bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutagenesis of the
Dengue Virus Type 2
NS3 Protein within and outside Helicase Motifs: Effects on Enzyme Activity and Virus Replication
Authors: Matusan Anita E. , Pryor Melinda J. , Davidson Andrew D. , Wright Peter J. 
Published: July 2002

ABSTRACT

The protein NS3 of
Dengue virus type 2
(DEN-2) is the second largest nonstructural protein specified by the virus and is known to possess multiple enzymatic activities, including a serine proteinase located in the N-terminal region and an NTPase-helicase in the remaining 70% of the protein. The latter region has seven conserved helicase motifs found in all members of the family
Flaviviridae
. DEN-2 NS3 lacking the proteinase region was synthesized as a fusion protein with glutathione
S-
transferase in
Escherichia coli.
The effects of 10 mutations on ATPase and RNA helicase activity were examined. Residues at four sites within enzyme motifs I, II, and VI were substituted, and six sites outside motifs were altered by clustered charged-to-alanine mutagenesis. The mutations were also tested for their effects on virus replication by incorporation into genomic-length cDNA. Two mutations, both in motif I (G198A and K199A) abolished both ATPase and helicase activity. Two further mutations, one in motif VI (R457A,R458A) and the other a clustered charged-to-alanine substitution at R
376
KNGK
380
, abolished helicase activity only. No virus was detected for any mutation which prevented helicase activity, demonstrating the requirement of this enzyme for virus replication. The remaining six mutations resulted in various levels of enzyme activities, and four permitted virus replication. For the two nonreplicating viruses encoding clustered changes at R
184
KR
186
and D
436
GEE
439
, we propose that the substituted residues are surface located and that the viruses are defective through altered interaction of NS3 with other components of the viral replication complex. Two of the replicating viruses displayed a temperature-sensitive phenotype. One contained a clustered mutation at D
334
EE
336
and grew too poorly for further characterization. However, virus with an M283F substitution in motif II was examined in a temperature shift experiment (33 to 37°C) and showed reduced RNA synthesis at the higher temperature.
Molecular Determinants of Species Specificity in the Coronavirus Receptor Aminopeptidase N (CD13): Influence of N-Linked Glycosylation
Authors: Wentworth David E. , Holmes Kathryn V. 
Published: July 2002

ABSTRACTAminopeptidase N (APN), a 150-kDa metalloprotease also called CD13, serves as a receptor for serologically related coronaviruses of humans (human coronavirus 229E [HCoV-229E]), pigs, and cats. These virus-receptor interactions can be highly species specific; for example, the human coronavirus can use human APN (hAPN) but not porcine APN (pAPN) as its cellular receptor, and porcine coronaviruses can use pAPN but not hAPN. Substitution of pAPN amino acids 283 to 290 into hAPN for the corresponding amino acids 288 to 295 introduced an N-glycosylation sequon at amino acids 291 to 293 that blocked HCoV-229E receptor activity of hAPN. Substitution of two amino acids that inserted an N-glycosylation site at amino acid 291 also resulted in a mutant hAPN that lacked receptor activity because it failed to bind HCoV-229E. Single amino acid revertants that removed this sequon at amino acids 291 to 293 but had one or five pAPN amino acid substitution(s) in this region all regained HCoV-229E binding and receptor activities. To determine if other N-linked glycosylation differences between hAPN, feline APN (fAPN), and pAPN account for receptor specificity of pig and cat coronaviruses, a mutant hAPN protein that, like fAPN and pAPN, lacked a glycosylation sequon at 818 to 820 was studied. This sequon is within the region that determines receptor activity for porcine and feline coronaviruses. Mutant hAPN lacking the sequon at amino acids 818 to 820 maintained HCoV-229E receptor activity but did not gain receptor activity for porcine or feline coronaviruses. Thus, certain differences in glycosylation between coronavirus receptors from different species are critical determinants in the species specificity of infection.
Replacement of the Epstein-Barr Virus Plasmid with the EBER Plasmid in Burkitt's Lymphoma Cells
Authors: Maruo Seiji , Nanbo Asuka , Takada Kenzo 
Published: July 2002

