bluuebunny/crossref_metadata_2025_split huggingface dataset:
U
L
31 and U
L
34 Proteins of Herpes Simplex Virus Type 1 Form a Complex That Accumulates at the Nuclear Rim and Is Required for Envelopment of Nucleocapsids
Authors: Reynolds Ashley E. , Ryckman Brent J. , Baines Joel D. , Zhou Yuping , Liang Li , Roller Richard J. 
Published: July 2002

ABSTRACT

The herpes simplex virus type 1 (HSV-1) U
L
34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the U
L
31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with U
L
34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing U
L
31 protein fused to glutathione-
S
-transferase (U
L
31-GST) and U
L
34 protein fused to GST (U
L
34-GST) were demonstrated to specifically recognize the U
L
31 and U
L
34 proteins of approximately 34,000 and 30,000 Da, respectively. The U
L
31 and U
L
34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. U
L
34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of U
L
31 protein at the nuclear rim required the presence of U
L
34 protein, inasmuch as cells infected with a U
L
34 null mutant virus contained U
L
31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of U
L
34 protein exclusively at the nuclear rim required the presence of the U
L
31 gene product, inasmuch as U
L
34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a U
L
31 null virus. When transiently expressed in the absence of other viral factors, U
L
31 protein localized diffusely in the nucleoplasm, whereas U
L
34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the U
L
31 and U
L
34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus lacking the U
S
3-encoded protein kinase, previously shown to phosphorylate the U
L
34 gene product, U
L
31 and U
L
34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, U
S
3 kinase is required for even distribution of U
L
31 and U
L
34 proteins throughout the nuclear rim. Taken together with the similar phenotypes of the U
L
31 and U
L
34 deletion mutants, these data strongly suggest that the U
L
31 and U
L
34 proteins form a complex that accumulates at the nuclear membrane and plays an important role in nucleocapsid envelopment at the inner nuclear membrane.
Human Cytomegalovirus Protein US2 Interferes with the Expression of Human HFE, a Nonclassical Class I Major Histocompatibility Complex Molecule That Regulates Iron Homeostasis
Authors: Ben-Arieh Sayeh Vahdati , Zimerman Baruch , Smorodinsky Nechama I. , Yaacubovicz Margalit , Schechter Chana , Bacik Igor , Gibbs Jim , Bennink Jack R. , Yewdell Jon W. , Coligan John E. , Firat Hüseyin , Lemonnier François , Ehrlich Rachel 
Published: July 2002

ABSTRACTHFE is a nonclassical class I major histocompatibility complex (MHC) molecule that is mutated in the autosomal recessive iron overload disease hereditary hemochromatosis. There is evidence linking HFE with reduced iron uptake by the transferrin receptor (TfR). Using a panel of HFE and TfR monoclonal antibodies to examine human HFE (hHFE)-expressing cell lines, we demonstrate the expression of stable and fully glycosylated TfR-free and TfR-associated hHFE/β2m complexes. We show that both the stability and assembly of hHFE complexes can be modified by the human cytomegalovirus (HCMV) viral protein US2, known to interfere with the expression of classical class I MHC molecules. HCMV US2, but not US11, targets HFE molecules for degradation by the proteasome. Whether this interference with the regulation of iron metabolism by a viral protein is a means of potentiating viral replication remains to be determined. The reduced expression of classical class I MHC and HFE complexes provides the virus with an efficient tool for altering cellular metabolism and escaping certain immune responses.
Insertions in the gG Gene of Pseudorabies Virus Reduce Expression of the Upstream Us3 Protein and Inhibit Cell-to-Cell Spread of Virus Infection
Authors: Demmin Gretchen L. , Clase Amanda C. , Randall Jessica A. , Enquist L. W. , Banfield Bruce W. 
Published: July 2002

