bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Major Determinant for Membrane Protein Interaction Localizes to the Carboxy-Terminal Domain of the Mouse Coronavirus Nucleocapsid Protein
Authors: Hurst Kelley R. , Kuo Lili , Koetzner Cheri A. , Ye Rong , Hsue Bilan , Masters Paul S. 
Published: October 2005

ABSTRACT
The two major constituents of coronavirus virions are the membrane (M) and nucleocapsid (N) proteins. The M protein is anchored in the viral envelope by three transmembrane segments flanked by a short amino-terminal ectodomain and a large carboxy-terminal endodomain. The M endodomain interacts with the viral nucleocapsid, which consists of the positive-strand RNA genome helically encapsidated by N protein monomers. In previous work with the coronavirus mouse hepatitis virus (MHV), a highly defective M protein mutant, MΔ2, was constructed. This mutant contained a 2-amino-acid carboxy-terminal truncation of the M protein. Analysis of second-site revertants of MΔ2 revealed mutations in the carboxy-terminal region of the N protein that compensated for the defect in the M protein. To seek further genetic evidence corroborating this interaction, we generated a comprehensive set of clustered charged-to-alanine mutants in the carboxy-terminal domain 3 of N protein. One of these mutants, CCA4, had a highly defective phenotype similar to that of MΔ2. Transfer of the CCA4 mutation into a partially diploid MHV genome showed that CCA4 was a loss-of-function mutation rather than a dominant-negative mutation. Analysis of multiple second-site revertants of CCA4 revealed mutations in both the M protein and the N protein that could compensate for the original lesion in N. These data more precisely define the region of the N protein that interacts with the M protein. Further, we found that fusion of domain 3 of the N protein to the carboxy terminus of a heterologous protein caused it to be incorporated into MHV virions.
In Nasopharyngeal Carcinoma Cells, Epstein-Barr Virus LMP1 Interacts with Galectin 9 in Membrane Raft Elements Resistant to Simvastatin
Authors: Pioche-Durieu Catherine , Keryer Cécile , Souquère Sylvie , Bosq Jacques , Faigle Wolfgang , Loew Damarys , Hirashima Mitsuomi , Nishi Nozomu , Middeldorp Jaap , Busson Pierre 
Published: October 2005

ABSTRACTNasopharyngeal carcinomas (NPC) are etiologically related to the Epstein-Barr virus (EBV), and malignant NPC cells have consistent although heterogeneous expression of the EBV latent membrane protein 1 (LMP1). LMP1 trafficking and signaling require its incorporation into membrane rafts. Conversely, raft environment is likely to modulate LMP1 activity. In order to investigate NPC-specific raft partners of LMP1, rafts derived from the C15 NPC xenograft were submitted to preparative immunoprecipitation of LMP1 combined with mass spectrometry analysis of coimmunoprecipitated proteins. Through this procedure, galectin 9, a beta-galactoside binding lectin and Hodgkin tumor antigen, was identified as a novel LMP1 partner. LMP1 interaction with galectin 9 was confirmed by coimmunoprecipitation and Western blotting in whole-cell extracts of NPC and EBV-transformed B cells (lymphoblastoid cell lines [LCLs]). Using mutant proteins expressed in HeLa cells, LMP1 was shown to bind galectin 9 in a TRAF3-independent manner. Galectin 9 is abundant in NPC biopsies as well as in LCLs, whereas it is absent in Burkitt lymphoma cells. In subsequent experiments, NPC cells were treated with Simvastatin, a drug reported to dissociate LMP1 from membrane rafts in EBV-transformed B cells. We found no significant effects of Simvastatin on the distribution of LMP1 and galectin 9 in NPC cell rafts. However, Simvastatin was highly cytotoxic for NPC cells, regardless of the presence or absence of LMP1. This suggests that Simvastatin is a potentially useful agent for the treatment of NPCs although it has distinct mechanisms of action in NPC and LCL cells.
Alpha/Beta Interferon Protects against Lethal West Nile Virus Infection by Restricting Cellular Tropism and Enhancing Neuronal Survival
Authors: Samuel Melanie A. , Diamond Michael S. 
Published: October 2005

