bluuebunny/crossref_metadata_2025_split huggingface dataset:
Palmitoylations on Murine Coronavirus Spike Proteins Are Essential for Virion Assembly and Infectivity
Authors: Thorp Edward B. , Boscarino Joseph A. , Logan Hillary L. , Goletz Jeffrey T. , Gallagher Thomas M. 
Published: January 2006

ABSTRACT
Coronavirus spike (S) proteins are palmitoylated at several cysteine residues clustered near their transmembrane-spanning domains. This is achieved by cellular palmitoyl acyltransferases (PATs), which can modify newly synthesized S proteins before they are assembled into virion envelopes at the intermediate compartment of the exocytic pathway. To address the importance of these fatty acylations to coronavirus infection, we exposed infected cells to 2-bromopalmitate (2-BP), a specific PAT inhibitor. 2-BP profoundly reduced the specific infectivities of murine coronaviruses at very low, nontoxic doses that were inert to alphavirus and rhabdovirus infections. 2-BP effected only two- to fivefold reductions in S palmitoylation, yet this correlated with reduced S complexing with virion membrane (M) proteins and consequent exclusion of S from virions. At defined 2-BP doses, underpalmitoylated S proteins instead trafficked to infected cell surfaces and elicited cell-cell membrane fusions, suggesting that the acyl chain adducts are more critical to virion assembly than to S-induced syncytial developments. These studies involving pharmacologic inhibition of S protein palmitoylation were complemented with molecular genetic analyses in which cysteine acylation substrates were mutated. Notably, some mutations (C1347F and C1348S) did not interfere with S incorporation into virions, indicating that only a subset of the cysteine-rich region provides the essential S-assembly functions. However, the C1347F/C1348S mutant viruses exhibited relatively low specific infectivities, similar to virions secreted from 2-BP-treated cultures. Our collective results indicate that the palmitate adducts on coronavirus S proteins are necessary in assembly and also in positioning the assembled envelope proteins for maximal infectivity.
West Nile Virus Discriminates between DC-SIGN and DC-SIGNR for Cellular Attachment and Infection
Authors: Davis Carl W. , Nguyen Hai-Yen , Hanna Sheri L. , Sánchez Melissa D. , Doms Robert W. , Pierson Theodore C. 
Published: January 2006

ABSTRACTThe C-type lectins DC-SIGN and DC-SIGNR bind mannose-rich glycans with high affinity. In vitro, cells expressing these attachment factors efficiently capture, and are infected by, a diverse array of appropriately glycosylated pathogens, including dengue virus. In this study, we investigated whether these lectins could enhance cellular infection by West Nile virus (WNV), a mosquito-borne flavivirus related to dengue virus. We discovered that DC-SIGNR promoted WNV infection much more efficiently than did DC-SIGN, particularly when the virus was grown in human cell types. The presence of a single N-linked glycosylation site on either the prM or E glycoprotein of WNV was sufficient to allow DC-SIGNR-mediated infection, demonstrating that uncleaved prM protein present on a flavivirus virion can influence viral tropism under certain circumstances. Preferential utilization of DC-SIGNR was a specific property conferred by the WNV envelope glycoproteins. Chimeras between DC-SIGN and DC-SIGNR demonstrated that the ability of DC-SIGNR to promote WNV infection maps to its carbohydrate recognition domain. WNV virions and subviral particles bound to DC-SIGNR with much greater affinity than DC-SIGN. We believe this is the first report of a pathogen interacting more efficiently with DC-SIGNR than with DC-SIGN. Our results should lead to the discovery of new mechanisms by which these well-studied lectins discriminate among ligands.
Important Role for the Transmembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Protein during Entry
Authors: Broer Rene , Boson Bertrand , Spaan Willy , Cosset François-Loïc , Corver Jeroen 
Published: January 2006

