bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phosphorylation of Structural Components Promotes Dissociation of the Herpes Simplex Virus Type 1 Tegument
Authors: Morrison Ewan E. , Wang Yi-Fen , Meredith David M. 
Published: December 2019

ABSTRACT
The role of phosphorylation in the dissociation of structural components of the herpes simplex virus type 1 (HSV-1) tegument was investigated, using an in vitro assay. Addition of physiological concentrations of ATP and magnesium to wild-type virions in the presence of detergent promoted the release of VP13/14 and VP22. VP1/2 and the UL13 protein kinase were not significantly solubilized. However, using a virus with an inactivated UL13 protein, we found that the release of VP22 was severely impaired. Addition of casein kinase II (CKII) to UL13 mutant virions promoted VP22 release. Heat inactivation of virions or addition of phosphatase inhibited the release of both proteins. Incorporation of radiolabeled ATP into the assay demonstrated the phosphorylation of VP1/2, VP13/14, VP16, and VP22. Incubation of detergent-purified, heat-inactivated capsid-tegument with recombinant kinases showed VP1/2 phosphorylation by CKII, VP13/14 phosphorylation by CKII, protein kinase A (PKA), and PKC, VP16 phosphorylation by PKA, and VP22 phosphorylation by CKII and PKC. Proteolytic mapping and phosphoamino acid analysis of phosphorylated VP22 correlated with previously published work. The phosphorylation of virion-associated VP13/14, VP16, and VP22 was demonstrated in cells infected in the presence of cycloheximide. Use of equine herpesvirus 1 in the in vitro release assay resulted in the enhanced release of VP10, the homolog of HSV-1 VP13/14. These results suggest that the dissociation of major tegument proteins from alphaherpesvirus virions in infected cells may be initiated by phosphorylation events mediated by both virion-associated and cellular kinases.
Interactions between the Structural Domains of the RNA Replication Proteins of Plant-Infecting RNA Viruses
Authors: O’Reilly Erin K. , Wang Zhaohui , French Roy , Kao C. Cheng 
Published: December 2019

ABSTRACT
Brome mosaic virus (BMV), a positive-strand RNA virus, encodes two replication proteins: the 2a protein, which contains polymerase-like sequences, and the 1a protein, with N-terminal putative capping and C-terminal helicase-like sequences. These two proteins are part of a multisubunit complex which is necessary for viral RNA replication. We have previously shown that the yeast two-hybrid assay consistently duplicated results obtained from in vivo RNA replication assays and biochemical assays of protein-protein interaction, thus permitting the identification of additional interacting domains. We now map an interaction found to take place between two 1a proteins. Using previously characterized 1a mutants, a perfect correlation was found between the in vivo phenotypes of these mutants and their abilities to interact with wild-type 1a (wt1a) and each other. Western blot analysis revealed that the stabilities of many of the noninteracting mutant proteins were similar to that of wt1a. Deletion analysis of 1a revealed that the N-terminal 515 residues of the 1a protein are required and sufficient for 1a-1a interaction. This intermolecular interaction between the putative capping domain and itself was detected in another tripartite RNA virus, cucumber mosaic virus (CMV), suggesting that the 1a-1a interaction is a feature necessary for the replication of tripartite RNA viruses. The boundaries for various activities are placed in the context of the predicted secondary structures of several 1a-like proteins of members of the alphavirus-like superfamily. Additionally, we found a novel interaction between the putative capping and helicase-like portions of the BMV and CMV 1a proteins. Our cumulative data suggest a working model for the assembly of the BMV RNA replicase.
Purified, Soluble Recombinant Mouse Hepatitis Virus Receptor, Bgp1b, and Bgp2 Murine Coronavirus Receptors Differ in Mouse Hepatitis Virus Binding and Neutralizing Activities
Authors: Zelus Bruce D. , Wessner David R. , Williams Richard K. , Pensiero Michael N. , Phibbs Fenna T. , deSouza Mark , Dveksler Gabriela S. , Holmes Kathryn V. 
Published: December 2019

