferon secretion. Recombinant BARF1 inhibited alpha interferon secretion by mononuclear cells in a dose-dependent fashion. The B cells transformed with mutant BARF1 EBV showed reduced inhibition of alpha interferon secretion by human mononuclear cells when compared with the B cells transformed with wild-type recombinant virus. These experiments indicate that BARF1 expressed from the EBV genome directly inhibits alpha interferon secretion, which may modulate the innate host response to the virus.
Murine Gammaherpesvirus 68 Encodes a Functional Regulator of Complement Activation
Authors: Kapadia Sharookh B. , Molina Hector , van Berkel Victor , Speck Samuel H. , Virgin Herbert W. 
Published: December 2019

ABSTRACTSequence analysis of the murine gammaherpesvirus 68 (γHV68) genome revealed an open reading frame (gene 4) which is homologous to a family of proteins known as the regulators of complement activation (RCA proteins) (H. W. Virgin, P. Latreille, P. Wamsley, K. Hallsworth, K. E. Weck, A. J. Dal Canto, and S. H. Speck, J. Virol. 71:5894–5904, 1997). The predicted gene 4 product has homology to other virally encoded RCA homologs, as well as to the complement-regulatory proteins decay-accelerating factor and membrane cofactor protein. Analyses by Northern blotting and rapid amplification of cDNA ends revealed that gene 4 is transcribed as a 5.2-kb bicistronic transcript of the late kinetic class. Three γHV68 RCA protein isoforms (60 to 65 kDa, 50 to 55 kDa, and 40 to 45 kDa) were detected by Western blotting of infected murine NIH 3T12 fibroblast cells. A soluble 40- to 45-kDa isoform was detected in the supernatants of virally infected cells. Flow cytometric analysis revealed that the γHV68 RCA protein was expressed on the surfaces of infected cells. Supernatants from virally infected cells contained an activity that inhibited murine complement activation as measured by inhibition of C3 deposition on activated zymosan particles. Recombinant γHV68 RCA protein, containing the four conserved short consensus repeats, inhibited murine C3 deposition on zymosan via both classical and alternative pathways and inhibited deposition of human C3 on activated zymosan particles. Expression of this inhibitor of complement activation, both at the cell surface and in the fluid phase, may be important for γHV68 pathogenesis via the inhibition of innate and adaptive immunity.
Cytopathogenic and Noncytopathogenic RNA Replicons of Classical Swine Fever Virus
Authors: Moser Christian , Stettler Peter , Tratschin Jon-Duri , Hofmann Martin A. 
Published: December 2019

ABSTRACT

To determine the minimal requirements for autonomous RNA replication of classical swine fever virus (CSFV), genomes having in-frame deletions within the genes for structural and flanking nonstructural proteins were constructed, based on an infectious cDNA clone of CSFV Alfort/187. RNA was transcribed in vitro from the respective plasmids and transfected into SK-6 swine kidney cells. The replication competence of the RNA was determined by immunostaining transfected cells for CSFV NS3 protein and by analysis of cell extracts for viral RNA, as well as protein synthesis at different times after transfection. The genes encoding N
pro
, C, E
rns
, E1, E2, p7, and NS2 proved to be dispensable for RNA replication, but the efficiency of replication varied strongly between individual constructs. RNA replicons containing the complete NS2-NS3 gene persisted in transfected cells and continued to replicate without causing any obvious morphological or functional damage to the cells, whereas genomes lacking the NS2 gene replicated more efficiently and induced a cytopathic effect. These findings suggest that NS2, although it is not essential for pestivirus RNA replication, has a regulatory function therein. Both cytopathogenic and noncytopathogenic replicons were packaged into virus particles provided in
trans
by a cotransfected full-length helper virus genome.
R5 Strains of Human Immunodeficiency Virus Type 1 from Rapid Progressors Lacking X4 Strains Do Not Possess X4-Type Pathogenicity in Human Thymus
Authors: Berkowitz Robert D. , van ’t Wout Angélique B. , Kootstra Neeltje A. , Moreno Mary E. , Linquist-Stepps Valerie D. , Bare Christopher , Stoddart Cheryl A. , Schuitemaker Hanneke , McCune Joseph M. 
Published: December 2019

ABSTRACTSome individuals infected with only R5 strains of human immunodeficiency virus type 1 progress to AIDS as quickly as individuals harboring X4 strains. We determined that three R5 viruses were much less pathogenic than an X4 virus in SCID-hu Thy/Liv mice, suggesting that R5 virus-mediated rapid disease progression is associated with host, not viral, factors.
Turnover of T Cells in Murine Gammaherpesvirus 68-Infected Mice
Authors: Hamilton-Easton Ann Marie , Christensen Jan P. , Doherty Peter C. 
Published: December 2019

ABSTRACTRespiratory challenge of C57BL/6 mice with murine gammaherpesvirus 68 induces proliferation of T lymphocytes early after infection, as evidenced by incorporation of the DNA precursor bromodeoxyuridine. Using pulse-chase analysis, splenic and peripheral blood activated T lymphocytes were found to continue dividing for at least a month after the initial virus challenge. The results are in accord with the idea that T cells are stimulated for a substantial time after the acute, lytic phase of virus infection is resolved.