ABSTRACT

The spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for receptor binding and membrane fusion. It contains a highly conserved transmembrane domain that consists of three parts: an N-terminal tryptophan-rich domain, a central domain, and a cysteine-rich C-terminal domain. The cytoplasmic tail of S has previously been shown to be required for assembly. Here, the roles of the transmembrane and cytoplasmic domains of S in the infectivity and membrane fusion activity of SARS-CoV have been studied. SARS-CoV S-pseudotyped retrovirus (SARSpp) was used to measure S-mediated infectivity. In addition, the cell-cell fusion activity of S was monitored by a
Renilla
luciferase-based cell-cell fusion assay. S
vsv-c
yt, an S chimera with a cytoplasmic tail derived from vesicular stomatitis virus G protein (VSV-G), and S
mhv-tmdc
yt, an S chimera with the cytoplasmic and transmembrane domains of mouse hepatitis virus, displayed wild-type-like activity in both assays. S
vsv-tmdc
yt, a chimera with the cytoplasmic and transmembrane domains of VSV-G, was impaired in the SARSpp and cell-cell fusion assays, showing 3 to 25% activity compared to the wild type, depending on the assay and the cells used. Examination of the oligomeric state of the chimeric S proteins in SARSpp revealed that S
vsv-tmdc
yt trimers were less stable than wild-type S trimers, possibly explaining the lowered fusogenicity and infectivity.
Dynamic Evolution of the Human Immunodeficiency Virus Type 1 Pathogenic Factor, Nef
Authors: O'Neill Eduardo , Kuo Lillian S. , Krisko John F. , Tomchick Diana R. , Garcia J. Victor , Foster John L. 
Published: January 2006

ABSTRACT
The human immunodeficiency virus type 1 (HIV-1) early gene product Nef is a multifunctional protein that alters numerous pathways of T-cell function, including endocytosis, signal transduction, vesicular trafficking, and immune modulation, and is a major determinant of pathogenesis. Individual Nef functions include PAK-2 activation, CD4 downregulation, major histocompatibility complex (MHC) class I downregulation, and enhancement of viral particle infectivity. How Nef accomplishes its multiple tasks presents a difficult problem of mechanistic analysis because of the complications associated with multiple, overlapping functional domains in the context of significant sequence variability. To address these issues we determined the conservation of each Nef residue based on 1,643 subtype B Nef sequences. Mutational analysis based on conservative substitutions and Nef sequence data allowed us to search for amino acids on the surface of Nef that are specifically required for PAK-2 activation. We found residues 85, 89, and 191 to be highly significant determinants for Nef's PAK-2 activation function but functionally unlinked to CD4 and MHC class I downregulation or enhancement of infectivity. These residues are not conserved across HIV-1 subtypes but are confined to separate sets of surface elements within a subtype. Thus, L85/H89/F191 and F85/F89/R191 are dominant in subtype B and subtype E or C, respectively. Our results provide support for developing subtype-specific interventions in HIV-1 disease.
Epstein-Barr Virus Protein Can Upregulate Cyclo-Oxygenase-2 Expression through Association with the Suppressor of Metastasis Nm23-H1
Authors: Kaul Rajeev , Verma Subhash C. , Murakami Masanao , Lan Ke , Choudhuri Tathagata , Robertson Erle S. 
Published: January 2006

ABSTRACT

Previous studies have demonstrated the interaction between the Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) and the metastatic suppressor Nm23-H1 both in vitro and in vivo (C. Subramanian, M. A. Cotter II, and E. S. Robertson, Nat. Med.
7:
350-355, 2001). EBNA3C can reverse the ability of Nm23-H1 to suppress migration of Burkitt's lymphoma and breast carcinoma cell lines in vitro. EBNA3C contributes to EBV-associated human cancers by regulating transcription of a number of cellular and viral promoters and by targeting and altering the transcription activities of the metastasis suppressor Nm23-H1. Cyclo-oxygenase-2 (COX-2), an inducible enzyme important in inflammation, is overexpressed in a variety of cancers and can influence cell migration. In this report we show that Nm23-H1 and EBNA3C can modulate expression of COX-2 in the context of EBV infection and transformation. The levels of COX-2 were consistently higher in EBV-positive cells than in EBV-negative cells. Additionally, we show that Nm23-H1 can upregulate the COX-2 promoter element in luciferase reporter assays, whereas EBNA3C alone did not affect the level of response but clearly contributed to an additive increase when coexpressed with Nm23-H1. The downstream effect of COX-2 expression was also evaluated and showed that prostaglandin E
2
levels increased with Nm23-H1 and that there was some level of cooperativity in the presence of EBNA3C. The majority of this response was mediated through the cyclic AMP response element and NF-κB sites. These studies suggest a potential role for COX-2 in EBV-associated human cancers.
Composition of Pseudorabies Virus Particles Lacking Tegument Protein US3, UL47, or UL49 or Envelope Glycoprotein E
Authors: Michael Kathrin , Klupp Barbara G. , Mettenleiter Thomas C. , Karger Axel 
Published: January 2006

