bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assembly and Maturation of the Flavivirus Kunjin Virus Appear To Occur in the Rough Endoplasmic Reticulum and along the Secretory Pathway, Respectively
Authors: Mackenzie Jason M. , Westaway Edwin G. 
Published: July 2002

ABSTRACT

The intracellular assembly site for flaviviruses in currently not known but is presumed to be located within the lumen of the rough endoplasmic reticulum (RER). Building on previous studies involving immunofluorescence (IF) and cryoimmunoelectron microscopy of Kunjin virus (KUN)-infected cells, we sought to identify the steps involved in the assembly and maturation of KUN. Thus, using antibodies directed against envelope protein E in IF analysis, we found the accumulation of E within regions coincident with the RER and endosomal compartments. Immunogold labeling of cryosections of infected cells indicated that E and minor envelope protein prM were localized to reticulum membranes continuous with KUN-induced convoluted membranes (CM) or paracrystalline arrays (PC) and that sometimes the RER contained immunogold-labeled virus particles. Both proteins were also observed to be labeled in membranes at the periphery of the induced CM or PC structures, but the latter were very seldom labeled internally. Utilizing drugs that inhibit protein and/or membrane traffic throughout the cell, we found that the secretion of KUN particles late in infection was significantly affected in the presence of brefeldin A and that the infectivity of secreted particles was severely affected in the presence of monensin and
N
-nonyl-deoxynojirimycin. Nocodazole did not appear to affect maturation, suggesting that microtubules play no role in assembly or maturation processes. Subsequently, we showed that the exit of intact virions from the RER involves the transport of individual virions within individual vesicles en route to the Golgi apparatus. The results suggest that the assembly of virions occurs within the lumen of the RER and that subsequent maturation occurs via the secretory pathway.
Secondary Structural Elements within the 3′ Untranslated Region of Mouse Hepatitis Virus Strain JHM Genomic RNA
Authors: Liu Qi , Johnson Reed F. , Leibowitz Julian L. 
Published: July 2002

ABSTRACT
Previously, we characterized two host protein binding elements located within the 3′-terminal 166 nucleotides of the mouse hepatitis virus (MHV) genome and assessed their functions in defective-interfering (DI) RNA replication. To determine the role of RNA secondary structures within these two host protein binding elements in viral replication, we explored the secondary structure of the 3′-terminal 166 nucleotides of the MHV strain JHM genome using limited RNase digestion assays. Our data indicate that multiple stem-loop and hairpin-loop structures exist within this region. Mutant and wild-type DIssEs were employed to test the function of secondary structure elements in DI RNA replication. Three stem structures were chosen as targets for the introduction of transversion mutations designed to destroy base pairing structures. Mutations predicted to destroy the base pairing of nucleotides 142 to 136 with nucleotides 68 to 74 exhibited a deleterious effect on DIssE replication. Destruction of base pairing between positions 96 to 99 and 116 to 113 also decreased DI RNA replication. Mutations interfering with the pairing of nucleotides 67 to 63 with nucleotides 52 to 56 had only minor effects on DIssE replication. The introduction of second complementary mutations which restored the predicted base pairing of positions 142 to 136 with 68 to 74 and nucleotides 96 to 99 with 116 to 113 largely ameliorated defects in replication ability, restoring DI RNA replication to levels comparable to that of wild-type DIssE RNA, suggesting that these secondary structures are important for efficient MHV replication. We also identified a conserved 23-nucleotide stem-loop structure involving nucleotides 142 to 132 and nucleotides 68 to 79. The upstream side of this conserved stem-loop is contained within a host protein binding element (nucleotides 166 to 129).
CREB-Binding Protein and Histone Deacetylase Regulate the Transcriptional Activity of Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 50
Authors: Gwack Yousang , Byun Hyewon , Hwang Seungmin , Lim Chunghun , Choe Joonho 
Published: July 2002

