bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutational Analyses of the Nonconserved Sequences in the Bunyamwera Orthobunyavirus S Segment Untranslated Regions
Authors: Lowen Anice C. , Elliott Richard M. 
Published: September 2005

ABSTRACT

Bunyamwera virus
(BUNV) is the prototype of the genus
Orthobunyavirus
and the family
Bunyaviridae
. BUNV has a tripartite genome of negative-sense RNA composed of small (S), medium (M), and large (L) segments. Partially complementary untranslated regions (UTRs) flank the coding region of each segment. The terminal 11 nucleotides of these UTRs are conserved between the three segments and throughout the genus, while the internal regions are unique to each segment and largely nonconserved between different viruses. To investigate the functions of the UTR sequences, we constructed a series of BUNV S segment cDNA clones with deletions in the 3′ and/or 5′ UTR and then attempted to rescue these segments into recombinant viruses. We found that the genomic 5′ UTR was much more sensitive to mutation than the 3′ UTR and, in general, sequences proximal to the termini were more important than those flanking the coding region. Northern blot analyses of infected-cell RNA showed that the internal, nonconserved sequences of the S segment 3′ UTR play a role in the regulation of transcription and replication and the balance between these two processes. In contrast, deletions in the 5′ UTR caused attenuation of the recombinant virus but did not specifically affect levels of S segment RNAs or the encoded nucleocapsid protein. Thus, the internal regions of both UTRs are functional: most of the 5′ UTR is essential to viral growth, and, while nonessential, the internal 3′ UTR is important to the regulation of viral RNA synthesis.
New Genes from Old: Redeployment of dUTPase by Herpesviruses
Authors: Davison Andrew J. , Stow Nigel D. 
Published: September 2005

ABSTRACT

Published work (D. J. McGeoch, Nucleic Acids Res.
18:
4105-4110, 1990; J. E. McGeehan, N. W. Depledge, and D. J. McGeoch, Curr. Protein Peptide Sci.
2:
325-333, 2001) has indicated that evolution of dUTPase in the class of herpesviruses that infect mammals and birds involved capture of a host gene followed by a duplication event that resulted in a coding region comprising two fused dUTPase domains. Some of the conserved residues required for enzyme activity were then lost, resulting in a dUTPase containing a single active site with different elements contributed by each half of the protein. Further conserved residues were lost in one subfamily (the
Betaherpesvirinae
), yielding a protein that is related to herpesvirus dUTPases but has a different and as yet unrecognized function. Evidence from sequence similarities and structural predictions now indicates that several additional genes were derived from the herpesvirus dUTPase gene, probably by duplication. These are UL31, UL82, UL83, and UL84 in human cytomegalovirus (and counterparts in other members of the
Betaherpesvirinae
) and ORF10 and ORF11 in human herpesvirus 8 (and counterparts in other members of the
Gammaherpesvirinae
). The findings clarify the evolutionary history of these genes and provide novel insights for structural and functional studies.
Hepatitis B Virus Large and Middle Glycoproteins Are Degraded by a Proteasome Pathway in Glucosidase-Inhibited Cells but Not in Cells with Functional Glucosidase Enzyme
Authors: Simsek Ender , Mehta Anand , Zhou Tianlun , Dwek Raymond A. , Block Timothy 
Published: September 2005

ABSTRACT
The secretion of hepatitis B virus (HBV) large (LHBs) and middle (MHBs) envelope polypeptides from tissue cultures requires proper protein folding and is prevented by inhibitors of the endoplasmic reticulum (ER) glucosidase. Using competitive inhibitors of the ER glucosidase, here it is shown that the amounts of glycosylated and unglycosylated forms of LHBs and MHBs proteins are all greatly reduced in tissue cultures producing HBV envelope glycoproteins. In contrast, the HBV small (SHBs) protein was not affected. The reduction in secretion of LHBs and MHBs proteins appears to be mediated by proteasomal degradation pathways, since it is prevented by either lactacystin or epoxomicin, two inhibitors of proteasomal degradation. Although there is no detectable proteasomal degradation of LHBs and MHBs in cells with functional glucosidase, the implications of the nearly quantitative sensitivity of glycosylated and unglycosylated forms of LHBs and MHBs proteins, with selective sparing of SHBs protein, in cells in which glucosidase is inhibited is surprising, and its implications are discussed.
T-Cell Tropism and the Role of ORF66 Protein in Pathogenesis of Varicella-Zoster Virus Infection
Authors: Schaap Anne , Fortin Jean-Francois , Sommer Marvin , Zerboni Leigh , Stamatis Shaye , Ku Chia-Chi , Nolan Garry P. , Arvin Ann M. 
Published: September 2005

