bluuebunny/crossref_metadata_2025_split huggingface dataset:
PrM- and Cell-Binding Domains of the Dengue Virus E Protein
Authors: Wang Songli , He Runtao , Anderson Robert 
Published: December 2019

ABSTRACT
The E-prM proteins of flaviviruses are unusual complexes which play important roles in virus assembly and fusion modulation and in potential immunity-inducing vaccines. Despite their importance, little is known about the biogenesis and structural organization of E-prM complexes. Pulse-chase radiolabeling of dengue virus-infected Vero cells demonstrated a rapid interassociation of E and prM proteins, and sucrose gradient sedimentation analysis suggested that E-prM complexes progressed from simple heteromers to more densely sedimenting structures indicating increased multimerization. E-prM heteromers of even higher complexity were observed in virus particles, suggesting an intracellular assembly process which results in the networking of E-prM subunits into a lattice-like structure found in virus particles. Trypsin cleavage of E-prM-containing virus particles resulted in the release of a soluble 45-kDa fragment of the E protein which retained cell-binding activity. The results suggest that E-prM interactions in dengue virus particles are largely mediated by domains in the carboxy-terminal anchoring domain of E, while cell-binding activity is retained in a trypsin-releasable ectodomain of the E protein.
Neoplastic Transformation-Associated Stimulation of the In Vitro Resolution of Concatemer Junction Fragments from Minute Virus of Mice DNA
Authors: Kuntz-Simon Gaëlle , Bashir Tarig , Rommelaere Jean , Willwand Kurt 
Published: December 2019

ABSTRACT
Minute virus of mice (MVM) shows an oncotropic behavior reflected by its ability to amplify its genome more efficiently in a number of transformed versus normal cells. In vivo and in vitro studies revealed that the major effect of cell transformation on MVM DNA replication occurs at the level of double-stranded replicative-form amplification. In particular, resolution of MVM DNA concatemers into monomers was found to be highly sensitive to neoplastic transformation.
Resistance of Interleukin-1β-Deficient Mice to Fatal Sindbis Virus Encephalitis
Authors: Liang Xiao Huan , Goldman James E. , Jiang Hui Hui , Levine Beth 
Published: December 2019

ABSTRACTInterleukin-1β (IL-1β) concentrations are frequently elevated in central nervous system (CNS) viral infections, but the pathophysiologic significance of such elevations is not known. To examine the role of IL-1β in CNS viral pathogenesis, we compared the natural histories of IL-1β-deficient and wild-type 129 SV(ev) mice infected with a neurovirulent viral strain, neuroadapted Sindbis virus (NSV). We found that the incidence of severe paralysis and death was markedly decreased in NSV-infected IL-1β−/−mice compared to NSV-infected wild-type mice (4 versus 88%,P< 0.001). Despite this marked difference in clinical outcome, no differences in numbers of apoptotic cells or presence of histopathologic lesions in the brains of moribund wild-type mice and those of clinically healthy IL-1β−/−mice could be detected. These results suggest that IL-1β deficiency is protective against fatal Sindbis virus infection by a mechanism that does not involve resistance to CNS virus-induced apoptosis or histopathology.
Opposing Effects of Human Immunodeficiency Virus Type 1 Matrix Mutations Support a Myristyl Switch Model of Gag Membrane Targeting
Authors: Paillart Jean-Christophe , Göttlinger Heinrich G. 
Published: December 2019

ABSTRACT

Targeting of the human immunodeficiency virus type 1 (HIV-1) Gag precursor Pr55

gag

to the plasma membrane, the site of virus assembly, is primarily mediated by the N-terminal matrix (MA) domain. N-myristylation of MA is essential for the stable association of Pr55

gag

with membranes and for virus assembly. We now show that single amino acid substitutions near the N terminus of MA can dramatically impair assembly without compromising myristylation. Subcellular fractionation demonstrated that Gag membrane binding was compromised to a similar extent as in the absence of the myristyl acceptor site, indicating that the myristyl group was not available for membrane insertion. Remarkably, the effects of the N-terminal modifications could be completely suppressed by second-site mutations in the globular core of MA. The compensatory mutations enhanced Gag membrane binding and increased viral particle yields above wild-type levels, consistent with an increase in the exposure of the myristyl group. Our results support a model in which the compact globular core of MA sequesters the myristyl group to prevent aberrant binding to intracellular membranes, while the N terminus is critical to allow the controlled exposure of the myristyl group for insertion into the plasma membrane.
A Brome Mosaic Virus Intergenic RNA3 Replication Signal Functions with Viral Replication Protein 1a To Dramatically Stabilize RNA In Vivo
Authors: Sullivan Michael L. , Ahlquist Paul 
Published: December 2019