ABSTRACT
Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) open reading frame 50 (ORF50) encodes a viral transcriptional activator, which binds to the KSHV promoter and stimulates the transcription of viral early and late genes, thus activating the lytic cycle of KSHV. We report here that KSHV ORF50 binds to the cellular proteins CREB-binding protein (CBP) and histone deacetylase (HDAC) and these binding events modulate ORF50-activated viral transcription. Binding of ORF50 to CBP and HDAC activates and represses, respectively, ORF50-mediated viral transcription. KSHV ORF50 was shown to bind to the C/H3 domain and the C-terminal transcriptional activation domain of CBP, while CBP bound to the amino-terminal basic domain and the carboxyl-terminal transactivation domain of ORF50. The LXXLL motif within the transcriptional activation domain of ORF50 is reminiscent of the CBP-binding sequence found in nuclear receptor proteins. The adenovirus E1A protein, which also binds to the C/H3 domain of CBP, repressed the transcriptional activation activity of ORF50. The cellular protein c-Jun, which binds to the kinase-induced activation domain of ORF50, stimulated ORF50-mediated viral transcription. The HDAC1-interacting domain of ORF50 was shown to be a central proline-rich sequence. Our data provide a framework for delineating the regulatory mechanisms used by KSHV to modulate its transcription and replication through interaction with both histone acetyltransferases and HDACs.
Entry of Human Parechovirus 1
Authors: Joki-Korpela Päivi , Marjomäki Varpu , Krogerus Camilla , Heino Jyrki , Hyypiä Timo 
Published: July 2002

ABSTRACT

Human parechovirus 1 (HPEV-1) is a prototype member of parechoviruses, a recently established picornavirus genus. Although there is preliminary evidence that HPEV-1 recognizes α
V
integrins as cellular receptors, our understanding of early events during HPEV-1 infection is still very limited. The aim of this study was to clarify the entry mechanisms of HPEV-1, including the attachment of the virus onto the host cell surface and subsequent internalization. In blocking experiments with monoclonal antibodies against different receptor candidates, antibodies against α
V
and β
3
integrin subunits, in particular in combination, appeared to be the most efficient ones in preventing the HPEV-1 infection. To find out whether HPEV-1 uses clathrin-coated vesicles or other routes for the entry into the host cell, we carried out double-labeling experiments of virus-infected cells with anti-HPEV-1 antibodies and antibodies against known markers of the clathrin and the caveolin routes. At the early phase of infection (5 min postinfection [p.i.]) HPEV-1 colocalized with EEA1 (early endosomes), and later, after 30 min p.i., it colocalized with mannose-6-phosphate receptor (late endosomes), whereas no colocalization with caveolin-1 was observed. The data indicate that HPEV-1 utilizes the clathrin-dependent endocytic pathway for entry into the host cells. Interestingly, endocytosed HPEV-1 capsid proteins were observed in the endoplasmic reticulum and
cis
-Golgi network 30 to 60 min p.i. Depolymerization of microtubules with nocodazole inhibited translocation of the virus to the late endosomes but did not block HPEV-1 replication, suggesting that the RNA genome may be released early during the entry process.
Self-Assembly of Nucleocapsid-Like Particles from Recombinant Hepatitis C Virus Core Protein
Authors: Kunkel Meghan , Lorinczi Marta , Rijnbrand René , Lemon Stanley M. , Watowich Stanley J. 
Published: July 2002

ABSTRACT
Little is known about the assembly pathway and structure of hepatitis C virus (HCV) since insufficient quantities of purified virus are available for detailed biophysical and structural studies. Here, we show that bacterially expressed HCV core proteins can efficiently self-assemble in vitro into nucleocapsid-like particles. These particles have a regular, spherical morphology with a modal distribution of diameters of approximately 60 nm. Self-assembly of nucleocapsid-like particles requires structured RNA molecules. The 124 N-terminal residues of the core protein are sufficient for self-assembly into nucleocapsid-like particles. Inclusion of the carboxy-terminal domain of the core protein modifies the core assembly pathway such that the resultant particles have an irregular outline. However, these particles are similar in size and shape to those assembled from the 124 N-terminal residues of the core protein. These results provide novel opportunities to delineate protein-protein and protein-RNA interactions critical for HCV assembly, to study the molecular details of HCV assembly, and for performing high-throughput screening of assembly inhibitors.
Capsid Structure of Kaposi's Sarcoma-Associated Herpesvirus, a Gammaherpesvirus, Compared to Those of an Alphaherpesvirus, Herpes Simplex Virus Type 1, and a Betaherpesvirus, Cytomegalovirus
Authors: Trus Benes L. , Heymann J. Bernard , Nealon Karin , Cheng Naiqian , Newcomb William W. , Brown Jay C. , Kedes Dean H. , Steven Alasdair C. 
Published: July 2002

