bluuebunny/crossref_metadata_2025_split huggingface dataset:
Physical and Functional Interactions between Vaccinia Virus F10 Protein Kinase and Virion Assembly Proteins A30 and G7
Authors: Szajner Patricia , Weisberg Andrea S. , Moss Bernard 
Published: December 2003

ABSTRACT
An early step in vaccinia virus morphogenesis, the association of crescent membranes with electron-dense granular material, is perturbed when expression of the viral protein encoded by the A30L or G7L open reading frame is repressed. Under these conditions, we found that phosphorylation of the A17 membrane protein, which is mediated by the F10 kinase, was severely reduced. Furthermore, A30 and G7 stimulated F10-dependent phosphorylation of A17 in the absence of other viral late proteins. Evidence for physical interactions between A30, G7, and F10 was obtained by their coimmunoprecipitation with antibody against A30 or F10. In addition, phosphorylation of A30 was dependent on the F10 kinase and autophosphorylation of F10 was stimulated by A30 and G7. Nevertheless, the association of A30, G7, and F10 occurred even with mutated, catalytically inactive forms of F10. Just as A30 and G7 are mutually dependent on each other for stability, F10 was nearly undetectable in the absence of A30 and G7. The reverse is not true, however, as repression of F10 did not diminish A30 or G7. Interaction of F10 with A30 and G7 presumably occurred within the virus factory areas of the cytoplasm, where each was concentrated. F10 localized predominantly in the cortical region of immature virions, beneath the membrane where A17 is located. F10 remained associated with the particulate core fraction of mature virions after treatment with a nonionic detergent and reducing agent. The formation of protein complexes such as the one involving A30, G7, and F10 may be a mechanism for the regulated packaging and processing of virion components.
The Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen 1 N Terminus Is Essential for Chromosome Association, DNA Replication, and Episome Persistence
Authors: Barbera Andrew J. , Ballestas Mary E. , Kaye Kenneth M. 
Published: December 2003

ABSTRACT

To persist in latently infected, proliferating cells, Kaposi's sarcoma-associated herpesvirus (KSHV) episomes must replicate and efficiently segregate to progeny nuclei. Episome persistence in uninfected cells requires latency-associated nuclear antigen 1 (LANA1) in
trans
and
cis
-acting KSHV terminal repeat (TR) DNA. The LANA1 C terminus binds TR DNA, and LANA1 mediates TR-associated DNA replication in transient assays. LANA1 also concentrates at sites of KSHV TR DNA episomes along mitotic chromosomes, consistent with a tethering role to efficiently segregate episomes to progeny nuclei. LANA1 amino acids 5 to 22 constitute a chromosome association region (Piolot et al., J. Virol.
75:
3948-3959, 2001). We now investigate LANA1 residues 5 to 22 with scanning alanine substitutions. Mutations targeting LANA1
5
GMR
7
,
8
LRS
10
, and
11
GRS
13
eliminated chromosome association, DNA replication, and episome persistence. LANA1 mutated at
14
TG
15
retained the ability to associate with chromosomes but was partially deficient in DNA replication and episome persistence. These results provide genetic support for a key role of the LANA1 N terminus in chromosome association, LANA1-mediated DNA replication, and episome persistence.
The ORF2 Protein of Hepatitis E Virus Binds the 5′ Region of Viral RNA
Authors: Surjit Milan , Jameel Shahid , Lal Sunil K. 
Published: December 2003