ABSTRACTMouse hepatitis virus receptor (MHVR) is a murine biliary glycoprotein (Bgp1a). Purified, soluble MHVR expressed from a recombinant vaccinia virus neutralized the infectivity of the A59 strain of mouse hepatitis virus (MHV-A59) in a concentration-dependent manner. Several anchored murine Bgps in addition to MHVR can also function as MHV-A59 receptors when expressed at high levels in nonmurine cells. To investigate the interactions of these alternative MHVR glycoproteins with MHV, we expressed and purified to apparent homogeneity the extracellular domains of several murine Bgps as soluble, six-histidine-tagged glycoproteins, using a baculovirus expression system. These include MHVR isoforms containing four or two extracellular domains and the corresponding Bgp1bglycoproteins from MHV-resistant SJL/J mice, as well as Bgp2 and truncation mutants of MHVR and Bgp1bcomprised of the first two immunoglobulin-like domains. The soluble four-domain MHVR glycoprotein (sMHVR[1-4]) had fourfold more MHV-A59 neutralizing activity than the corresponding soluble Bgp1b(sBgp1b) glycoprotein and at least 1,000-fold more neutralizing activity than sBgp2. Although virus binds to the N-terminal domain (domain 1), soluble truncation mutants of MHVR and Bgp1bcontaining only domains 1 and 2 bound virus poorly and had 10- and 300-fold less MHV-A59 neutralizing activity than the corresponding four-domain glycoproteins. In contrast, the soluble MHVR glycoprotein containing domains 1 and 4 (sMHVR[1,4]) had as much neutralizing activity as the four-domain glycoprotein, sMHVR[1-4]. Thus, the virus neutralizing activity of MHVR domain 1 appears to be enhanced by domain 4. The sBgp1b[1-4] glycoprotein had 500-fold less neutralizing activity for MHV-JHM than for MHV-A59. Thus, MHV strains with differences in S-glycoprotein sequence, tissue tropism, and virulence can differ in the ability to utilize the various murine Bgps as receptors.
Polyomavirus Large T Antigen Binds Cooperatively to Its Multiple Binding Sites in the Viral Origin of DNA Replication
Authors: Peng Yu-Cai , Acheson Nicholas H. 
Published: December 2019

ABSTRACT
Polyomavirus large T antigen binds to multiple 5′-G(A/G)GGC-3′ pentanucleotide sequences in sites 1/2, A, B, and C within and adjacent to the origin of viral DNA replication on the polyomavirus genome. We asked whether the binding of large T antigen to one of these sites could influence binding to other sites. We discovered that binding to origin DNA is substantially stronger at pH 6 to 7 than at pH 7.4 to 7.8, a range often used in DNA binding assays. Large T antigen-DNA complexes formed at pH 6 to 7 were stable, but a fraction of these complexes dissociated at pH 7.6 and above upon dilution or during electrophoresis. Increased binding at low pH is therefore due at least in part to increased stability of protein-DNA complexes, and binding at higher pH values is reversible. Binding to fragments of origin DNA in which one or more sites were deleted or inactivated by point mutations was measured by nitrocellulose filter binding and DNase I footprinting. The results showed that large T antigen binds cooperatively to its four binding sites in viral DNA, suggesting that the binding of this protein to one of these sites stabilizes its binding to other sites via protein-protein contacts. Sites A, B, and C may therefore augment DNA replication by facilitating the binding of large T antigen to site 1/2 at the replication origin. ATP stabilized large T antigen-DNA complexes against dissociation in the presence, but not the absence, of site 1/2, and ATP specifically enhanced protection against DNase I digestion in the central 10 to 12 bp of site 1/2, at which hexamers are believed to form and begin unwinding DNA. We propose that large T antigen molecules bound to these multiple sites on origin DNA interact with each other to form a compact protein-DNA complex and, furthermore, that ATP stimulates their assembly into hexamers at site 1/2 by a “handover” mechanism mediated by these protein-protein contacts.
Infectivity of a Pseudorabies Virus Mutant Lacking Attachment Glycoproteins C and D
Authors: Karger Axel , Schmidt Jerg , Mettenleiter Thomas C. 
Published: December 2019