ABSTRACT

West Nile virus (WNV) is a mosquito-borne flavivirus that is neurotropic in humans, birds, and other animals. While adaptive immunity plays an important role in preventing WNV spread to the central nervous system (CNS), little is known about how alpha/beta interferon (IFN-α/β) protects against peripheral and CNS infection. In this study, we examine the virulence and tropism of WNV in IFN-α/β receptor-deficient (IFN- α/βR
−/−
) mice and primary neuronal cultures. IFN-α/βR
−/−
mice were acutely susceptible to WNV infection through subcutaneous inoculation, with 100% mortality and a mean time to death (MTD) of 4.6 ± 0.7 and 3.8± 0.5 days after infection with 10
0
and 10
2
PFU, respectively. In contrast, congenic wild-type 129Sv/Ev mice infected with 10
2
PFU showed 62% mortality and a MTD of 11.9 ± 1.9 days. IFN-α/βR
−/−
mice developed high viral loads by day 3 after infection in nearly all tissues assayed, including many that were not infected in wild-type mice. IFN-α/βR
−/−
mice also demonstrated altered cellular tropism, with increased infection in macrophages, B cells, and T cells in the spleen. Additionally, treatment of primary wild-type neurons in vitro with IFN-β either before or after infection increased neuronal survival independent of its effect on WNV replication. Collectively, our data suggest that IFN-α/β controls WNV infection by restricting tropism and viral burden and by preventing death of infected neurons.
Kelp Fly Virus: a Novel Group of Insect Picorna-Like Viruses as Defined by Genome Sequence Analysis and a Distinctive Virion Structure
Authors: Hartley C. J. , Greenwood D. R. , Gilbert R. J. C. , Masoumi A. , Gordon K. H. J. , Hanzlik T. N. , Fry E. E. , Stuart D. I. , Scotti P. D. 
Published: October 2005

ABSTRACT

The complete genomic sequence of kelp fly virus (KFV), originally isolated from the kelp fly,
Chaetocoelopa sydneyensis
, has been determined. Analyses of its genomic and structural organization and phylogeny show that it belongs to a hitherto undescribed group within the picorna-like virus superfamily. The single-stranded genomic RNA of KFV is 11,035 nucleotides in length and contains a single large open reading frame encoding a polypeptide of 3,436 amino acids with 5′ and 3′ untranslated regions of 384 and 343 nucleotides, respectively. The predicted amino acid sequence of the polypeptide shows that it has three regions. The N-terminal region contains sequences homologous to the baculoviral inhibitor of apoptosis repeat domain, an inhibitor of apoptosis commonly found in animals and in viruses with double-stranded DNA genomes. The second region contains at least two capsid proteins. The third region has three sequence motifs characteristic of replicase proteins of many plant and animal viruses, including a helicase, a 3C chymotrypsin-like protease, and an RNA-dependent RNA polymerase. Phylogenetic analysis of the replicase motifs shows that KFV forms a distinct and distant taxon within the picorna-like virus superfamily. Cryoelectron microscopy and image reconstruction of KFV to a resolution of 15 Å reveals an icosahedral structure, with each of its 12 fivefold vertices forming a turret from the otherwise smooth surface of the 20-Å-thick capsid. The architecture of the KFV capsid is unique among the members of the picornavirus superfamily for which structures have previously been determined.
The nsp2 Replicase Proteins of Murine Hepatitis Virus and Severe Acute Respiratory Syndrome Coronavirus Are Dispensable for Viral Replication
Authors: Graham Rachel L. , Sims Amy C. , Brockway Sarah M. , Baric Ralph S. , Denison Mark R. 
Published: October 2005

ABSTRACT

The positive-stranded RNA genome of the coronaviruses is translated from ORF1 to yield polyproteins that are proteolytically processed into intermediate and mature nonstructural proteins (nsps). Murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) polyproteins incorporate 16 protein domains (nsps), with nsp1 and nsp2 being the most variable among the coronaviruses and having no experimentally confirmed or predicted functions in replication. To determine if nsp2 is essential for viral replication, MHV and SARS-CoV genome RNA was generated with deletions of the nsp2 coding sequence (MHVΔnsp2 and SARSΔnsp2, respectively). Infectious MHVΔnsp2 and SARSΔnsp2 viruses recovered from electroporated cells had 0.5 to 1 log
10
reductions in peak titers in single-cycle growth assays, as well as a reduction in viral RNA synthesis that was not specific for any positive-stranded RNA species. The Δnsp2 mutant viruses lacked expression of both nsp2 and an nsp2-nsp3 precursor, but cleaved the engineered chimeric nsp1-nsp3 cleavage site as efficiently as the native nsp1-nsp2 cleavage site. Replication complexes in MHVΔnsp2-infected cells lacked nsp2 but were morphologically indistinguishable from those of wild-type MHV by immunofluorescence. nsp2 expressed in cells by stable retroviral transduction was specifically recruited to viral replication complexes upon infection with MHVΔnsp2. These results demonstrate that while nsp2 of MHV and SARS-CoV is dispensable for viral replication in cell culture, deletion of the nsp2 coding sequence attenuates viral growth and RNA synthesis. These findings also provide a system for the study of determinants of nsp targeting and function.
Liver-Directed Gamma Interferon Gene Delivery in Chronic Hepatitis C
Authors: Shin Eui-Cheol , Protzer Ulrike , Untergasser Andreas , Feinstone Stephen M. , Rice Charles M. , Hasselschwert Dana , Rehermann Barbara 
Published: October 2005