ABSTRACT

The alphaherpesvirus Us4 gene encodes glycoprotein G (gG), which is conserved in most viruses of the alphaherpesvirus subfamily. In the swine pathogen pseudorabies virus (PRV), mutant viruses with internal deletions and insertions in the gG gene have shown no discernible phenotypes. We report that insertions in the gG locus of the attenuated PRV strain Bartha show reduced virulence in vivo and are defective in their ability to spread from cell to cell in a cell-type-specific manner. Similar insertions in the gG locus of the wild-type PRV strain Becker had no effect on the ability of virus infection to spread between cells. Insertions in the gG locus of the virulent NIA-3 strain gave results similar to those found with the Bartha strain. To examine the role of gG in cell-to-cell spread, a nonsense mutation in the gG signal sequence was constructed and crossed into the Bartha strain. This mutant, PRV157, failed to express gG yet had cell-to-cell spread properties indistinguishable from those of the parental Bartha strain. These data indicated that, while insertions in the gG locus result in decreased cell-to-cell spread, the phenotype was not due to loss of gG expression as first predicted. Analysis of gene expression upstream and downstream of gG revealed that expression of the upstream Us3 protein is reduced by insertion of
lacZ
or
egfp
at the gG locus. By contrast, expression of the gene immediately downstream of gG, Us6, which encodes glycoprotein gD, was not affected by insertions in gG. These data indicate that DNA insertions in gG have polar effects and suggest that the serine/threonine kinase encoded by the Us3 gene, and not gG, functions in the spread of viral infection between cells.
Broadly Neutralizing Antibodies Targeted to the Membrane-Proximal External Region of Human Immunodeficiency Virus Type 1 Glycoprotein gp41
Authors: Zwick Michael B. , Labrijn Aran F. , Wang Meng , Spenlehauer Catherine , Saphire Erica Ollmann , Binley James M. , Moore John P. , Stiegler Gabriela , Katinger Hermann , Burton Dennis R. , Parren Paul W. H. I. 
Published: July 2002

ABSTRACT

The identification and epitope mapping of broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies (Abs) is important for vaccine design, but, despite much effort, very few such Abs have been forthcoming. Only one broadly neutralizing anti-gp41 monoclonal Ab (MAb), 2F5, has been described. Here we report on two MAbs that recognize a region immediately C-terminal of the 2F5 epitope. Both MAbs were generated from HIV-1-seropositive donors, one (Z13) from an antibody phage display library, and one (4E10) as a hybridoma. Both MAbs recognize a predominantly linear and relatively conserved epitope, compete with each other for binding to synthetic peptide derived from gp41, and bind to HIV-1
MN
virions. By flow cytometry, these MAbs appear to bind relatively weakly to infected cells and this binding is not perturbed by pretreatment of the infected cells with soluble CD4. Despite the apparent linear nature of the epitopes of Z13 and 4E10, denaturation of recombinant envelope protein reduces the binding of these MAbs, suggesting some conformational requirements for full epitope expression. Most significantly, Z13 and 4E10 are able to neutralize selected primary isolates from diverse subtypes of HIV-1 (e.g., subtypes B, C, and E). The results suggest that a rather extensive region of gp41 close to the transmembrane domain is accessible to neutralizing Abs and could form a useful target for vaccine design.
Origin-Independent Assembly of Kaposi's Sarcoma-Associated Herpesvirus DNA Replication Compartments in Transient Cotransfection Assays and Association with the ORF-K8 Protein and Cellular PML
Authors: Wu Frederick Y. , Ahn Jin-Hyun , Alcendor Donald J. , Jang Won-Jong , Xiao Jinsong , Hayward S. Diane , Hayward Gary S. 
Published: July 2002