ABSTRACT

Initiation of herpesvirus infection requires attachment of virions to the host cell followed by fusion of virion envelope and cellular cytoplasmic membrane during penetration. In several alphaherpesviruses, glycoprotein C (gC) is the primary attachment protein, interacting with cell-surface heparan sulfate proteoglycans. Secondary binding is mediated by gD, which, normally, is also required for penetration. Recently, we described the isolation of a gD-negative infectious pseudorabies virus (PrV) mutant, PrV gD
−
Pass (J. Schmidt, B. G. Klupp, A. Karger, and T. C. Mettenleiter, J. Virol. 71:17–24, 1997). In PrV gD
−
Pass, attachment and penetration occur in the absence of gD. To assess the importance of specific attachment for infectivity of PrV gD
−
Pass, the gene encoding gC was deleted, resulting in mutant PrV gCD
−
Pass. Deletion of both known attachment proteins reduced specific infectivity compared to wild-type PrV by more than 10,000-fold. Surprisingly, the virus mutant still retained significant infectivity and could be propagated on normal noncomplementing cells, indicating the presence of another receptor-binding virion protein. Selection of bovine kidney (MDBK) cells resistant to infection by PrV gCD
−
Pass resulted in the isolation of a cell clone, designated NB, which was susceptible to infection by wild-type PrV but refractory to infection by either PrV gCD
−
Pass or PrV gD
−
Pass, a defect which could partially be overcome by polyethylene glycol (PEG)-induced membrane fusion. However, even after PEG-induced infection plaque formation of PrV gCD
−
Pass or PrV gD
−
Pass did not ensue in NB cells. Also, phenotypic gD complementation of PrV gCD
−
Pass or PrV gD
−
Pass rescued the defect in infection of NB cells but did not restore plaque formation. Glycosaminoglycan analyses of MDBK and NB cells yielded identical results, and NB cells were normally susceptible to infection by other alphaherpesviruses as well as vesicular stomatitis virus. Infectious center assays after PEG-induced infection of NB cells with PrV gD
−
Pass on MDBK cells indicated efficient exit of virions from infected NB cells. Together, our data suggest the presence of another receptor and receptor-binding virion protein which can mediate PrV entry and cell-to-cell spread in MDBK cells.
Human Cytomegalovirus Glycoprotein B Contains Autonomous Determinants for Vectorial Targeting to Apical Membranes of Polarized Epithelial Cells
Authors: Tugizov Sharof , Maidji Ekaterina , Xiao Jianqiao , Zheng Zhenwei , Pereira Lenore 
Published: December 2019

ABSTRACT

We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is vectorially transported to apical membranes of CMV-infected polarized human retinal pigment epithelial cells propagated on permeable filter supports and that virions egress predominantly from the apical membrane domain. In the present study, we investigated whether gB itself contains autonomous information for apical transport by expressing the molecule in stably transfected Madine-Darby canine kidney (MDCK) cells grown on permeable filter supports. Laser scanning confocal immunofluorescence microscopy and domain-selective biotinylation of surface membrane domains showed that CMV gB was transported to apical membranes independently of other envelope glycoproteins and that it colocalized with proteins in transport vesicles of the biosynthetic and endocytic pathways. Determinants for trafficking to apical membranes were located by evaluating the targeting of gB derivatives with deletions in the lumen, transmembrane (TM) anchor, and carboxyl terminus. Derivative gB(Δ717-747), with an internal deletion in the luminal juxtamembrane sequence that preserved the
N
- and
O
-glycosylation sites, retained vectorial transport to apical membranes. In contrast, derivatives that lacked the TM anchor and cytosolic domain (gBΔ646-906) or the TM anchor alone (gBΔ751-771) underwent considerable basolateral targeting. Likewise, derivatives lacking the entire cytosolic domain (gBΔ772-906) or the last 73 amino acids (gBΔ834-906) showed disrupted apical transport. Site-specific mutations that deleted or altered the cluster of acidic residues with a casein kinase II phosphorylation site at the extreme carboxyl terminus, which can serve as an internalization signal, caused partial missorting of gB to basolateral membranes. Our studies indicate that CMV gB contains autonomous information for apical targeting in luminal, TM anchor, and cytosolic domain sequences, forming distinct structural elements that cooperate in vectorial transport in polarized epithelial cells.
CD40 Ligand-Mediated Interactions Are Involved in the Generation of Memory CD8+Cytotoxic T Lymphocytes (CTL) but Are Not Required for the Maintenance of CTL Memory following Virus Infection
Authors: Borrow Persephone , Tough David F. , Eto Danelle , Tishon Antoinette , Grewal Iqbal S. , Sprent Jonathan , Flavell Richard A. , Oldstone Michael B. A. 
Published: December 2019