ABSTRACT
Gamma interferon (IFN-γ) has been shown to inhibit replication of subgenomic and genomic hepatitis C virus (HCV) RNAs in vitro and to noncytolytically suppress hepatitis B virus (HBV) replication in vivo. IFN-γ is also known for its immunomodulatory effects and as a marker of a successful cellular immune response to HCV. Therapeutic expression of IFN-γ in the liver may therefore facilitate resolution of chronic hepatitis C, an infection that is rarely resolved spontaneously. To analyze immunomodulatory and antiviral effects of liver-specific IFN-γ expression in vivo, we intravenously injected two persistently HCV-infected chimpanzees twice with a recombinant, replication-deficient HBV vector and subsequently with a recombinant adenoviral vector. These vectors expressed human IFN-γ under control of HBV- and liver-specific promoters, respectively. Gene transfer resulted in a transient increase of intrahepatic IFN-γ mRNA, without increase in serum alanine aminotransferase levels. Ex vivo analysis of peripheral blood lymphocytes demonstrated enhanced CD16 expression on T cells and upregulation of the liver-homing marker CXCR3. Moreover, an increased frequency of HCV-specific T cells was detected ex vivo in the peripheral blood and in vitro in liver biopsy-derived, antigen-nonspecifically expanded T-cell lines. None of these immunologic effects were observed in the third chimpanzee injected with an HBV control vector. Despite these immunologic effects of the experimental vector, however, IFN-γ gene transfer did not result in a significant and long-lasting decrease of HCV titers. In conclusion, liver-directed IFN-γ gene delivery resulted in HCV-specific and nonspecific activation of cellular immune responses but did not result in effective control of HCV replication.
VP24 of Marburg Virus Influences Formation of Infectious Particles
Authors: Bamberg Sandra , Kolesnikova Larissa , Möller Peggy , Klenk Hans-Dieter , Becker Stephan 
Published: October 2005

ABSTRACT
The highly pathogenic enveloped Marburg virus (MARV) is composed of seven
structural proteins and the nonsegmented negative-sense viral RNA
genome. Four proteins (NP, VP35, VP30, and L) make up the helical
nucleocapsid, which is surrounded by a matrix that is composed of the
viral proteins VP40 and VP24. VP40 is functionally homologous to the
matrix proteins of other nonsegmented negative-strand RNA viruses. As
yet, the function of VP24 remains elusive. In the present study we
found that VP24 colocalized with inclusions in MARV-infected cells that
contain preformed nucleocapsids and with nucleocapsids outside the
inclusions. Coexpression studies revealed that VP24 is recruited into
the inclusions by the presence of NP. Furthermore, VP24 displayed
membrane-binding properties and was recruited into filamentous
virus-like particles (VLPs) that are induced by VP40. The incorporation
of VP24 altered neither the morphology of VLPs nor the budding
efficiency of VLPs. When VP24 was silenced in MARV-infected cells by
small interfering RNA technology, the release of viral particles was
significantly reduced while viral transcription and replication were
unimpaired. Our data support the idea that VP24 is essential for a
process that takes place after replication and transcription and before
budding of virus progeny. It is presumed that VP24 is necessary for the
formation of transport-competent nucleocapsids and/or the interaction
between the nucleocapsids and the budding sites at the plasma
membrane.
Simian Virus 5 V Protein Acts as an Adaptor, Linking DDB1 to STAT2, To Facilitate the Ubiquitination of STAT1
Authors: Precious B. , Childs K. , Fitzpatrick-Swallow V. , Goodbourn S. , Randall R. E. 
Published: October 2005