ABSTRACT

Hepatitis E virus (HEV) is a major human pathogen in much of the developing world. It is a plus-strand RNA virus with a 7.2-kb polyadenylated genome consisting of three open reading frames,
ORF1
,
ORF2
, and
ORF3
. Of these,
ORF2
encodes the major capsid protein of the virus and
ORF3
encodes a small protein of unknown function. Using the yeast three-hybrid system and traditional biochemical techniques, we have studied the RNA binding activities of ORF2 and ORF3, two proteins encoded in the 3′ structural part of the genome. Since the genomic RNA from HEV has been postulated to contain secondary structures at the 5′ and 3′ ends, we used these two terminal regions, besides other regions within the genome, in this study. Experiments were designed to test for interactions between the genomic RNA fusion constructs with ORF2 and ORF3 hybrid proteins in a yeast cellular environment. We show here that the ORF2 protein contains RNA binding activity. The ORF2 protein specifically bound the 5′ end of the HEV genome. Deletion analysis of this protein showed that its RNA binding activity was lost when deletions were made beyond the N-terminal 111 amino acids. Finer mapping of the interacting RNA revealed that a 76-nucleotide (nt) region at the 5′ end of the HEV genome was responsible for binding the ORF2 protein. This 76-nt region included the 51-nt HEV sequence, conserved across alphaviruses. Our results support the requirement of this conserved sequence for interaction with ORF2 and also indicate an increase in the strength of the RNA-protein interaction when an additional 44 bases downstream of this 76-nt region were included. Secondary-structure predictions and the location of the ORF2 binding region within the HEV genome indicate that this interaction may play a role in viral encapsidation.
Characterization of the Interaction between P143 and LEF-3 from Two Different Baculovirus Species:
Choristoneura fumiferana
Nucleopolyhedrovirus LEF-3 Can Complement
Autographa californica
Nucleopolyhedrovirus LEF-3 in Supporting DNA Replication
Authors: Chen Tricia , Sahri Daniela , Carstens Eric B. 
Published: December 2003

ABSTRACT

The baculovirus protein P143 is essential for viral DNA replication in vivo, likely as a DNA helicase. We have demonstrated that another viral protein, LEF-3, first described as a single-stranded DNA binding protein, is required for transporting P143 into the nuclei of insect cells. Both of these proteins, along with several other early viral proteins, are also essential for DNA replication in transient assays. We now describe the identification, nucleotide sequences, and transcription patterns of the
Choristoneura fumiferana
nucleopolyhedrovirus (Cf
M
NPV) homologues of
p143
and
lef-3
and demonstrate that Cf
M
NPV LEF-3 is also responsible for P143 localization to the nucleus. We predicted that the interaction between P143 and LEF-3 might be critical for cross-species complementation of DNA replication. Support for this hypothesis was generated by substitution of heterologous P143 and LEF-3 between two different baculovirus species,
Autographa californica
nucleopolyhedrovirus and Cf
M
NPV, in transient DNA replication assays. The results suggest that the P143-LEF-3 complex is an important baculovirus replication factor.
Functional Analysis of Epstein-Barr Virus SM Protein: Identification of Amino Acids Essential for Structure, Transactivation, Splicing Inhibition, and Virion Production
Authors: Ruvolo Vivian , Sun Liang , Howard Karilynn , Sung Seung , Delecluse Henri-Jacques , Hammerschmidt Wolfgang , Swaminathan Sankar 
Published: December 2003

ABSTRACT
The Epstein-Barr virus (EBV) SM protein is a posttranscriptional regulator of cellular and viral gene expression that binds and stabilizes target mRNAs and shuttles from nucleus to cytoplasm. SM enhances expression of several EBV genes required for lytic replication and is essential for virion production. SM increases accumulation of specific mRNAs but also inhibits expression of several intron-containing transcripts. The mechanism by which SM inhibits gene expression is poorly understood. The experiments described here had several aims: to determine whether specific domains of SM were responsible for activation or inhibition function; whether these functions could be separated; and whether one or more of these functions were essential for virion production. A mutational analysis of SM was performed, focusing on amino acids in SM that are evolutionarily conserved among SM homologs in other herpesviruses. Mutation of the carboxy-terminal region of SM revealed a region that is likely to be structurally important for SM protein conformation. In addition, several amino acids were identified that are critical for activation and inhibition function. A specific mutation of a highly conserved cysteine residue revealed that it was essential for gene inhibition but not for transactivation, indicating that these two functions operate through independent mechanisms. Furthermore, the ability of wild-type SM and the inability of the mutant to inhibit gene expression were shown to correlate with the ability to inhibit splicing of a human target gene and thereby prevent accumulation of its processed mRNA. Surprisingly, some mutations which preserved both activation and inhibition functions in vitro nevertheless abolished virion production, suggesting that other SM functions or protein-protein interactions are also required for lytic replication.
Spatial and Temporal Organization of Adeno-Associated Virus DNA Replication in Live Cells
Authors: Fraefel Cornel , Bittermann Anne Greet , Büeler Hansruedi , Heid Irma , Bächi Thomas , Ackermann Mathias 
Published: December 2003