ABSTRACTCD8+cytotoxic T lymphocytes (CTL) play a key role in the control of many virus infections, and the need for vaccines to elicit strong CD8+T-cell responses in order to provide optimal protection in such infections is increasingly apparent. However, the mechanisms involved in the induction and maintenance of CD8+CTL memory are currently poorly understood. In this study, we investigated the involvement of CD40 ligand (CD40L)-mediated interactions in these processes by analyzing the memory CTL response of CD40L-deficient mice following infection with lymphocytic choriomeningitis virus (LCMV). The maintenance of memory CD8+CTL precursors (CTLp) at stable frequencies over time was not impaired in CD40L-deficient mice. By contrast, the initial generation of memory CTLp was affected. CD40L-deficient mice produced lower levels of CD8+CTLp during the primary immune response to LCMV than did wild-type controls, despite the fact that the LCMV-specific effector CTL response of CD40L-deficient mice was indistinguishable from that of control animals. The differentiation of naı̈ve CD8+T cells into effector and memory CTL thus involves pathways that can be discriminated from each other by their requirement for CD40L-mediated interactions. Expression of CD40L by CTLp themselves was not an essential step during their expansion and differentiation from naı̈ve CD8+cells into memory CTLp; instead, the reduction in memory CTLp generation in CD40L-deficient mice was likely a consequence of defects in the CD4+T-cell response mounted by these animals. These results thus suggest a previously unappreciated role for CD40L in the generation of CD8+memory CTLp, the probable nature of which is discussed.
Characterization of Unusual Escape Variants of Hepatitis B Virus Isolated from a Hepatitis B Surface Antigen-Negative Subject
Authors: Grethe Stefanie , Monazahian Masyar , Böhme Ingo , Thomssen Reiner 
Published: December 2019

ABSTRACT
Hepatitis B virus DNA was extracted from serial serum samples of a hepatitis B surface antigen-negative patient with antibodies to the core protein as the only marker of an infection with hepatitis B virus. This patient showed no symptoms of hepatic injury. Sequencing of the amplified viral DNA demonstrated multiple amino acid changes clustering in surface-exposed regions of the surface protein. Synthesis and association of the middle (M) and small (S) surface proteins could be shown in vitro. The variant surface antigens were recognized neither by monoclonal antibodies to the surface antigen nor by the vaccinee’s sera. Consequences for hepatitis B surface antigen testing and vaccine development are discussed.
Identification ofNitric Oxide Synthase 2as an Innate Resistance Locus against Ectromelia Virus Infection
Authors: Karupiah Gunasegaran , Chen Jian-He , Nathan Carl F. , Mahalingam Surendran , MacMicking John D. 
Published: December 2019

ABSTRACTTo assess whethernitric oxide synthase 2(NOS2) fulfills the criteria of an innate resistance locus against an acute viral infection, we inoculated genetically deficient NOS2−/− mice with virulent ectromelia virus (EV), the causative agent of mousepox. NOS2−/− mice proved highly susceptible to EV yet showed no diminution in other well-characterized anti-EV immune responses, i.e., gamma interferon secretion and NK cell and EV-specific cytotoxic T lymphocyte activities. Thus, theNOS2locus can be considered a critical monogenic determinant of EV resistance that contributes to host survival.
Genetic Dissociation of the Encapsidation and Reverse Transcription Functions in the 5′ R Region of Human Immunodeficiency Virus Type 1
Authors: Clever Jared L. , Eckstein Daniel A. , Parslow Tristram G. 
Published: December 2019