ABSTRACT
The pathogenesis of varicella-zoster virus (VZV) involves a cell-associated viremia during which infectious virus is carried from sites of respiratory mucosal inoculation to the skin. We now demonstrate that VZV infection of T cells is associated with robust virion production and modulation of the apoptosis and interferon pathways within these cells. The VZV serine/threonine protein kinase encoded by ORF66 is essential for the efficient replication of VZV in T cells. Preventing ORF66 protein expression by stop codon insertion (pOka66S) impaired the growth of the parent Oka (pOka) strain in T cells in SCID-hu T-cell xenografts in vivo and reduced formation of VZV virions. The lack of ORF66 protein also increased the susceptibility of infected T cells to apoptosis and reduced the capacity of the virus to interfere with induction of the interferon (IFN) signaling pathway following exposure to IFN-γ. However, preventing ORF66 protein expression only slightly reduced growth in melanoma cells in culture and did not diminish virion formation in these cells. The pOka66S virus showed only a slight defect in growth in SCID-hu skin implants compared with intact pOka. These observations suggest that the ORF66 kinase plays a unique role during infection of T cells and supports VZV T-cell tropism by contributing to immune evasion and enhancing survival of infected T cells.
De Novo Generation of Escape Variant-Specific CD8+T-Cell Responses following Cytotoxic T-Lymphocyte Escape in Chronic Human Immunodeficiency Virus Type 1 Infection
Authors: Allen Todd M. , Yu Xu G. , Kalife Elizabeth T. , Reyor Laura L. , Lichterfeld Mathias , John Mina , Cheng Michael , Allgaier Rachel L. , Mui Stanley , Frahm Nicole , Alter Galit , Brown Nancy V. , Johnston Mary N. , Rosenberg Eric S. , Mallal Simon A. , Brander Christian , Walker Bruce D. , Altfeld Marcus 
Published: September 2005

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) evades CD8+T-cell responses through mutations within targeted epitopes, but little is known regarding its ability to generate de novo CD8+T-cell responses to such mutants. Here we examined gamma interferon-positive, HIV-1-specific CD8+T-cell responses and autologous viral sequences in an HIV-1-infected individual for more than 6 years following acute infection. Fourteen optimal HIV-1 T-cell epitopes were targeted by CD8+T cells, four of which underwent mutation associated with dramatic loss of the original CD8+response. However, following the G357S escape in the HLA-A11-restricted Gag349-359epitope and the decline of wild-type-specific CD8+T-cell responses, a novel CD8+T-cell response equal in magnitude to the original response was generated against the variant epitope. CD8+T cells targeting the variant epitope did not exhibit cross-reactivity against the wild-type epitope but rather utilized a distinct T-cell receptor Vβ repertoire. Additional studies of chronically HIV-1-infected individuals expressing HLA-A11 demonstrated that the majority of the subjects targeted the G357S escape variant of the Gag349-359epitope, while the wild-type consensus sequence was significantly less frequently recognized. These data demonstrate that de novo responses against escape variants of CD8+T-cell epitopes can be generated in chronic HIV-1 infection and provide the rationale for developing vaccines to induce CD8+T-cell responses directed against both the wild-type and variant forms of CD8 epitopes to prevent the emergence of cytotoxic T-lymphocyte escape variants.
Isolation of Cell Lines That Show Novel, Murine Leukemia Virus-Specific Blocks to Early Steps of Retroviral Replication
Authors: Bruce James W. , Bradley Kenneth A. , Ahlquist Paul , Young John A. T. 
Published: September 2005