ABSTRACT

Proteins located in the tegument layer of herpesvirus particles play important roles in the replicative cycle at both early and late times after infection. As major constituents of the virion, they execute important functions in particular during formation of progeny virions. These functions have mostly been elucidated by construction and analysis of mutant viruses deleted in single or multiple tegument protein-encoding genes (reviewed in the work of T. C. Mettenleiter, Virus Res. 106:167-180, 2004). However, since tegument proteins have been shown to be involved in numerous protein-protein interactions, the impact of single protein deletions on the composition of the virus particle is unknown, but they could impair correct interpretation of the results. To analyze how the absence of single virion constituents influences virion composition, we established a procedure to assay relative amounts of virion structural proteins in deletion mutants of the alphaherpesvirus
Pseudorabies virus
(PrV) in comparison to wild-type particles. The assay is based on the mass spectrometric quantitation of virion protein-derived peptides carrying stable isotope mass tags. After deletion of the US3, UL47, UL49, or glycoprotein E gene, relative amounts of a capsid protein (UL38), a capsid-associated protein (UL25), several tegument proteins (UL36 and UL47, if present), and glycoprotein H were unaffected, whereas the content of other tegument proteins (UL46, UL48, and UL49, if present) varied significantly. In the case of the UL48 gene product, a specific increase in incorporation of a smaller isoform was observed after deletion of the UL47 or UL49 gene, whereas a larger isoform remained unaffected. The cellular protein actin was enriched in virions of mutants deficient in any of the tegument proteins UL47, UL49, or US3. By two-dimensional gel electrophoresis multiple isoforms of host cell-derived heat shock protein 70 and annexins A1 and A2 were also identified as structural components of PrV virions.
Antibodies against West Nile Virus Nonstructural Protein NS1 Prevent Lethal Infection through Fc γ Receptor-Dependent and -Independent Mechanisms
Authors: Chung Kyung Min , Nybakken Grant E. , Thompson Bruce S. , Engle Michael J. , Marri Anantha , Fremont Daved H. , Diamond Michael S. 
Published: January 2006

ABSTRACTThe flavivirus nonstructural protein NS1 is a highly conserved secreted glycoprotein that does not package with the virion. Immunization with NS1 elicits a protective immune response against yellow fever, dengue, and tick-borne encephalitis flaviviruses through poorly defined mechanisms. In this study, we purified a recombinant, secreted form of West Nile virus (WNV) NS1 glycoprotein from baculovirus-infected insect cells and generated 22 new NS1-specific monoclonal antibodies (MAbs). By performing competitive binding assays and expressing truncated NS1 proteins on the surface of yeast (Saccharomyces cerevisiae) and in bacteria, we mapped 21 of the newly generated MAbs to three NS1 fragments. Prophylaxis of C57BL/6 mice with any of four MAbs (10NS1, 14NS1, 16NS1, and 17NS1) strongly protected against lethal WNV infection (75 to 95% survival, respectively) compared to saline-treated controls (17% survival). In contrast, other anti-NS1 MAbs of the same isotype provided no significant protection. Notably, 14NS1 and 16NS1 also demonstrated marked efficacy as postexposure therapy, even when administered as a single dose 4 days after infection. Virologic analysis showed that 17NS1 protects at an early stage in infection through a C1q-independent and Fc γ receptor-dependent pathway. Interestingly, 14NS1, which maps to a distinct region on NS1, protected through a C1q- and Fc γ receptor-independent mechanism. Overall, our data suggest that distinct regions of NS1 can elicit protective humoral immunity against WNV through different mechanisms.
Stable Growth of Wild-Type Hepatitis A Virus in Cell Culture
Authors: Konduru Krishnamurthy , Kaplan Gerardo G. 
Published: January 2006