ABSTRACT

The efficient packaging of genomic RNA into virions of human immunodeficiency virus type 1 (HIV-1) is directed by
cis
-acting encapsidation signals, which have been mapped to particular RNA stem-loop structures near the 5′ end of the genome. Earlier studies have shown that three such stem-loops, located adjacent to the major 5′ splice donor, are required for optimal packaging; more recent reports further suggest a requirement for the TAR and poly(A) hairpins of the 5′ R region. In the present study, we have compared the phenotypes that result from mutating these latter elements in the HIV-1 provirus. Using a single-round infectivity assay, we find that mutations which disrupt base pairing in either the TAR or poly(A) stems cause profound defects in both packaging and viral replication. Decreased genomic packaging in a given mutant was always accompanied by increased packaging of spliced viral RNAs. Compensatory mutations that restored base pairing also restored encapsidation, indicating that the secondary structures of the TAR and poly(A) stems, rather than their primary sequences, are important for packaging activity. Despite having normal RNA contents, however, viruses with compensatory mutations at the base of the TAR stem were severely replication defective, owing to a defect in proviral DNA synthesis. Our findings thus confirm that the HIV-1 TAR stem-loop is required for at least three essential viral functions (transcriptional activation, RNA packaging, and reverse transcription) and reveal that its packaging and reverse transcription activities can be dissociated genetically by mutations at the base of the TAR stem.
Sendai Virus Infection Induces Apoptosis through Activation of Caspase-8 (FLICE) and Caspase-3 (CPP32)
Authors: Bitzer Michael , Prinz Florian , Bauer Manuel , Spiegel Martin , Neubert Wolfgang J. , Gregor Michael , Schulze-Osthoff Klaus , Lauer Ulrich 
Published: December 2019

ABSTRACT
Sendai virus (SV) infection and replication lead to a strong cytopathic effect with subsequent death of host cells. We now show that SV infection triggers an apoptotic program in target cells. Incubation of infected cells with the peptide inhibitor z-VAD-fmk abrogated SV-induced apoptosis, indicating that proteases of the caspase family were involved. Moreover, proteolytic activation of two distinct caspases, CPP32/caspase-3 and, as shown for the first time in virus-infected cells, FLICE/caspase-8, could be detected. So far, activation of FLICE/caspase-8 has been described in apoptosis triggered by death receptors, including CD95 and tumor necrosis factor (TNF)-R1. In contrast, we could show that SV-induced apoptosis did not require TNF or CD95 ligand. We further found that apoptosis of infected cells did not influence the maturation and budding of SV progeny. In conclusion, SV-induced cell injury is mediated by CD95- and TNF-R1-independent activation of caspases, leading to the death of host cells without impairment of the viral life cycle.
Human Herpesvirus 6 Latently Infects Early Bone Marrow Progenitors In Vivo
Authors: Luppi Mario , Barozzi Patrizia , Morris Christine , Maiorana Antonio , Garber Richard , Bonacorsi Goretta , Donelli Amedea , Marasca Roberto , Tabilio Antonio , Torelli Giuseppe 
Published: December 2019

ABSTRACT
We have studied the in vivo tropism of human herpesvirus 6 (HHV-6) for hemopoietic cells in patients with latent HHV-6 infection. Having used a variety of cell purification, molecular, cytogenetic, and immunocytochemical procedures, we report the first evidence that HHV-6 latently infects early bone marrow progenitor cells and that HHV-6 may be transmitted longitudinally to cells which differentiate along the committed pathways.
Search for the Mechanism of Genetic Variation in the
pro
Gene of Human Immunodeficiency Virus
Authors: Rouzine I. M. , Coffin J. M. 
Published: December 2019

ABSTRACT

To study the mechanism of evolution of the human immunodeficiency virus (HIV) protease gene (
pro
), we analyzed a database of 213
pro
sequences isolated from 11 HIV type 1-infected patients who had not been treated with protease inhibitors. Variation in
pro
is restricted to rare variable bases which are highly diverse and differ in location among individuals; an average variable base appears in about 16% of individuals. The average intrapatient distance per individual variable site, 27%, is similar for synonymous and nonsynonymous sites, although synonymous sites are twice as abundant. The latter observation excludes selection for diversity as an important, permanently acting factor in the evolution of
pro
and leaves purifying selection as the only kind of selection. Based on this, we developed a model of evolution, both within individuals and along the transmission chain, which explains variable sites as slightly deleterious mutants slowly reverting to the better-fit variant during individual infection. In the case of a single-source transmission, genetic bottlenecks at the moment of transmission effectively suppress selection, allowing mutants to accumulate along the transmission chain to high levels. However, even very rare coinfections from independent sources are, as we show, able to counteract the bottleneck effect. Therefore, there are two possible explanations for the high mutant frequency. First, the frequency of coinfection in the natural host population may be quite low. Alternatively, a strong variation of the best-adapted sequence between individuals could be caused by a combination of an immune response present in early infection and coselection.