ABSTRACT
Six predicted Kaposi's sarcoma virus herpesvirus (KSHV) proteins have homology with other well-characterized herpesvirus core DNA replication proteins and are expected to be essential for viral DNA synthesis. Intact Flag-tagged protein products from all six were produced from genomic expression vectors, although the ORF40/41 transcript encoding a primase-helicase component proved to be spliced with a 127-bp intron. The intracellular localization of these six KSHV replication proteins and the mechanism of their nuclear translocation were investigated. SSB (single-stranded DNA binding protein, ORF6) and PPF (polymerase processivity factor, ORF59) were found to be intrinsic nuclear proteins, whereas POL (polymerase, ORF9), which localized in the cytoplasm on its own, was translocated to the nucleus when cotransfected with PPF. PAF (primase-associated factor, ORF40/41), a component of the primase-helicase tripartite subcomplex together with PRI (primase, ORF56) and HEL (helicase, ORF44), required the presence of all five other replication proteins for efficient nuclear translocation. Surprisingly, even in the absence of a lytic cycle replication origin (ori-Lyt) and any known initiator or origin binding protein, the protein products of all six KSHV core replication genes cooperated in a transient cotransfection assay to form large globular shaped pseudo-replication compartments (pseudo-RC), which excluded cellular DNA. These pseudo-RC structures were confirmed to include POL, SSB, PRI, and PAF but did not contain any newly synthesized DNA. Similar to the human cytomegalovirus system, the peripheries of these KSHV pre-RC were also found to be surrounded by punctate PML oncogenic domains (PODs). Furthermore, by transient cotransfection, the six KSHV core replication machinery proteins successfully replicated a plasmid containing EBV ori-Lyt in the presence of the Epstein-Barr virus-encoded DNA binding initiator protein, ZTA. The KSHV-encoded K8 (ORF-K8) protein, which is a distant evolutionary homologue to ZTA, was incorporated into pseudo-RC structures formed by transient cotransfection with the six core KSHV replication genes. However, unlike ZTA, K8 displayed a punctate nuclear pattern both in transfected cells and at early stages of lytic infection and colocalized with the cellular PML proteins in PODs. Finally, K8 was also found to accumulate in functional viral RC, detected by incorporation of pulse-labeled bromodeoxyuridine into newly synthesized DNA in both tetradecanoyl phorbol acetate-induced JSC-1 primary effusion lymphoblasts and in KSHV lytically infected endothelial cells.
Adaptation of Influenza A Viruses to Cells Expressing Low Levels of Sialic Acid Leads to Loss of Neuraminidase Activity
Authors: Hughes Mark T. , McGregor Martha , Suzuki Takashi , Suzuki Yasuo , Kawaoka Yoshihiro 
Published: July 2002

ABSTRACT

Influenza A viruses possess two virion surface proteins, hemagglutinin (HA) and neuraminidase (NA). The HA binds to sialyloligosaccharide viral receptors, while the NA removes sialic acids from the host cell and viral sialyloligosaccarides. Alterations of the HA occur during adaptation of influenza viruses to new host species, as in the 1957 and 1968 influenza pandemics. To gain a better understanding of the contributions of the HA and possibly the NA to this process, we generated cell lines expressing reduced levels of the influenza virus receptor determinant, sialic acid, by selecting Madin-Darby canine kidney cells resistant to a lectin specific for sialic acid linked to galactose by α(2-3) or α(2-6) linkages. One of these cell lines had less than 1/10 as much
N
-acetylneuraminic acid as its parent cell line. When serially passaged in this cell line, human H3N2 viruses lost sialidase activity due to a large internal deletion in the NA gene, without alteration of the HA gene. These findings indicate that NA mutations can contribute to the adaptation of influenza A virus to new host environments and hence may play a role in the transmission of virus across species.
Biogenesis of the Semliki Forest Virus RNA Replication Complex
Authors: Kujala Pekka , Ikäheimonen Anne , Ehsani Neda , Vihinen Helena , Auvinen Petri , Kääriäinen Leevi 
Published: July 2002