ABSTRACT
The V protein of simian virus 5 (SV5) facilitates the ubiquitination and subsequent proteasome-mediated degradation of STAT1. Here we show, by visualizing direct protein-protein interactions and by using the yeast two-hybrid system, that while the SV5 V protein fails to bind to STAT1 directly, it binds directly and independently to both DDB1 and STAT2, two cellular proteins known to be essential for SV5-mediated degradation of STAT1. We also demonstrate that STAT1 and STAT2 interact independently of SV5 V and show that SV5 V protein acts as an adaptor molecule linking DDB1 to STAT2/STAT1 heterodimers, which in the presence of additional accessory cellular proteins, including Cullin 4a, can ubiquitinate STAT1. Additionally, we show that the avidity of STAT2 for V is relatively weak but is significantly enhanced by the presence of both STAT1 and DDB1, i.e., the complex of STAT1, STAT2, DDB1, and SV5 V is more stable than a complex of STAT2 and V. From these studies we propose a dynamic model in which SV5 V acts as a bridge, bringing together a DDB1/Cullin 4a-containing ubiquitin ligase complex and STAT1/STAT2 heterodimers, which leads to the degradation of STAT1. The loss of STAT1 results in a decrease in affinity of binding of STAT2 for V such that STAT2 either dissociates from V or is displaced from V by STAT1/STAT2 complexes, thereby ensuring the cycling of the DDB1 and SV5 V containing E3 complex for continued rounds of STAT1 ubiquitination and degradation.
The Retroviral Capsid Domain Dictates Virion Size, Morphology, and Coassembly of Gag into Virus-Like Particles
Authors: Ako-Adjei Danso , Johnson Marc C. , Vogt Volker M. 
Published: October 2005

ABSTRACT
The retroviral structural protein, Gag, is capable of independently assembling into virus-like particles (VLPs) in living cells and in vitro. Immature VLPs of human immunodeficiency virus type 1 (HIV-1) and of Rous sarcoma virus (RSV) are morphologically distinct when viewed by transmission electron microscopy (TEM). To better understand the nature of the Gag-Gag interactions leading to these distinctions, we constructed vectors encoding several RSV/HIV-1 chimeric Gag proteins for expression in either insect cells or vertebrate cells. We used TEM, confocal fluorescence microscopy, and a novel correlative scanning EM (SEM)-confocal microscopy technique to study the assembly properties of these proteins. Most chimeric proteins assembled into regular VLPs, with the capsid (CA) domain being the primary determinant of overall particle diameter and morphology. The presence of domains between matrix and CA also influenced particle morphology by increasing the spacing between the inner electron-dense ring and the VLP membrane. Fluorescently tagged versions of wild-type RSV, HIV-1, or murine leukemia virus Gag did not colocalize in cells. However, wild-type Gag proteins colocalized extensively with chimeric Gag proteins bearing the same CA domain, implying that Gag interactions are mediated by CA. A dramatic example of this phenomenon was provided by a nuclear export-deficient chimera of RSV Gag carrying the HIV-1 CA domain, which by itself localized to the nucleus but relocalized to the cytoplasm in the presence of wild type HIV-1 Gag. Wild-type and chimeric Gag proteins were capable of coassembly into a single VLP as viewed by correlative fluorescence SEM if, and only if, the CA domain was derived from the same virus. These results imply that the primary selectivity of Gag-Gag interactions is determined by the CA domain.
CD8 T Cells Require Gamma Interferon To Clear Borna Disease Virus from the Brain and Prevent Immune System-Mediated Neuronal Damage
Authors: Hausmann Jürgen , Pagenstecher Axel , Baur Karen , Richter Kirsten , Rziha Hanns-Joachim , Staeheli Peter 
Published: October 2005