ABSTRACT

The capsid of Kaposi's sarcoma-associated herpesvirus (KSHV) was visualized at 24-Å resolution by cryoelectron microscopy. Despite limited sequence similarity between corresponding capsid proteins, KSHV has the same T=16 triangulation number and much the same capsid architecture as herpes simplex virus (HSV) and cytomegalovirus (CMV). Its capsomers are hexamers and pentamers of the major capsid protein, forming a shell with a flat, close-packed, inner surface (the “floor”) and chimney-like external protrusions. Overlying the floor at trigonal positions are (αβ
2
) heterotrimers called triplexes. The floor structure is well conserved over all three viruses, and the most variable capsid features reside on the outer surface, i.e., in the shapes of the protrusions and triplexes, in which KSHV resembles CMV and differs from HSV. Major capsid protein sequences from the three subfamilies have some similarity, which is closer between KSHV and CMV than between either virus and HSV. The triplex proteins are less highly conserved, but sequence analysis identifies relatively conserved tracts. In alphaherpesviruses, the α-subunit (VP19c in HSV) has a 100-residue N-terminal extension and an insertion near the C terminus. The small basic capsid protein sequences are highly divergent: whereas the HSV and CMV proteins bind only to hexons, difference mapping suggests that the KSHV protein, ORF65, binds around the tips of both hexons and pentons.
Virus-Specific and Bystander CD8+T-Cell Proliferation in the Acute and Persistent Phases of a Gammaherpesvirus Infection
Authors: Belz Gabrielle T. , Doherty Peter C. 
Published: July 2002

ABSTRACTThe cycling characteristics of CD8+T cells specific for two lytic-phase epitopes of murine gammaherpesvirus 68 (γHV68) have been analyzed for mice with high or low levels of virus persistence. The extent of cell division is generally reflective of the antigen load and suggests that γHV68 may be regularly reactivating from latency for some months after the resolution of the acute phase of the infectious process. Although γHV68 infection is also associated with massive proliferation of lymphocytes that are not obviously specific for the virus, the level of “bystander-induced” cycling in a population of influenza virus-specific CD8+T cells was generally fourfold lower than the extent of cell division seen for the antigen-driven, γHV68-specific response. The overall conclusion is that turnover rates substantially in excess of 5 to 10% over 6 days for CD8+“memory” T-cell populations are likely to be reflective of continued antigenic exposure.
Neither
LAT
nor Open Reading Frame
P
Mutations Increase Expression of Spliced or Intron-Containing
ICP0
Transcripts in Mouse Ganglia Latently Infected with Herpes Simplex Virus
Authors: Chen Shun-Hua , Lee Lily Yeh , Garber David A. , Schaffer Priscilla A. , Knipe David M. , Coen Donald M. 
Published: July 2002

ABSTRACT

Latent infections by herpes simplex virus are characterized by repression of productive-cycle gene expression. Several hypotheses to explain this repression involve inhibition of expression of the immediate-early gene activator ICP0 during latency. To address these hypotheses, we developed quantitative reverse transcriptase-PCR assays that detected spliced and intron-containing
ICP0
transcripts in mouse ganglia latently infected with wild-type virus. In these ganglia, the numbers of spliced
ICP0
transcripts correlated better with the numbers of transcripts from the immediate-early gene encoding ICP4 than with those from the early gene encoding thymidine kinase. There were fewer spliced than intron-containing
ICP0
transcripts on average, with considerable ganglion-to-ganglion variation. We then investigated whether
ICP0
expression in latently infected ganglia is reduced by the latency-associated transcripts (LATs) and whether splicing of
ICP0
transcripts is inhibited by the product of open reading frame (ORF) P. A
LAT
deletion mutation which essentially eliminates expression of the major LATs did not appreciably increase levels of
ICP0
transcripts.
LAT
deletion mutants did, however, appear to express reduced levels of intron-containing
ICP0
transcripts.
ORF P
mutations did not alter levels of
ICP0
transcripts in a manner consistent with inhibition of
ICP0
splicing by ORF P. Although these results argue against antisense inhibition of
ICP0
expression by LATs or inhibition of
ICP0
splicing by ORF P, they are consistent with the possibilities of a block between immediate-early and early gene expression and regulation of spliced versus intron-containing
ICP0
transcripts in latently infected ganglia.
Eukaryotic Elongation Factor 1A Interacts with the Upstream Pseudoknot Domain in the 3′ Untranslated Region of Tobacco Mosaic Virus RNA
Authors: Zeenko Vladimir V. , Ryabova Lyubov A. , Spirin Alexander S. , Rothnie Helen M. , Hess Daniel , Browning Karen S. , Hohn Thomas 
Published: July 2002