ABSTRACTBrome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication proteins. The 1a protein has putative helicase and RNA-capping domains, whereas 2a contains a polymerase-like domain.Saccharomyces cerevisiaeexpressing 1a and 2a is capable of replicating a BMV RNA3 template produced by in vivo transcription of a DNA copy of RNA3. Although insufficient for RNA3 replication, the expression of 1a protein alone results in a dramatic and specific stabilization of the RNA3 template in yeast. As one step toward understanding 1a-induced stabilization of RNA3, the interactions involved, and its possible relation to RNA replication, we have identified thecis-acting sequences required for this effect. We find that 1a-induced stabilization is mediated by a 150- to 190-base segment of the RNA3 intergenic region corresponding to a previously identified enhancer of RNA3 replication. Moreover, this segment is sufficient to confer 1a-induced stability on a heterologous β-globin RNA. Within this intergenic segment, partial deletions that inhibited 1a-induced stabilization in yeast expressing 1a alone resulted in parallel decreases in the levels of negative- and positive-strand RNA3 replication products in yeast expressing 1a and 2a. In particular, a small deletion encompassing a motif corresponding to the box B element of RNA polymerase III promoters dramatically reduced the ability of RNAs to respond to 1a or 1a and 2a. These and other findings suggest that 1a-induced stabilization likely reflects an early template selection step in BMV RNA replication.
The Transmembrane Domain of Hepatitis C Virus Glycoprotein E1 Is a Signal for Static Retention in the Endoplasmic Reticulum
Authors: Cocquerel Laurence , Duvet Sandrine , Meunier Jean-Christophe , Pillez André , Cacan René , Wychowski Czeslaw , Dubuisson Jean 
Published: December 2019

ABSTRACT
Hepatitis C virus (HCV) glycoproteins E1 and E2 assemble to form a noncovalent heterodimer which, in the cell, accumulates in the endoplasmic reticulum (ER). Contrary to what is observed for proteins with a KDEL or a KKXX ER-targeting signal, the ER localization of the HCV glycoprotein complex is due to a static retention in this compartment rather than to its retrieval from the cis-Golgi region. A static retention in the ER is also observed when E2 is expressed in the absence of E1 or for a chimeric protein containing the ectodomain of CD4 in fusion with the transmembrane domain (TMD) of E2. Although they do not exclude the presence of an intracellular localization signal in E1, these data do suggest that the TMD of E2 is an ER retention signal for HCV glycoprotein complex. In this study chimeric proteins containing the ectodomain of CD4 or CD8 fused to the C-terminal hydrophobic sequence of E1 were shown to be localized in the ER, indicating that the TMD of E1 is also a signal for ER localization. In addition, these chimeric proteins were not processed by Golgi enzymes, indicating that the TMD of E1 is responsible for true retention in the ER, without recycling through the Golgi apparatus. Together, these data suggest that at least two signals (TMDs of E1 and E2) are involved in ER retention of the HCV glycoprotein complex.
Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein
Authors: Urabe Masashi , Hasumi Yoko , Kume Akihiro , Surosky Richard T. , Kurtzman Gary J. , Tobita Kiyotake , Ozawa Keiya 
Published: December 2019