ABSTRACT
Transfection of an Epstein-Barr virus (EBV)-encoded plasmid containing EBER caused a substantial decrease in the level of plasmid containing EBV in Akata and Mutu Burkitt's lymphoma (BL) lines, but failed to do so in other BL lines. The results suggest that EBER could replace the role of EBV, but other EBV products also play a role in the growth of BL.
Human Immunodeficiency Virus Type 1 (HIV-1) Non-B Subtypes Are Similar to HIV-1 Subtype B in that Coreceptor Specificity Is a Determinant of Cytopathicity in Human Lymphoid Tissue Infected Ex Vivo
Authors: Malkevich Nina , Womack Chad , Pandya Punita , Grivel Jean-Charles , Fauci Anthony S. , Margolis Leonid 
Published: July 2002

ABSTRACT

We sought to determine the relationship between virus-mediated CD4
+
T-lymphocyte cytopathicity and viral coreceptor preference among various human immunodeficiency virus type 1 (HIV-1) subtypes in an ex vivo-infected human lymphoid tissue model. Our data show that all R5 HIV-1 infections resulted in mild depletion of CD4
+
T lymphocytes, whereas all X4 HIV-1 infections caused severe depletion of CD4
+
T lymphocytes regardless of their subtype origin. Thus, at least for the viruses within subtypes A, B, C, and E that were tested, coreceptor specificity is a critical factor that determines the ability of HIV-1 to deplete CD4
+
T cells in human lymphoid tissue infected ex vivo.
The Replicator of the Epstein-Barr Virus Latent Cycle Origin of DNA Replication,
oriP
, Is Composed of Multiple Functional Elements
Authors: Koons Michelle D. , Van Scoy Sarah , Hearing Janet 
Published: July 2002

ABSTRACT

Replication of the Epstein-Barr virus genome initiates at one of several sites in latently infected, dividing cells. One of these replication origins is close to the viral DNA maintenance element, and, together, this replication origin and the maintenance element are referred to as
oriP
. The replicator of
oriP
contains four binding sites for Epstein-Barr virus nuclear antigen 1 (EBNA-1), the sole viral protein required for the replication and maintenance of
oriP
plasmids. We showed previously that these EBNA-1 sites function in pairs and that mutational inactivation of one pair does not eliminate replicator function. In this study we characterized the contribution of each EBNA-1 site within the replicator and flanking sequences through the use of an internally controlled replication assay. We present evidence that shows that all four EBNA-1 sites are required for an
oriP
plasmid to be replicated in every cell cycle. Results from these experiments also show that the paired EBNA-1 binding sites are not functionally equivalent and that the low affinity of sites 2 and 3 compared to that of sites 1 and 4 is not essential for replicator function. Our results suggest that a host cell protein(s) binds sequences flanking the EBNA-1 sites and that interactions between EBNA-1 and this protein(s) are critical for replicator function. Finally, we present evidence that shows that the minimal replicator of
oriP
consists of EBNA-1 sites 3 and 4 and two copies of a 14-bp repeat that is present in inverse orientation flanking these EBNA-1 sites. EBNA-1 sites 1 and 2, together with an element(s) within nucleotides 9138 to 9516, are ancillary elements required for full replicator activity.
Replication from
oriP
of Epstein-Barr Virus Requires Exact Spacing of Two Bound Dimers of EBNA1 Which Bend DNA
Authors: Bashaw Jacqueline M. , Yates John L. 
Published: July 2002