ABSTRACT
The genomic RNA of tobacco mosaic virus (TMV), like that of other positive-strand RNA viruses, acts as a template for both translation and replication. The highly structured 3′ untranslated region (UTR) of TMV RNAs plays an important role in both processes; it is not polyadenylated but ends with a tRNA-like structure (TLS) preceded by a conserved upstream pseudoknot domain (UPD). The TLS of tobamoviral RNAs can be specifically aminoacylated and, in this state, can interact with eukaryotic elongation factor 1A (eEF1A)/GTP with high affinity. Using a UV cross-linking assay, we detected another specific binding site for eEF1A/GTP, within the UPDs of TMV and crucifer-infecting tobamovirus (crTMV), that does not require aminoacylation. A mutational analysis revealed that UPD pseudoknot conformation and some conserved primary sequence elements are required for this interaction. Its possible role in the regulation of tobamovirus gene expression and replication is discussed.
Chemokine Expression in the Central Nervous System of Mice with a Viral Disease Resembling Multiple Sclerosis: Roles of CD4+and CD8+T Cells and Viral Persistence
Authors: Ransohoff R. M. , Wei T. , Pavelko K. D. , Lee J.-C. , Murray P. D. , Rodriguez M. 
Published: July 2002

ABSTRACTDuring the first 45 days after intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV), the levels of mRNAs encoding chemokines MCP-1/CCL2, RANTES/CCL5, and IP-10/CXCL10 in the central nervous system (CNS) are closely related to the sites of virus gene expression and tissue inflammation. In the present study, these chemokines were monitored during the latter 135 days of a 6-month course of TMEV-induced disease in susceptible (PLJ) or resistant (C57BL/6) mice that possessed or lacked either CD4+or CD8+T cells. These data were additionally correlated to mouse genotype, virus persistence in the CNS, antiviral antibody titers, mortality, and the severity of neurological disease. Surprisingly, the major determinant of chemokine expression was virus persistence: the factors of susceptible or resistant genotype, severity of neuropathology, and presence or absence of regulatory T cells exerted minimal effects. Our observations indicated that chemokine expression in the CNS in this chronic viral disorder was intrinsic to the CNS innate immune response to infection and was not governed by elements of the adaptive immune system.
Mechanism of Human Immunodeficiency Virus-Induced Complement Expression in Astrocytes and Neurons
Authors: Speth Cornelia , Schabetsberger Thomas , Mohsenipour Iradji , Stöckl Gabriele , Würzner Reinhard , Stoiber Heribert , Lass-Flörl Cornelia , Dierich Manfred P. 
Published: July 2002