ABSTRACT
Human wild-type (wt) hepatitis A virus (HAV), the causative agent of acute hepatitis, barely grows in cell culture and in the process accumulates attenuating and cell culture-adapting mutations. This genetic instability of wt HAV in cell culture is a major roadblock to studying HAV pathogenesis and producing live vaccines that are not overly attenuated for humans. To develop a robust cell culture system capable of supporting the efficient growth of wt HAV, we transfected different cell lines with in vitro RNA transcripts of wt HAV containing the blasticidin resistance gene. Blasticidin-resistant colonies grew only in transfected Huh7 cells and produced infectious virus. HAV was genetically stable in Huh7 cells for at least nine serial passages and did not accumulate attenuating or cell culture-adapting mutations. Treatment with alpha interferon A/D cured the blasticidin-resistant Huh7 cells of the HAV infection. The cured cells, termed Huh7-A-I cells, did not contain virus or HAV antigens and were sensitive to blasticidin. Huh7-A-I cells were more permissive than parental cells for wt HAV infection, including a natural isolate from a human stool sample, and produced 10-fold-more infectious particles. This is the first report of a cell line that allows the genetically stable growth of human wt HAV. The viral vectors and cells described here should allow better insight into the pathogenesis of HAV and the development of attenuated vaccines. The cell lines susceptible to wt HAV growth may also be used to detect and isolate infectious virus from patient and environmental samples.
Identification of Gangliosides GD1b and GT1b as Receptors for BK Virus
Authors: Low Jonathan A. , Magnuson Brian , Tsai Billy , Imperiale Michael J. 
Published: January 2006

ABSTRACT
Gangliosides have been shown to be plasma membrane receptors for both murine polyomavirus and SV40, while JC virus uses serotonin receptors. In contrast, little is known of the membrane receptor and entry pathway for BK virus (BKV), which can cause severe disease in immunosuppressed bone marrow and renal transplant patients. Using sucrose flotation assays, we investigated BKV binding to and interaction with human erythrocyte membranes and determined that this interaction was dependent on a neuraminidase-sensitive, proteinase K-resistant molecule. BKV was found to interact with the gangliosides GT1b and GD1b. The terminal α2-8-linked disialic acid motif, present in both of these gangliosides, is likely to be important for this interaction. We also determined that the addition of GD1b and GT1b to LNCaP cells, which are normally resistant to BKV infection, made them susceptible to the virus. In addition, BKV interacted with membranes extracted from the endoplasmic reticulum (ER) and infection was blocked by the addition of brefeldin A, which interferes with transport from the ER to the Golgi apparatus. These data demonstrate that BKV uses the gangliosides GT1b and GD1b as receptors and passes through the ER on the way to the nucleus.
Divergent Patterns of Recent Retroviral Integrations in the Human and Chimpanzee Genomes:Probable Transmissions between Other Primates and Chimpanzees
Authors: Jern Patric , Sperber Göran O. , Blomberg Jonas 
Published: January 2006

ABSTRACT

The
human genome is littered by endogenous retrovirus sequences (HERVs),
which constitute up to 8% of the total genomic sequence. The sequencing
of the human (
Homo sapiens
) and chimpanzee (
Pan
troglodytes
) genomes has facilitated the evolutionary study of
ERVs and related sequences. We screened both the human genome (version
hg16) and the chimpanzee genome (version PanTro1) for ERVs and
conducted a phylogenetic analysis of recent integrations. We found a
number of recent integrations within both genomes. They segregated into
four groups. Two larger gammaretrovirus-like groups (PtG1 and PtG2)
occurred in chimpanzees but not in humans. The PtG sequences were most
similar to two baboon ERVs and a macaque sequence but
neither to other chimpanzee ERVs nor to any human gammaretrovirus-like
ERVs. The pattern was consistent with cross-species transfer via
predation. This appears to be an example of horizontal transfer of
retroviruses with occasional fixation in the germ
line.
Adenovirus Virus-Associated RNA Is Processed to Functional Interfering RNAs Involved in Virus Production
Authors: Aparicio Oscar , Razquin Nerea , Zaratiegui Mikel , Narvaiza Iñigo , Fortes Puri 
Published: January 2006