ABSTRACT

The adeno-associated virus (AAV) Rep78 and Rep68 proteins are required for site-specific integration of the AAV genome into the AAVS1 locus (19q13.3-qter) as well as for viral DNA replication. Rep78 and Rep68 bind to the GAGC motif on the inverted terminal repeat (ITR) and cut at the
trs
(terminal resolution site). A similar reaction is believed to occur in AAVS1 harboring an analogous GAGC motif and a
trs
homolog, followed by integration of the AAV genome. To elucidate the functional domains of Rep proteins at the amino acid level, we performed charged-to-alanine scanning mutagenesis of the N terminus (residues 1 to 240) of Rep78, where DNA binding and nicking domains are thought to exist. Mutants were analyzed for their abilities to bind the GAGC motif, nick at the
trs
homolog, and integrate an ITR-containing plasmid into AAVS1 by electrophoretic mobility shift assay,
trs
endonuclease assay, and PCR-based integration assay. We identified the residues responsible for DNA binding: R107A, K136A, and R138A mutations completely abolished the binding activity. The H90A or H92A mutant, carrying a mutation in a putative metal binding site, lost nicking activity while retaining binding activity. Mutations affecting DNA binding or
trs
nicking also impaired the site-specific integration, except for E66A and E239A. These results provide important information on the structure-function relationship of Rep proteins. We also describe an aberrant nicking of Rep78. We found that Rep78 cuts predominantly at the
trs
homolog not only between the T residues (GGT/TGG), but also between the G and T residues (GG/TTGG), which may be influenced by the sequence surrounding the GAGC motif.
Identification of CXCR4 Domains That Support Coreceptor and Chemokine Receptor Functions
Authors: Doranz Benjamin J. , Orsini Michael J. , Turner Julie D. , Hoffman Trevor L. , Berson Joanne F. , Hoxie James A. , Peiper Stephen C. , Brass Lawrence F. , Doms Robert W. 
Published: December 2019

ABSTRACTThe interaction of the chemokine stromal cell-derived factor 1 (SDF-1) with its receptor CXCR4 is vital for cell trafficking during development, is capable of inhibiting human immunodeficiency virus type 1 (HIV-1) utilization of CXCR4 as a coreceptor, and has been implicated in delaying disease progression to AIDS in vivo. Because of the importance of this chemokine-chemokine receptor pair to both development and disease, we investigated the molecular basis of the interaction between CXCR4 and its ligands SDF-1 and HIV-1 envelope. Using CXCR4 chimeras and mutants, we determined that SDF-1 requires the CXCR4 amino terminus for binding and activates downstream signaling pathways by interacting with the second extracellular loop of CXCR4. SDF-1-mediated activation of CXCR4 required the Asp-Arg-Tyr motif in the second intracellular loop of CXCR4, was pertussis toxin sensitive, and did not require the distal C-terminal tail of CXCR4. Several CXCR4 mutants that were not capable of binding SDF-1 or signaling still supported HIV-1 infection, indicating that the ability of CXCR4 to function as a coreceptor is independent of its ability to signal. Direct binding studies using the X4 gp120s HXB, BH8, and MN demonstrated the ability of HIV-1 gp120 to bind directly and specifically to the chemokine receptor CXCR4 in a CD4-dependent manner, using a conformationally complex structure on CXCR4. Several CXCR4 variants that did not support binding of soluble gp120 could still function as viral coreceptors, indicating that detectable binding of monomeric gp120 is not always predictive of coreceptor function.
Detection and Induction of Equine Infectious Anemia Virus-Specific Cytotoxic T-Lymphocyte Responses by Use of Recombinant Retroviral Vectors
Authors: Lonning S. M. , Zhang W. , Leib S. R. , McGuire T. C. 
Published: December 2019

ABSTRACTCytotoxic T lymphocytes (CTL) appear to be critical in resolving or reducing the severity of lentivirus infections. Retroviral vectors expressing the Gag/Pr or SU protein of the lentivirus equine infectious anemia virus (EIAV) were constructed and used to evaluate EIAV-specific CTL responses in horses. Three promoters, cytomegalovirus, simian virus SV40, and Moloney murine sarcoma virus (MoMSV) long terminal repeat (LTR), were used, and there was considerable variation in their ability to direct expression of Gag/Pr and SU. Vectors expressing EIAV proteins under the direction of MoMSV LTR and using the gibbon ape leukemia virus (GALV) Env for internalization were efficient at transducing equine kidney (EK) target cells and were effective targets for EIAV-specific CTL lysis. CTL from EIAV-infected horses caused lysis of retroviral vector-transduced EK cells expressing either Gag/Pr or SU in an ELA-A-restricted manner. In contrast, lysis of recombinant vaccinia virus-infected EK cells expressing Gag/Pr and SU/TM was often non-LA-A restricted. Five horses were immunized by direct intramuscular injection with a mixture of retroviral vectors expressing Gag/Pr or SU, and one responded with EIAV-specific CTL. This result indicates that retroviral vector stimulation of CTL in horses needs to be optimized, perhaps by inclusion of appropriate cytokine genes in the constructs. However, the studies demonstrated that retroviral vector-transduced target cells were very effective for in vitro dissection of EIAV-specific CTL responses.
Hepatitis C Virus Core Protein Interacts with Cellular Putative RNA Helicase
Authors: You Li-Ru , Chen Chun-Ming , Yeh Tien-Shun , Tsai Tzung-Yuan , Mai Ru-Tsun , Lin Chi-Hung , Lee Yan-Hwa Wu 
Published: December 2019