ABSTRACT

Upon cell entry, the genomes of herpes simplex virus type 1 (HSV-1) and adenovirus (Ad) associate with distinct nuclear structures termed ND10 or promyelocytic leukemia (PML) nuclear bodies (NBs). PML NB morphology is altered or disrupted by specific viral proteins as replication proceeds. We examined whether adeno-associated virus (AAV) replication compartments also associate with PML NBs, and whether modification or disruption of these by HSV-1 or Ad, both of which are helper viruses for AAV, is necessary at all. Furthermore, to add a fourth dimension to our present view of AAV replication, we established an assay that allows visualization of AAV replication in live cells. A recombinant AAV containing 40
lac
repressor binding sites between the AAV inverted terminal repeats was constructed. AAV Rep protein and helper virus-mediated replication of this recombinant AAV genome was visualized by binding of enhanced yellow fluorescent protein-
lac
repressor fusion protein to double-stranded AAV replication intermediates. We demonstrate in live cells that AAV DNA replication occurs in compartments which colocalize with AAV Rep. Early after infection, the replication compartments were small and varied in numbers from 2 to more than 40 per cell nucleus. Within 4 to 8 h, individual small replication compartments expanded and fused to larger structures which filled out much of the cell nucleus. We also show that AAV replication compartments can associate with modified PML NBs in Ad-infected cells. In wild-type HSV-1-infected cells, AAV replication compartments and PML NBs did not coexist, presumably because PML was completely disrupted by the HSV-1 ICP0 protein. However, alteration or disruption of PML appears not to be a prerequisite for AAV replication, as the formation of replication compartments was normal when the ICP0 mutants HSV-1 dl1403 and HSV-1 FXE, which do not affect PML NBs, were used as the helper viruses; under these conditions, AAV replication compartments did not associate with PML NBs.
Complete, Annotated Sequence of the Pseudorabies Virus Genome
Authors: Klupp Barbara G. , Hengartner Christoph J. , Mettenleiter Thomas C. , Enquist Lynn W. 
Published: December 2003

ABSTRACT
We have obtained the complete DNA sequence of pseudorabies virus (PRV), an alphaherpesvirus also known as Aujeszky's disease virus or suid herpesvirus 1, using sequence fragments derived from six different strains (Kaplan, Becker, Rice, Indiana-Funkhauser, NIA-3, and TNL). The assembled PRV genome sequence comprises 143,461 nucleotides. As expected, it matches the predicted gene arrangement, genome size, and restriction enzyme digest patterns. More than 70 open reading frames were identified with homologs in related alphaherpesviruses; none were unique to PRV. RNA polymerase II transcriptional control elements in the PRV genome, including core promoters, splice sites, and polyadenylation sites, were identified with computer prediction programs. The correlation between predicted and experimentally determined transcription start and stop sites was excellent. The transcriptional control architecture is characterized by three key features: core transcription elements shared between genes, yielding divergent transcripts and a large number of coterminal transcripts; bifunctional transcriptional elements, yielding head-to-tail transcripts; and short repetitive sequences that could function as insulators against improperly terminated transcripts. Many of these features are conserved in the alphaherpesvirus subfamily and have important implications for gene array analyses.
The Adenovirus E3-6.7K Protein Adopts Diverse Membrane Topologies following Posttranslational Translocation
Authors: Moise Alexander R. , Grant Jason R. , Lippé Roger , Gabathuler Reinhard , Jefferies Wilfred A. 
Published: December 2003