ABSTRACT
Posttranscriptional gene silencing allows sequence-specific control of gene expression. Specificity is guaranteed by small antisense RNAs such as microRNAs (miRNAs) or small interfering RNAs (siRNAs). Functional miRNAs derive from longer double-stranded RNA (dsRNA) molecules that are cleaved to pre-miRNAs in the nucleus and are transported by exportin 5 (Exp 5) to the cytoplasm. Adenovirus-infected cells express virus-associated (VA) RNAs, which are dsRNA molecules similar in structure to pre-miRNAs. VA RNAs are also transported by Exp 5 to the cytoplasm, where they accumulate. Here we show that small RNAs derived from VA RNAs (svaRNAs), similar to miRNAs, can be found in adenovirus-infected cells. VA RNA processing to svaRNAs requires neither viral replication nor viral protein expression, as evidenced by the fact that svaRNA accumulation can be detected in cells transfected with VA sequences. svaRNAs are efficiently bound by Argonaute 2, the endonuclease of the RNA-induced silencing complex, and behave as functional siRNAs, in that they inhibit the expression of reporter genes with complementary sequences. Blocking svaRNA-mediated inhibition affects efficient adenovirus production, indicating that svaRNAs are required for virus viability. Thus, svaRNA-mediated silencing could represent a novel mechanism used by adenoviruses to control cellular or viral gene expression.
Transcriptional Regulation of the Kaposi's Sarcoma-Associated Herpesvirus K15 Gene
Authors: Wong Emily L. , Damania Blossom 
Published: January 2006

ABSTRACT

The K15 gene product of Kaposi's sarcoma-associated herpesvirus (KSHV) is a transmembrane protein that is encoded by the last open reading frame of the KSHV genome. The K15 protein has been implicated in modulation of B-cell signal transduction and activation of the Ras/mitogen-activated protein kinase and NF-κB signal transduction pathways. Here we report the identification of the transcriptional start site of the full-length K15 gene in KSHV-positive BCBL-1 cells. We have mapped the K15 transcriptional start site to a position 152 nucleotides upstream from the translation start site by rapid amplification of cDNA ends and RNase protection assays. We have also characterized the K15 promoter element. To analyze the
cis
-acting elements necessary to regulate K15 gene expression, a series of 5′ promoter deletion constructs were generated and subcloned upstream of the luciferase reporter gene. Transcriptional assays with these mutant promoters demonstrated that chemical induction in latently infected KSHV-positive BCBL-1 cells activated K15 transcription. In addition, K15 promoter transactivation was also mediated by the viral immediate-early protein Orf50/Rta, suggesting that the K15 gene is actively transcribed during lytic replication.
Nuclear Export of African Swine Fever Virus p37 Protein Occurs through Two Distinct Pathways and Is Mediated by Three Independent Signals
Authors: Eulálio Ana , Nunes-Correia Isabel , Carvalho Ana Luísa , Faro Carlos , Citovsky Vitaly , Salas José , Salas Maria L. , Simões Sérgio , de Lima Maria C. Pedroso 
Published: January 2006

ABSTRACT
Nucleocytoplasmic shuttling activity of the African swine fever virus p37 protein, a major structural protein of this highly complex virus, has been recently reported. The systematic characterization of the nuclear export ability of this protein constituted the major purpose of the present study. We report that both the N- and C-terminal regions of p37 protein are actively exported from the nucleus to the cytoplasm of yeast and mammalian cells. Moreover, experiments using leptomycin B and small interfering RNAs targeting the CRM1 receptor have demonstrated that the export of p37 protein is mediated by both the CRM1-dependent and CRM1-independent nuclear export pathways. Two signals responsible for the CRM1-mediated nuclear export of p37 protein were identified at the N terminus of the protein, and an additional signal was identified at the C-terminal region, which mediates the CRM1-independent nuclear export. Interestingly, site-directed mutagenesis revealed that hydrophobic amino acids are critical to the function of these three nuclear export signals. Overall, our results demonstrate that two distinct pathways contribute to the strong nuclear export of full-length p37 protein, which is mediated by three independent nuclear export signals. The existence of overlapping nuclear export mechanisms, together with our observation that p37 protein is localized in the nucleus at early stages of infection and exclusively in the cytoplasm at later stages, suggests that the nuclear transport ability of this protein may be critical to the African swine fever virus replication cycle.