ABSTRACT

The nucleocapsid core protein of hepatitis C virus (HCV) has been shown to
trans
-act on several viral or cellular promoters. To get insight into the
trans
-action mechanism of HCV core protein, a yeast two-hybrid cloning system was used for identification of core protein-interacting cellular protein. One such cDNA clone encoding the DEAD box family of putative RNA helicase was obtained. This cellular putative RNA helicase, designated CAP-Rf, exhibits more than 95% amino acid sequence identity to other known RNA helicases including human DBX and DBY, mouse mDEAD3, and PL10, a family of proteins generally involved in translation, splicing, development, or cell growth. In vitro binding or in vivo coimmunoprecipitation studies demonstrated the direct interaction of the full-length/matured form and C-terminally truncated variants of HCV core protein with this targeted protein. Additionally, the protein’s interaction domains were delineated at the N-terminal 40-amino-acid segment of the HCV core protein and the C-terminal tail of CAP-Rf, which encompassed its RNA-binding and ATP hydrolysis domains. Immunoblotting or indirect immunofluorescence analysis revealed that the endogenous CAP-Rf was mainly localized in the nucleus and to a lesser extent in the cytoplasm, and when fused with FLAG tag, it colocalized with the HCV core protein either in the cytoplasm or in the nucleus. Similar to other RNA helicases, this cellular RNA helicase has nucleoside triphosphatase-deoxynucleoside triphosphatase activity, but this activity is inhibited by various forms of homopolynucleotides and enhanced by the HCV core protein. Moreover, transient expression of HCV core protein in human hepatoma HuH-7 cells significantly potentiated the
trans
-activation effect of FLAG-tagged CAP-Rf or untagged CAP-Rf on the luciferase reporter plasmid activity. All together, our results indicate that CAP-Rf is involved in regulation of gene expression and that HCV core protein promotes the
trans
-activation ability of CAP-Rf, likely via the complex formation and the modulation of the ATPase-dATPase activity of CAP-Rf. These findings provide evidence that HCV may have evolved a distinct mechanism in alteration of host cellular gene expression regulation via the interaction of its nucleocapsid core protein and cellular putative RNA helicase known to participate in all aspects of cellular processes involving RNA metabolism. This feature of core protein may impart pleiotropic effects on host cells, which may partially account for its role in HCV pathogenesis.
Primary and Secondary Structural Elements Required for Synthesis of Barley Yellow Dwarf Virus Subgenomic RNA1
Authors: Koev Gennadiy , Mohan B. R. , Miller W. Allen 
Published: December 2019