ABSTRACTBorna disease virus (BDV) frequently causes meningoencephalitis and fatal neurological disease in young but not old mice of strain MRL. Disease does not result from the virus-induced destruction of infected neurons. Rather, it is mediated byH-2k-restricted antiviral CD8 T cells that recognize a peptide derived from the BDV nucleoprotein N. Persistent BDV infection in mice is not spontaneously cleared. We report here that N-specific vaccination can protect wild-type MRL mice but not mutant MRL mice lacking gamma interferon (IFN-γ) from persistent infection with BDV. Furthermore, we observed a significant degree of resistance of old MRL mice to persistent BDV infection that depended on the presence of CD8 T cells. We found that virus initially infected hippocampal neurons around 2 weeks after intracerebral infection but was eventually cleared in most wild-type MRL mice. Unexpectedly, young as well as old IFN-γ-deficient MRL mice were completely susceptible to infection with BDV. Moreover, neurons in the CA1 region of the hippocampus were severely damaged in most diseased IFN-γ-deficient mice but not in wild-type mice. Furthermore, large numbers of eosinophils were present in the inflamed brains of IFN-γ-deficient mice but not in those of wild-type mice, presumably because of increased intracerebral synthesis of interleukin-13 and the chemokines CCL1 and CCL11, which can attract eosinophils. These results demonstrate that IFN-γ plays a central role in host resistance against infection of the central nervous system with BDV and in clearance of BDV from neurons. They further indicate that IFN-γ may function as a neuroprotective factor that can limit the loss of neurons in the course of antiviral immune responses in the brain.
Nonrandom Packaging of Host RNAs in Moloney Murine Leukemia Virus
Authors: Onafuwa-Nuga Adewunmi A. , King Steven R. , Telesnitsky Alice 
Published: October 2005

ABSTRACT

Moloney murine leukemia virus (MLV) particles contain both viral genomic RNA and an assortment of host cell RNAs. Packaging of virus-encoded RNA is selective, with virions virtually devoid of spliced
env
mRNA and highly enriched for unspliced genome. Except for primer tRNA, it is unclear whether packaged host RNAs are randomly sampled from the cell or specifically encapsidated. To address possible biases in host RNA sampling, the relative abundances of several host RNAs in MLV particles and in producer cells were compared. Using 7SL RNA as a standard, some cellular RNAs, such as those of the Ro RNP, were found to be enriched in MLV particles in that their ratios relative to 7SL differed little, if at all, from their ratios in cells. Some RNAs were underrepresented, with ratios relative to 7SL several orders of magnitude lower in virions than in cells, while others displayed intermediate values. At least some enriched RNAs were encapsidated by genome-defective nucleocapsid mutants. Virion RNAs were not a random sample of the cytosol as a whole, since some cytoplasmic RNAs like tRNA
Met
were vastly underrepresented, while U6 spliceosomal RNA, which functions in the nucleus, was enriched. Real-time PCR demonstrated that
env
mRNA, although several orders of magnitude less abundant than unspliced viral RNA, was slightly enriched relative to actin mRNA in virions. These data demonstrate that certain host RNAs are nearly as enriched in virions as genomic RNA and suggest that Ψ
−
mRNAs and some other host RNAs may be specifically excluded from assembly sites.
β-Adrenoreceptors Reactivate Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication via PKA-Dependent Control of Viral RTA
Authors: Chang Margaret , Brown Helen J. , Collado-Hidalgo Alicia , Arevalo Jesusa M. , Galic Zoran , Symensma Tonia L. , Tanaka Lena , Deng Hongyu , Zack Jerome A. , Sun Ren , Cole Steve W. 
Published: October 2005

ABSTRACTReactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication is mediated by the viral RTA transcription factor, but little is known about the physiological processes controlling its expression or activity. Links between autonomic nervous system activity and AIDS-associated Kaposi's sarcoma led us to examine the potential influence of catecholamine neurotransmitters. Physiological concentrations of epinephrine and norepinephrine efficiently reactivated lytic replication of KSHV in latently infected primary effusion lymphoma cells via β-adrenergic activation of the cellular cyclic AMP/protein kinase A (PKA) signaling pathway. Effects were blocked by PKA antagonists and mimicked by pharmacological and physiological PKA activators (prostaglandin E2and histamine) or overexpression of the PKA catalytic subunit. PKA up-regulatedRTAgene expression, enhanced activity of theRTApromoter, and posttranslationally enhanced RTA'strans-activating capacity for its own promoter and heterologous lytic promoters (e.g., the viralPANgene). Mutation of predicted phosphorylation targets at RTA serines 525 and 526 inhibited PKA-mediated enhancement of RTAtrans-activating capacity. Given the high catecholamine levels at sites of KSHV latency such as the vasculature and lymphoid organs, these data suggest that β-adrenergic control of RTA might constitute a significant physiological regulator of KSHV lytic replication. These findings also suggest novel therapeutic strategies for controlling the activity of this oncogenic gammaherpesvirus in vivo.