ABSTRACT
In order to identify cellular proteins required for early stages of retroviral replication, a high volume screening with mammalian somatic cells was performed. Ten pools of chemically mutagenized Chinese hamster ovary (CHO-K1) cells were challenged with a murine leukemia virus (MLV) vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G), and cells that failed to be transduced were enriched by cell sorting. Each pool yielded a clonally derived cell line with a 5-fold or greater resistance to virus infection, and five cell lines exhibited a >50-fold resistance. These five cell lines were efficiently infected by a human immunodeficiency virus vector pseudotyped with VSV-G. When engineered to express the TVA receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), the five cell lines were resistant to infection with a MLV vector pseudotyped with the ASLV-A envelope protein but were fully susceptible to infection with an ASLV-A vector. Thus, the defect in these cells resides after virus-cell membrane fusion and, unlike those in other mutant cell lines that have been described, is specific for the MLV core. To identify the specific stages of MLV infection that are impaired in the resistant cell lines, real-time quantitative PCR analyses were employed and two phenotypic groups were identified. Viral infection of three cell lines was restricted before reverse transcription; in the other two cell lines, it was blocked after reverse transcription, nuclear localization, and two-long terminal repeat circle formation but before integration. These data provide genetic evidence that at least two distinct intracellular gene products are required specifically for MLV infection. These cell lines are important tools for the biochemical and genetic analysis of early stages in retrovirus infection.
Full-Breadth Analysis of CD8+T-Cell Responses in Acute Hepatitis C Virus Infection and Early Therapy
Authors: Lauer Georg M. , Lucas Michaela , Timm Joerg , Ouchi Kei , Kim Arthur Y. , Day Cheryl L. , zur Wiesch Julian Schulze , Paranhos-Baccala Glaucia , Sheridan Isabelle , Casson Deborah R. , Reiser Markus , Gandhi Rajesh T. , Li Bin , Allen Todd M. , Chung Raymond T. , Klenerman Paul , Walker Bruce D. 
Published: September 2005

ABSTRACTMultispecific CD8+T-cell responses are thought to be important for the control of acute hepatitis C virus (HCV) infection, but to date little information is actually available on the breadth of responses at early time points. Additionally, the influence of early therapy on these responses and their relationships to outcome are controversial. To investigate this issue, we performed comprehensive analysis of the breadth and frequencies of virus-specific CD8+T-cell responses on the single epitope level in eight acutely infected individuals who were all started on early therapy. During the acute phase, responses against up to five peptides were identified. During therapy, CD8+T-cell responses decreased rather than increased as virus was controlled, and no new specificities emerged. A sustained virological response following completion of treatment was independent of CD8+T-cell responses, as well as CD4+T-cell responses. Rapid recrudescence also occurred despite broad CD8+T-cell responses. Importantly, in vivo suppression of CD3+T cells using OKT3 in one subject did not result in recurrence of viremia. These data suggest that broad CD8+T-cell responses alone may be insufficient to contain HCV replication, and also that early therapy is effective independent of such responses.
Analysis of a Foot-and-Mouth Disease Virus Type A
24
Isolate Containing an SGD Receptor Recognition Site In Vitro and Its Pathogenesis in Cattle
Authors: Rieder Elizabeth , Henry Tina , Duque Hernando , Baxt Barry 
Published: September 2005

ABSTRACT

Foot-and-mouth disease virus (FMDV) initiates infection by binding to integrin receptors via an Arg-Gly-Asp (RGD) sequence found in the G-H loop of the structural protein VP1. Following serial passages of a type A
24
Cruzeiro virus (A
24
Cru) in bovine, via tongue inoculation, a virus was generated which contained an SGD sequence in the cell receptor-binding site and expressed a turbid plaque phenotype in BHK-21 cells. Propagation of this virus in these cells resulted in the rapid selection of viruses that grew to higher titers, produced clear plaques, and now contained an RGD sequence in place of the original SGD. To study the role of the SGD sequence in FMDV receptor recognition and bovine virulence, we assembled an infectious cDNA clone of an RGD-containing A
24
Cru and derived mutant clones containing either SGD with a single nucleotide substitution in the R
144
codon or double substitutions at this position to prevent mutation of the S to an R. The SGD viruses grew poorly in BHK-21 cells and stably maintained the sequence during propagation in BHK-21 cells expressing the bovine α
V
β
6
integrin (BHK3-α
V
β
6
), as well as in experimentally infected and contact steers. While all the SGD-containing viruses used only the bovine α
V
β
6
integrin as a cellular receptor with relatively high efficiency, the revertant RGD viruses utilized either the α
V
β
1
or α
V
β
3
bovine integrins with higher efficiency than α
V
β
6
and grew well in BHK-21 cells. Replacing the R at the −1 SGD position with either K or E showed that this residue did not contribute to integrin utilization in vitro. These results illustrate the rapid evolution of FMDV with alteration in receptor specificity and suggest that viruses with sequences other than RGD, but closely related to it, can still infect via integrin receptors and induce and transmit the disease to susceptible animals.
Essential Elements of the Capsid Protein for Self-Assembly into Empty Virus-Like Particles of Hepatitis E Virus
Authors: Li Tian-Cheng , Takeda Naokazu , Miyamura Tatsuo , Matsuura Yoshiharu , Wang Joseph C. Y. , Engvall Helena , Hammar Lena , Xing Li , Cheng R. Holland 
Published: September 2005