ABSTRACT

The E3 region of adenovirus codes for several membrane proteins, most of which are involved in immune evasion and prevention of host cell apoptosis. We explored the topology and targeting mechanisms of E3-6.7K, the most recently described member of this group, by using an in vitro translation system supplemented with microsomes. Here, we present evidence that E3-6.7K, one of the smallest signal-anchor proteins known, translocates across the membrane of the endoplasmic reticulum in a posttranslational, ribosome-independent, yet ATP-dependent manner, reminiscent of the translocation of tail-anchored proteins. Our analysis also demonstrated that E3-6.7K could achieve several distinct topological fates. In addition to the previously postulated type III orientation (N-luminal/C-cytoplasmic, termed
Ntm
E3-6.7K), we detected a tail-anchored form adopting the opposite orientation (N-cytoplasmic/C-luminal, termed
Ctm
E3-6.7K) as well as the possibility of a fully translocated form (N and C termini are both translocated, termed
NC
E3-6.7K). Due to the translocation of a positively charged domain, both the
Ctm
E3-6.7K and
NC
E3-6.7K topologies of E3-6.7K constitute exceptions to the “positive inside” rule. The
Ntm
E3-6.7K and
NC
E3-6.7K are the first examples of posttranslationally translocated proteins in higher eukaryotes that are not tail anchored. Distinct topological forms were also found in transfected cells, as both N and C termini of E3-6.7K were detected on the extracellular surface of transfected cells. The demonstration of unexpected topological forms and translocation mechanisms for E3-6.7K defies conventional thinking about membrane protein topogenesis and advises that both the mode of targeting and topology of signal-anchor proteins should be determined experimentally.
Pseudorabies Virus UL3 Gene Codes for a Nuclear Protein Which Is Dispensable for Viral Replication
Authors: Klupp Barbara G. , Granzow Harald , Fuchs Walter , Mundt Egbert , Mettenleiter Thomas C. 
Published: December 2003

ABSTRACT

Many of the products of the ca. 80 genes encoded by alphaherpesviruses have already been identified and, at least tentatively, functionally characterized. Among the least characterized proteins are the products of the genes homologous to herpes simplex virus UL3, which are present only in the subfamily
Alphaherpesvirinae
. To identify the UL3 protein of the porcine alphaherpesvirus pseudorabies virus (PrV), the complete PrV UL3 open reading frame was cloned, expressed in
Escherichia coli
as a glutathione
S
-transferase fusion protein, and used for immunization of a rabbit. In Western blots, the generated antiserum specifically detected a 34-kDa protein in PrV-infected cells, which was absent from purified virus preparations, indicating that PrV UL3 encodes a nonstructural protein. In indirect immunofluorescence analysis, the anti-UL3 serum produced predominantly nuclear staining in transfected as well as in infected cells, which was not altered in the absence of other virus-encoded nuclear proteins such as the UL31 and UL34 gene products. To investigate UL3 function, a deletion mutant, PrV-ΔUL3F2, was constructed and characterized. This mutant replicated and formed plaques on noncomplementing cells indistinguishable from wild-type PrV, demonstrating that PrV UL3 is not required for virus propagation in cultured cells. Moreover, ultrastructural examinations revealed no impairment of capsid formation in the nucleus, nuclear egress of capsids, virion maturation in the cytoplasm, or virus release. Thus, the overall properties of PrV UL3 are similar to those described for the homologous herpes simplex virus proteins which may be indicative of a common, yet hitherto unknown, function in alphaherpesvirus replication. However, based on our studies, an involvement of the UL3 homologs in virion formation appears unlikely.
A Point Mutation in the Binding Subunit of a Retroviral Envelope Protein Arrests Virus Entry at Hemifusion
Authors: Zavorotinskaya Tatiana , Qian Zhaohui , Franks John , Albritton Lorraine M. 
Published: December 2003

ABSTRACT
The transmembrane subunits of viral envelope proteins are thought to perform all of the functions required for membrane fusion during entry of enveloped viruses. However, changes in a conserved SPHQ motif near the N terminus of the receptor binding subunit of a murine leukemia virus (MLV) envelope protein block infection and induction of cell-cell fusion but not receptor binding. Here we report evidence that a histidine-to-arginine change at position 8 (H8R) in the SPHQ motif of Moloney MLV blocks infection by arresting virus-cell fusion at the hemifusion state. In cell-cell fusion assays, H8R envelope protein induced mixing of membrane outer leaflet lipids but did not lead to content mixing, a finding indicative of fusion pore formation. Kinetic studies of virus-cell fusion showed that lipid mixing of H8R virus membranes begins much later than for wild-type virus. The length of the delay in lipid mixing decreased upon addition of two second-site changes that increase H8R virus infection to 100-fold less than the wild-type virus. Finally, chlorpromazine, dibucaine, and trifluoperazine, agents that induce pores in an arrested hemifusion state, rescued infection by H8R virus to within 2.5-fold of the level of wild-type virus infection and cell-cell fusion to half that mediated by wild-type envelope protein. We interpret these results to indicate that fusion progressed to the hemifusion intermediate but fusion pore formation was inhibited. These results establish that membrane fusion of Moloney MLV occurs via a hemifusion intermediate. We also interpret these findings as evidence that histidine 8 is a key switch-point residue between the receptor-induced conformation changes that expose fusion peptide and those that lead to six-helix bundle formation.
Hepatitis C Virus Subgenomic Replicons in the Human Embryonic Kidney 293 Cell Line
Authors: Ali Samir , Pellerin Charles , Lamarre Daniel , Kukolj George 
Published: December 2003