ABSTRACT
Barley yellow dwarf luteovirus (BYDV) generates three 3′-coterminal subgenomic RNAs (sgRNAs) in infected cells. The promoter of sgRNA1 is a putative hot spot for RNA recombination in luteovirus evolution. The sgRNA1 transcription start site was mapped previously to either nucleotide 2670 or nucleotide 2769 of BYDV genomic RNA (gRNA) in two independent studies. Our data support the former initiation site. The boundaries of the sgRNA1 promoter map between nucleotides 2595 and 2692 on genomic RNA. Computer prediction, phylogenetic comparison, and structural probing revealed two stem-loops (SL1 and SL2) in the sgRNA1 promoter region on the negative strand. Promoter function was analyzed by inoculating protoplasts with a full-length infectious clone of the BYDV genome containing mutations in the sgRNA promoter. Because the promoter is located in an essential coding region of the replicase gene, we duplicated it in a nonessential part of the genome from which a new sgRNA was expressed. Mutational analysis revealed that secondary structure, but not the nucleotide sequence, was important at the base of SL1. Regions with both RNA primary and secondary structural features that contributed to transcription initiation were found at the top of SL1. Primary sequence, but not the secondary structure, was required in SL2, which includes the initiation site. Disruption of base pairing near the sgRNA1 start site increased the level of transcription three- to fourfold. We propose that both primary and secondary structures of the sgRNA1 promoter of BYDV play unique roles in sgRNA1 promoter recognition and transcription initiation.
A Proline-Rich Motif within the Matrix Protein of Vesicular Stomatitis Virus and Rabies Virus Interacts with WW Domains of Cellular Proteins: Implications for Viral Budding
Authors: Harty Ronald N. , Paragas Jason , Sudol Marius , Palese Peter 
Published: December 2019

ABSTRACT

The matrix (M) protein of rhabdoviruses has been shown to play a key role in virus assembly and budding; however, the precise mechanism by which M mediates these processes remains unclear. We have associated a highly conserved, proline-rich motif (PPxY or PY motif, where P denotes proline, Y represents tyrosine, and x denotes any amino acid) of rhabdoviral M proteins with a possible role in budding mediated by the M protein. Point mutations that disrupt the PY motif of the M protein of vesicular stomatitis virus (VSV) have no obvious effect on membrane localization of M but instead lead to a decrease in the amount of M protein released from cells in a functional budding assay. Interestingly, the PPxY sequence within rhabdoviral M proteins is identical to that of the ligand which interacts with WW domains of cellular proteins. Indeed, results from two in vitro binding assays demonstrate that amino acids 17 through 33 and 29 through 44, which contain the PY motifs of VSV and rabies virus M proteins, respectively, mediate interactions with WW domains of specific cellular proteins. Point mutations that disrupt the consensus PY motif of VSV or rabies virus M protein result in a significant decrease in their ability to interact with the WW domains. These properties of the PY motif of rhabdovirus M proteins are strikingly analogous to those of the late (L) budding domain identified in the
gag
-specific protein p2b of Rous sarcoma virus. Thus, it is possible that rhabdoviruses may usurp host proteins to facilitate the budding process and that late stages in the budding process of rhabdoviruses and retroviruses may have features in common.
Comparative Characterization of Rep Proteins from the Helper-Dependent Adeno-Associated Virus Type 2 and the Autonomous Goose Parvovirus
Authors: Smith Deborah H. , Ward Peter , Linden R. Michael 
Published: December 2019

ABSTRACT
Adeno-associated viruses (AAVs) are nonautonomous human parvoviruses in that they are dependent on helper functions supplied by other viruses or on genotoxic stimuli for conditions permissive for replication. In the absence of helper, AAV type 2 enters latency by integration into a specific site on human chromosome 19. This feature of AAV, in combination with a lack of pathogenicity, makes AAV an attractive candidate vector for human gene therapy. Goose parvovirus (GPV) is both autonomous and pathogenic yet is highly homologous to AAV. To address the molecular bases for the different viral lifestyles, we compare the AAV and GPV nonstructural proteins, Rep78 and Rep1, respectively. We find that Rep78 and Rep1 possess several biochemical activities in common, including (i) high-affinity DNA binding for sequences that constitute the minimal DNA replication origin; (ii) nucleoside triphosphate-dependent DNA helicase activity; and (iii) origin-specific replication of double-stranded linear DNA. These experiments also establish a specific 38-bp DNA sequence as the minimal GPV DNA replication origin. It is noteworthy that although the proposed Rep binding sites of GPV and AAV are highly similar, Rep1 and Rep78 show a high degree of specificity for their respective origins, in both binding and replication assays. One significant difference was observed; with the minimal replication origin in adenovirus-uninfected extracts, Rep78-mediated replication exhibited low processivity, as previously reported. In contrast, Rep1 efficiently replicated full-length template. Overall, our studies indicate that GPV Rep1 and AAV Rep78 support a comparable mode of replication. Thus, a comparison of the two proteins provides a model system with which to determine the contribution of Rep in the regulation of dependence and autonomy at the level of DNA replication.