ABSTRACT

oriP
is a 1.7-kb region of the Epstein-Barr virus (EBV) chromosome that supports replication and stable maintenance of plasmids in human cells that contain EBV-encoded protein EBNA1. Plasmids that depend on
oriP
are replicated once per cell cycle by cellular factors. The replicator of
oriP
is an ∼120-bp region called DS which depends on either of two pairs of closely spaced EBNA1 binding sites. Here we report that changing the distance between the EBNA1 sites of a functional pair by inserting or deleting 1 or 2 bp abolished replication activity. The results indicated that, while the distance separating the binding sites is critical, the specific nucleotide sequence between them is unlikely to be important. The use of electrophoretic mobility shift assays to investigate binding by EBNA1 to the sites with normal or altered spacing revealed that EBNA1 induces DNA to bend significantly when it binds, with the center of bending coinciding with the center of binding. EBNA1 binding to a functional pair of sites which are spaced 21 bp apart center to center and which thus are in helical phase induces a larger symmetrical bend, which based on electrophoretic mobility approximates the sum of two separate EBNA1-induced DNA bends. The results imply that replication from
oriP
requires a precise structure in which DNA forms a large bend around two EBNA1 dimers.
Genome Sequence of a Baculovirus Pathogenic for
Culex nigripalpus
Authors: Afonso C. L. , Tulman E. R. , Lu Z. , Balinsky C. A. , Moser B. A. , Becnel J. J. , Rock D. L. , Kutish G. F. 
Published: July 2002

ABSTRACT

In this report we describe the complete genome sequence of a nucleopolyhedrovirus that infects larval stages of the mosquito
Culex nigripalpus
(CuniNPV). The CuniNPV genome is a circular double-stranded DNA molecule of 108,252 bp and is predicted to contain 109 genes. Although 36 of these genes show homology to genes from other baculoviruses, their orientation and order exhibit little conservation relative to the genomes of lepidopteran baculoviruses. CuniNPV genes homologous to those from other baculoviruses include genes involved in early and late gene expression (
lef-4
,
lef-5
,
lef-8
,
lef-9
,
vlf-1
, and
p47
), DNA replication (
lef-1
,
lef-2
,
helicase-1
, and
dna-pol
), and structural functions (
vp39
,
vp91
,
odv-ec27
,
odv-e56
,
p6.9
,
gp41
,
p74
, and
vp1054
). Auxiliary genes include homologues of genes encoding the p35 antiapoptosis protein and a novel insulin binding-related protein. In contrast to these conserved genes, CuniNPV lacks apparent homologues of baculovirus genes essential (
ie-1
and
lef-3
) or stimulatory (
ie-2
,
lef-7
,
pe38
) for DNA replication. Also, baculovirus genes essential or stimulatory for early-late (
ie-1
,
ie-2
), early (
ie-0
and
pe-38
), and late (
lef-6
,
lef-11
, and
pp31
) gene transcription are not identifiable. In addition, CuniNPV lacks homologues of genes involved in the formation of virogenic stroma (
pp31
), nucleocapsid (
orf1629
,
p87
, and
p24
), envelope of occluded virions (
odv-e25
,
odv-e66
,
odv-e18
), and polyhedra (
polyhedrin/granulin
,
p10
,
pp34
, and
fp25k
). A homologue of gp64, a budded virus envelope fusion protein, was also absent, although a gene related to the other category of baculovirus budded virus envelope proteins, Ld130, was present. The absence of homologues of occlusion-derived virion (ODV) envelope proteins and occlusion body (OB) protein (polyhedrin) suggests that both CuniNPV ODV and OB may be structurally and compositionally different from those found in terrestrial lepidopteran hosts. The striking difference in genome organization, the low level of conservation of homologous genes, and the lack of many genes conserved in other baculoviruses suggest a large evolutionary distance between CuniNPV and lepidopteran baculoviruses.
Epidermal Powder Immunization Induces both Cytotoxic T-Lymphocyte and Antibody Responses to Protein Antigens of Influenza and Hepatitis B Viruses
Authors: Chen Dexiang , Weis Kathleen F. , Chu Qili , Erickson Cherie , Endres Ryan , Lively Chris R. , Osorio Jorge , Payne Lendon G. 
Published: July 2002