ABSTRACTThe cerebral complement system is hypothesized to contribute to neurodegeneration in the pathogenesis of AIDS-associated neurological disorders. Our former results have shown that the human immunodeficiency virus (HIV) strongly induces the synthesis of complement factor C3 in astrocytes. This upregulation explains in vivo data showing elevated complement levels in the cerebrospinal fluid of patients with AIDS-associated neurological symptoms. Since inhibition of complement synthesis and activation in the brain may represent a putative therapeutic goal to prevent virus-induced damage, we analyzed in detail the mechanisms of HIV-induced modulation of C3 expression. HIV-1 increased the C3 levels in astrocyte culture supernatants from 30 to up to 400 ng/ml; signal transduction studies revealed that adenylate cyclase activation with upregulation of cyclic AMP is the central signaling pathway to mediate that increase. Furthermore, activity of protein kinase C is necessary for HIV induction of C3, since inhibition of protein kinase C by prolonged exposure to the phorbol ester tetradecanoyl phorbol acetate partly abolished the HIV effect. The cytokines tumor necrosis factor alpha and gamma interferon were not involved in mediating the HIV-induced C3 upregulation, since neutralizing antibodies had no effect. Besides whole HIV virions, the purified viral proteins Nef and gp41 are biologically active in upregulating C3, whereas Tat, gp120, and gp160 were not able to modulate C3 synthesis. Further experiments revealed that neurons were also able to respond on incubation with HIV with increased C3 synthesis, although the precise pattern was slightly different from that in astrocytes. This strengthens the hypothesis that HIV-induced complement synthesis represents an important mechanism for the pathogenesis of AIDS in the brain.
RNA Replication of Mouse Hepatitis Virus Takes Place at Double-Membrane Vesicles
Authors: Gosert Rainer , Kanjanahaluethai Amornrat , Egger Denise , Bienz Kurt , Baker Susan C. 
Published: July 2002

ABSTRACT
The replication complexes (RCs) of positive-stranded RNA viruses are intimately associated with cellular membranes. To investigate membrane alterations and to characterize the RC of mouse hepatitis virus (MHV), we performed biochemical and ultrastructural studies using MHV-infected cells. Biochemical fractionation showed that all 10 of the MHV gene 1 polyprotein products examined pelleted with the membrane fraction, consistent with membrane association of the RC. Furthermore, MHV gene 1 products p290, p210, and p150 and the p150 cleavage product membrane protein 1 (MP1, also called p44) were resistant to extraction with Triton X-114, indicating that they are integral membrane proteins. The ultrastructural analysis revealed double-membrane vesicles (DMVs) in the cytoplasm of MHV-infected cells. The DMVs were found either as separate entities or as small clusters of vesicles. To determine whether MHV proteins and viral RNA were associated with the DMVs, we performed immunocytochemistry electron microscopy (IEM). We found that the DMVs were labeled using an antiserum directed against proteins derived from open reading frame 1a of MHV. By electron microscopy in situ hybridization (ISH) using MHV-specific RNA probes, DMVs were highly labeled for both gene 1 and gene 7 sequences. By combined ISH and IEM, positive-stranded RNA and viral proteins localized to the same DMVs. Finally, viral RNA synthesis was detected by labeling with 5-bromouridine 5′-triphosphate. Newly synthesized viral RNA was found to be associated with the DMVs. We conclude from these data that the DMVs carry the MHV RNA replication complex and are the site of MHV RNA synthesis.
Minute Virus of Mice NS1 Interacts with the SMN Protein, and They Colocalize in Novel Nuclear Bodies Induced by Parvovirus Infection
Authors: Young Philip J. , Jensen Klaus T. , Burger Lisa R. , Pintel David J. , Lorson Christian L. 
Published: July 2002

ABSTRACT

The human
survival motor neuron
(
SMN
) gene is the spinal muscular atrophy-determining gene, and a knockout of the murine
Smn
gene results in preembryonic lethality. Here we show that SMN can directly interact in vitro and in vivo with the large nonstructural protein NS1 of the autonomous parvovirus minute virus of mice (MVM), a protein essential for viral replication and a potent transcriptional activator. Typically, SMN localizes within nuclear Cajal bodies and diffusely in the cytoplasm. Following transient NS1expression, SMN and NS1 colocalize within Cajal bodies. At early time points following parvovirus infection, NS1 fails to colocalize with SMN within Cajal bodies; however, during the course of MVM infection, dramatic nuclear alterations occur. Formerly distinct nuclear bodies such as Cajal bodies, promyelocytic leukemia gene product (PML) oncogenic domains (PODs), speckles, and autonomous parvovirus-associated replication (APAR) bodies are seen aggregating at later points in infection. These newly formed large nuclear bodies (termed SMN-associated APAR bodies) are active sites of viral replication and viral capsid assembly. These results highlight the transient nature of nuclear bodies and their contents and identify a novel nuclear body formed during infection. Furthermore, simple transient expression of the viral nonstructural proteins is insufficient to induce this nuclear reorganization, suggesting that this event is induced specifically by a step in the viral infection process.