ABSTRACT
Hepatitis E virus (HEV) is a noncultivable virus that causes acute liver failure in humans. The virus's major capsid protein is encoded by an open reading frame 2 (ORF2) gene. When the recombinant protein consisting of amino acid (aa) residues 112 to 660 of ORF2 is expressed with a recombinant baculovirus, the protein self-assembles into virus-like particles (VLPs) (T.-C. Li, Y. Yamakawa, K. Suzuki, M. Tatsumi, M. A. Razak, T. Uchida, N. Takeda, and T. Miyamura, J. Virol. 71:7207-7213, 1997). VLPs can be found in the culture medium of infected Tn5 cells but not in that of Sf9 cells, and the major VLPs have lost the C-terminal 52 aa. To investigate the protein requirement for HEV VLP formation, we prepared 14 baculovirus recombinants to express the capsid proteins truncated at the N terminus, the C terminus, or both. The capsid protein consisting of aa residues 112 to 608 formed VLPs in Sf9 cells, suggesting that particle formation is dependent on the modification process of the ORF2 protein. In the present study, electron cryomicroscopy and image processing of VLPs produced in Sf9 and Tn5 cells indicated that they possess the same configurations and structures. Empty VLPs were found in both Tn5 and Sf9 cells infected with the recombinant containing an N-terminal truncation up to aa residue 125 and C-terminal to aa residue 601, demonstrating that the aa residues 126 to 601 are the essential elements required for the initiation of VLP assembly. The recombinant HEV VLPs are potential mucosal vaccine carrier vehicles for the presentation of foreign antigenic epitopes and may also serve as vectors for the delivery of genes to mucosal tissue for DNA vaccination and gene therapy. The results of the present study provide useful information for constructing recombinant HEV VLPs having novel functions.
Identification of an RNA Silencing Suppressor from a Plant Double-Stranded RNA Virus
Authors: Cao Xuesong , Zhou Peng , Zhang Xiaoming , Zhu Shifeng , Zhong Xuehua , Xiao Qi , Ding Biao , Li Yi 
Published: September 2005

ABSTRACT

RNA silencing is a mechanism which higher plants and animals have evolved to defend against viral infection in addition to regulation of gene expression for growth and development. As a counterdefense, many plant and some animal viruses studied to date encode RNA silencing suppressors (RSS) that interfere with various steps of the silencing pathway. In this study, we report the first identification of an RSS from a plant double-stranded RNA (dsRNA) virus. Pns10, encoded by S10 of
Rice dwarf phytoreovirus
(RDV), exhibited RSS activity in coinfiltration assays with the reporter green fluorescent protein (GFP) in transgenic
Nicotiana benthamiana
line 16c carrying GFP. The other gene segments of the RDV genome did not have such a function. Pns10 suppressed local and systemic silencing induced by sense RNA but did not interfere with local and systemic silencing induced by dsRNA. Expression of Pns10 also increased the expression of β-glucuronidase in transient assays and enhanced
Potato virus X
pathogenicity in
N. benthamiana
. Collectively, our results establish Pns10 as an RSS encoded by a plant dsRNA virus and further suggest that Pns10 targets an upstream step of dsRNA formation in the RNA silencing pathway.
Herpes Simplex Virus 1 Envelopment Follows Two Diverse Pathways
Authors: Leuzinger Helene , Ziegler Urs , Schraner Elisabeth M. , Fraefel Cornel , Glauser Daniel L. , Heid Irma , Ackermann Mathias , Mueller Martin , Wild Peter 
Published: September 2005