ABSTRACT
The nonstructural (ns) proteins nsP1 to -4, the components of Semliki Forest virus (SFV) RNA polymerase, were localized in infected cells by confocal microscopy using double labeling with specific antisera against the individual ns proteins. All ns proteins were associated with large cytoplasmic vacuoles (CPV), the inner surfaces of which were covered by small invaginations, or spherules, typical of alphavirus infection. All ns proteins were localized by immuno-electron microscopy (EM) to the limiting membranes of CPV and to the spherules, together with newly labeled viral RNA. Along with earlier observations by EM-autoradiography (P. M. Grimley, I. K. Berezesky, and R. M. Friedman, J. Virol. 2:326–338, 1968), these results suggest that individual spherules represent template-associated RNA polymerase complexes. Immunoprecipitation of radiolabeled ns proteins showed that each antiserum precipitated the other three ns proteins, implying that they functioned as a complex. Double labeling with organelle-specific and anti-ns-protein antisera showed that CPV were derivatives of late endosomes and lysosomes. Indeed, CPV frequently contained endocytosed bovine serum albumin-coated gold particles, introduced into the medium at different times after infection. With time, increasing numbers of spherules were also observed on the cell surfaces; they were occasionally released into the medium, probably by secretory lysosomes. We suggest that the spherules arise by primary assembly of the RNA replication complexes at the plasma membrane, guided there by nsP1, which has affinity to lipids specific for the cytoplasmic leaflet of the plasma membrane. Endosomal recycling and fusion of CPV with the plasma membrane can circulate spherules between the plasma membrane and the endosomal-lysosomal compartment.
The SH Integral Membrane Protein of the Paramyxovirus Simian Virus 5 Is Required To Block Apoptosis in MDBK Cells
Authors: He Biao , Lin Grace Y. , Durbin Joan E. , Durbin Russell K. , Lamb Robert A. 
Published: July 2002

ABSTRACTIn some cell types the paramyxovirus simian virus 5 (SV5) causes little cytopathic effect (CPE) and infection continues productively for long periods of time; e.g., SV5 can be produced from MDBK cells for up to 40 days with little CPE. SV5 differs from most paramyxoviruses in that it encodes a small (44-amino-acid) hydrophobic integral membrane protein (SH). When MDBK cells were infected with a recombinant SV5 containing a deletion of the SH gene (rSV5ΔSH), the MDBK cells exhibited an increase in CPE compared to cells infected with wild-type SV5 (recovered from cDNA; rSV5). The increased CPE correlated with an increase in apoptosis in rSV5ΔSH-infected cells over mock-infected and rSV5-infected cells when assayed for annexin V binding, DNA content (propidium iodide staining), and DNA fragmentation (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay). In rSV5ΔSH-infected MDBK cells an increase in caspase-2 and caspase-3 activities was observed. By using peptide inhibitors of individual caspases it was found that caspase-2 and caspase-3 were activated separately in rSV5ΔSH-infected cells. Expression of caspase-2 and -3 in rSV5ΔSH-infected MDBK cells appeared not to require STAT1 protein, as STAT1 protein could not be detected in SV5-infected MDBK cells. When mutant mice homologous for a targeted disruption ofSTAT1were used as a model animal system and infected with the viruses it was found that rSV5ΔSH caused less mortality than wild-type rSV5, consistent with the notion of clearance of apoptotic cells in a host species.
Hepatitis B Virus HBx Protein Activation of Cyclin A–Cyclin-Dependent Kinase 2 Complexes and G
1
Transit via a Src Kinase Pathway
Authors: Bouchard Michael , Giannakopoulos Stavros , Wang Edith H. , Tanese Naoko , Schneider Robert J. 
Published: July 2002

ABSTRACT

Numerous studies have demonstrated that the hepatitis B virus HBx protein stimulates signal transduction pathways and may bind to certain transcription factors, particularly the cyclic AMP response element binding protein, CREB. HBx has also been shown to promote early cell cycle progression, possibly by functionally replacing the TATA-binding protein-associated factor 250 (TAF
II
250), a transcriptional coactivator, and/or by stimulating cytoplasmic signal transduction pathways. To understand the basis for early cell cycle progression mediated by HBx, we characterized the molecular mechanism by which HBx promotes deregulation of the G
0
and G
1
cell cycle checkpoints in growth-arrested cells. We demonstrate that TAF
II
250 is absolutely required for HBx activation of the cyclin A promoter and for promotion of early cell cycle transit from G
0
through G
1
. Thus, HBx does not functionally replace TAF
II
250 for transcriptional activity or for cell cycle progression, in contrast to a previous report. Instead, HBx is shown to activate the cyclin A promoter, induce cyclin A–cyclin-dependent kinase 2 complexes, and promote cycling of growth-arrested cells into G
1
through a pathway involving activation of Src tyrosine kinases. HBx stimulation of Src kinases and cyclin gene expression was found to force growth-arrested cells to transit through G
1
but to stall at the junction with S phase, which may be important for viral replication.
Herpes Simplex Virus IE63 (ICP27) Protein Interacts with Spliceosome-Associated Protein 145 and Inhibits Splicing prior to the First Catalytic Step
Authors: Bryant H. E. , Wadd S. E. , Lamond A. I. , Silverstein S. J. , Clements J. B. 
Published: July 2002