ABSTRACT

Hepatitis C virus (HCV) infects liver cells and its replication in other cells is incompletely defined. Human hepatoma Huh-7 cells harboring subgenomic HCV replicons were used in somatic cell fusion experiments with human embryonic kidney 293 cells as a means of examining the permissiveness of 293 cells for HCV subgenomic RNA replication. 293 cells were generally not permissive for replication of Huh-7 cell-adapted replicons. However, upon coculturing of the two cell lines, we selected rare replicon-containing cells, termed 293Rep cells, that resembled parental 293 cells. Direct metabolic labeling of cells with
33
P in the presence of actinomycin D and Northern blotting to detect the negative strand of the replicon demonstrated functional RNA replicons in 293Rep cells. Furthermore, Western blots revealed that 293Rep cells expressed the HCV nonstructural proteins as well as markers of the naïve 293 cells but not Huh-7 cells. Propidium iodide staining and fluorescence-activated cell sorting analysis of 293Rep cells revealed that clone 293Rep17 closely resembled naïve 293 cells. Transfection of total RNA from 293Rep17 into naïve 293 cells produced replicon-containing 293 cell lines with characteristics distinct from those of Huh-7-derived replicon cell lines. Relative to Huh-7 replicons, the 293 cell replicons were less sensitive to inhibition by alpha interferon and substantially more sensitive to inhibition by poly(I)-poly(C) double-stranded RNA. This study established HCV subgenomic replicons in nonhepatic 293 cells and demonstrated their utility in expanding the study of cellular HCV RNA replication.
Restrictions to the Adaptation of Influenza A Virus H5 Hemagglutinin to the Human Host
Authors: Harvey Ruth , Martin Andrew C. R. , Zambon Maria , Barclay Wendy S. 
Published: December 2003

ABSTRACT
The binding specificities of a panel of avian influenza virus subtype H5 hemagglutinin (HA) proteins bearing mutations at key residues in the receptor binding site were investigated. The results demonstrate that two simultaneous mutations in the receptor binding site resulted in H5 HA binding in a pattern similar to that shown by human viruses. Coexpression of the ion channel protein, M2, from most avian and human strains tested protected H5 HA conformation during trafficking, indicating that no genetic barrier to the reassortment of the H5 surface antigen gene with internal genes of human viruses existed at this level.
An Aromatic Side Chain Is Required at Residue 8 of SU for Fusion of Ecotropic Murine Leukemia Virus
Authors: Qian Zhaohui , Albritton Lorraine M. 
Published: December 2003

ABSTRACT
The surface glycoprotein (SU) of most gammaretroviruses contains a conserved histidine at its amino terminus. In ecotropic murine leukemia virus SU, replacement of histidine 8 with arginine (H8R) or deletion of H8 (H8del) abolishes infection and cell-cell fusion but has no effect on binding to the cellular receptor. We report here that an aromatic ring side chain is essential to the function of residue 8. The size of the aromatic ring appears to be important, as does its ability to form a hydrogen bond. In addition, infection by all of the nonaromatic amino acid substitutions could be partially rescued by the addition of two suppressor mutations (glutamine 227 to arginine [Q227R] and aspartate 243 to tyrosine [D243Y]) or by exposure to chlorpromazine, an agent that induces fusion pores in hemifusion intermediates to complete fusion, suggesting that, like the previously described H8R mutant, the mutants reported here also arrest membrane fusion at the hemifusion state. We propose that H8 is a key switch-point residue in the conformation changes that lead to membrane fusion and present a possible mechanism for how its substitution arrests fusion at the hemifusion state.