ABSTRACT

Herpesvirus envelopment is assumed to follow an uneconomical pathway including primary envelopment at the inner nuclear membrane, de-envelopment at the outer nuclear membrane, and reenvelopment at the
trans
-Golgi network. In contrast to the hypothesis of de-envelopment by fusion of the primary envelope with the outer nuclear membrane, virions were demonstrated to be transported from the perinuclear space to rough endoplasmic reticulum (RER) cisternae. Here we show by high-resolution microscopy that herpes simplex virus 1 envelopment follows two diverse pathways. First, nuclear envelopment includes budding of capsids at the inner nuclear membrane into the perinuclear space whereby tegument and a thick electron dense envelope are acquired. The substance responsible for the dense envelope is speculated to enable intraluminal transportation of virions via RER into Golgi cisternae. Within Golgi cisternae, virions are packaged into transport vacuoles containing one or several virions. Second, for cytoplasmic envelopment, capsids gain direct access from the nucleus to the cytoplasm via impaired nuclear pores. Cytoplasmic capsids could bud at the outer nuclear membrane, at membranes of RER, Golgi cisternae, and large vacuoles, and at banana-shaped membranous entities that were found to continue into Golgi membranes. Envelopes originating by budding at the outer nuclear membrane and RER membrane also acquire a dense substance. Budding at Golgi stacks, designated wrapping, results in single virions within small vacuoles that contain electron-dense substances between envelope and vacuolar membranes.
Dissection of a Novel Nuclear Localization Signal in Open Reading Frame 29 of Varicella-Zoster Virus
Authors: Stallings Christina L. , Silverstein Saul 
Published: September 2005

ABSTRACT

Open reading frame 29 (ORF29) of varicella-zoster virus (VZV) encodes a 120-kDa single-stranded DNA binding protein (ORF29p) that is not packaged in the virion and is expressed during latency. During lytic infection, ORF29p is localized primarily to infected cell nuclei. In contrast, ORF29p is found exclusively in the cytoplasm in neurons of the dorsal root ganglia obtained at autopsy from seropositive latently infected patients. ORF29p accumulates in the nuclei of neurons in dorsal root ganglia obtained at autopsy from patients with active zoster. The localization of this protein is, therefore, tightly correlated with the proposed VZV lytic/latent switch. In this report, we have investigated the nuclear import mechanism of ORF29p. We identified a novel nuclear targeting domain bounded by amino acids 9 to 154 of ORF29p that functions independent of other VZV-encoded factors. In vitro import assays in digitonin-permeabilized HeLa cells reveal that ORF29p is transported into the nucleus by a Ran-, karyopherin α- and β-dependent mechanism. These data are further supported by the demonstration that a glutathione
S
-transferase-karyopherin α fusion interacts with ORF29p, but not with a protein containing a point mutation in its nuclear localization signal (NLS). Therefore, the region of ORF29p responsible for its nuclear targeting is also involved in the association with karyopherin α. As a result of this interaction, this noncanonical NLS appears to hijack the classical cellular nuclear import machinery. Elucidation of the mechanisms governing ORF29p nuclear targeting could shed light on the VZV reactivation process.
Herpes Simplex Virus Tegument Protein VP22 Contains an Internal VP16 Interaction Domain and a C-Terminal Domain That Are Both Required for VP22 Assembly into the Virus Particle
Authors: Hafezi Wali , Bernard Emmanuelle , Cook Rachelle , Elliott Gillian 
Published: September 2005

ABSTRACT
Many steps along the herpesvirus assembly and maturation pathway remain unclear. In particular, the acquisition of the virus tegument is a poorly understood process, and the molecular interactions involved in tegument assembly have not yet been defined. Previously we have shown that the two major herpes simplex virus tegument proteins VP22 and VP16 are able to interact, although the relevance of this to virus assembly is not clear. Here we have constructed a number of recombinant viruses expressing N- and C-terminal truncations of VP22 and have used them to identify regions of the protein involved in its assembly into the virus structure. Analysis of the packaging of these VP22 variants into extracellular virions revealed that the C terminus of VP22 is absolutely required for this process, with removal of the C-terminal 89 residues abrogating its incorporation. However, while these 89 residues alone were sufficient for specific incorporation of small amounts of VP22 into the tegument, efficient packaging of VP22 to the levels of full-length protein required an additional 52 residues of the protein. Coimmunoprecipitation assays indicated that these 52 residues also contained the interaction domain for VP16. Furthermore, analysis of the subcellular localization of the mutant forms of VP22 revealed that only those truncations that were efficiently assembled formed characteristic cytoplasmic trafficking complexes, suggesting that these complexes may represent the cellular location for VP22 assembly into the virus. Taken together, these results suggest that there are two determinants involved in the packaging of VP22—a C-terminal domain and an internal VP16 interaction domain, both of which are required for the efficient recruitment of VP22 to sites of virus assembly.