ABSTRACT
The multifunctional herpes simplex virus type 1 (HSV-1) protein IE63 (ICP27) interacts with the essential pre-mRNA splicing factor, spliceosome-associated protein 145 (SAP145), and in infected cells IE63 and SAP145 colocalize. This interaction was reduced or abrogated completely using extracts from cells infected with IE63 viral mutants, with mutations in IE63 KH and Sm homology domains, which do not exhibit host shutoff or inhibit splicing. In the presence of IE63, splicing in vitro was inhibited prior to the first catalytic step and the B/C complex formed during splicing was shifted up in mobility and reduced in intensity. With the use of splicing extracts, IE63 and SAP145 both comigrated with the B/C complex, suggesting that they interact within this complex to inhibit B/C complex formation or conversion. The inhibition of splicing may facilitate the export of viral or cellular transcripts, possibly via other protein partners of IE63. These data provide important new insights into how IE63 influences pre-mRNA processing during HSV-1 infection.
A Small Molecule Inhibits and Misdirects Assembly of Hepatitis B Virus Capsids
Authors: Zlotnick Adam , Ceres Pablo , Singh Sushmita , Johnson Jennifer M. 
Published: July 2002

ABSTRACT
Hepatitis B virus (HBV) capsids play an important role in viral nucleic acid metabolism and other elements of the virus life cycle. Misdirection of capsid assembly (leading to formation of aberrant particles) may be a powerful approach to interfere with virus production. HBV capsids can be assembled in vitro from the dimeric capsid protein. We show that a small molecule, bis-ANS, binds to capsid protein, inhibiting assembly of normal capsids and promoting assembly of noncapsid polymers. Using equilibrium dialysis to investigate binding of bis-ANS to free capsid protein, we found that only one bis-ANS molecule binds per capsid protein dimer, with an association energy of −28.0 ± 2.0 kJ/mol (−6.7 ± 0.5 kcal/mol). Bis-ANS inhibited in vitro capsid assembly induced by ionic strength as observed by light scattering and size exclusion chromatography. The binding energy of bis-ANS for capsid protein calculated from assembly inhibition data was −24.5 ± 0.9 kJ/mol (−5.9 ± 0.2 kcal/mol), essentially the same binding energy observed in studies of unassembled protein. These data indicate that capsid protein bound to bis-ANS did not participate in assembly; this mechanism of assembly inhibition is analogous to competitive or noncompetitive inhibition of enzymes. While assembly of normal capsids is inhibited, our data suggest that bis-ANS leads to formation of noncapsid polymers. Evidence of aberrant polymers was identified by light scattering and electron microscopy. We propose that bis-ANS acts as a molecular “wedge” that interferes with normal capsid protein geometry and capsid formation; such wedges may represent a new class of antiviral agent.
Fatty Acid Modification of the Coxsackievirus and Adenovirus Receptor
Authors: van't Hof Wouter , Crystal Ronald G. 
Published: July 2002

ABSTRACT
Membrane-proximal cysteines 259 and 260 in the cytoplasmic tail of the coxsackievirus and adenovirus receptor (CAR) are known to be essential for the tumor suppression activity of CAR. We demonstrate that these residues provide an S-acylation motif for modification of CAR with the fatty acid palmitate. Substitution of alanine for cysteines 259 and 260 results in the additional localization of CAR in perinuclear compartments with no effect on the efficiency of adenovirus infection. The results indicate that palmitylation is important for stable plasma membrane expression and biological activity of CAR but is not critical for adenovirus receptor performance.