ABSTRACTCytotoxic T lymphocytes (CTL) play a vital role in host defense against viral and intracellular bacterial infections. However, nonreplicating vaccines administered by intramuscular injection using a syringe and needle elicit predominantly humoral responses and not CTL responses. Here we report that epidermal powder immunization (EPI), a technology that delivers antigens on 1.5- to 2.5-μm gold particles to the epidermis using a needle-free powder delivery system, elicits CTL responses to nonreplicating antigens. Following EPI, a majority of the antigen-coated gold particles were found in the viable epidermis in the histological sections of the target skin. Further studies using transmission electron microscopy revealed the intracellular localization of the gold particles. Many Langerhans cells (LCs) at the vaccination site contained antigen-coated particles, as revealed by two-color immunofluorescence microscopy, and these cells were found in the draining lymph nodes 20 h later. Immune responses to several viral protein antigens after EPI were studied in mice. EPI with hepatitis B surface antigen (HBsAg) and a synthetic peptide of influenza virus nucleoprotein (NP peptide) elicited antigen-specific CTL responses as well as antibody responses. In an in vitro cell depletion experiment, we demonstrated that the CTL activity against HBsAg elicited by EPI was attributed to CD8+, not CD4+, T cells. As controls, needle injections of HBsAg or the NP peptide into deeper tissues elicited solely antibody, not CTL, responses. We further demonstrated that EPI with inactivated A/Aichi/68 (H3N2) or A/Sydney/97 (H3N2) influenza virus elicited complete protection against a mouse-adapted A/Aichi/68 virus. In summary, EPI directly delivers protein antigens to the cytosol of the LCs in the skin and elicits both cellular and antibody responses.
Differential Histopathology and Chemokine Gene Expression in Lung Tissues following Respiratory Syncytial Virus (RSV) Challenge of Formalin-Inactivated RSV- or BBG2Na-Immunized Mice
Authors: Power Ultan F. , Huss Thierry , Michaud Vincent , Plotnicky-Gilquin Hélène , Bonnefoy Jean-Yves , Nguyen Thien Ngoc 
Published: July 2002

ABSTRACTA BALB/c mouse model of enhanced pulmonary pathology following vaccination with formalin-inactivated alum-adsorbed respiratory syncytial virus (FI-RSV) and live RSV challenge was used to determine the type and kinetics of histopathologic lesions induced and chemokine gene expression profiles in lung tissues. These data were compared and contrasted with data generated following primary and/or secondary RSV infection or RSV challenge following vaccination with a promising subunit vaccine, BBG2Na. Severe peribronchiolitis and perivascularitis coupled with alveolitis and interstitial inflammation were the hallmarks of lesions in the lungs of FI-RSV-primed mice, with peak histopathology evident on days 5 and 9. In contrast, primary RSV infection resulted in no discernible lesions, while challenge of RSV-primed mice resulted in rare but mild peribronchiolitis and perivascularitis, with no evidence of alveolitis or interstitial inflammation. Importantly, mice vaccinated with a broad dose range (20 to 0.02 μg) of a clinical formulation of BBG2Na in aluminium phosphate demonstrated histopathology similar to that observed in secondary RSV infection. At the molecular level, FI-RSV priming was characterized by a rapid and strong up-regulation of eotaxin and monocyte chemotactic protein 3 (MCP-3) relative gene expression (potent lymphocyte and eosinophil chemoattractants) that was sustained through late time points, early but intermittent up-regulation of GRO/melanoma growth stimulatory activity gene and inducible protein 10 gene expression, while macrophage inflammatory protein 2 (MIP-2) and especially MCP-1 were up-regulated only at late time points. By comparison, primary RSV infection or BBG2Na priming resulted in considerably lower eotaxin and MCP-3 gene expression increases postchallenge, while expression of lymphocyte or monocyte chemoattractant chemokine genes (MIP-1β, MCP-1, and MIP-2) were of higher magnitude and kinetics at early, but not late, time points. Our combined histopathologic and chemokine gene expression data provide a basis for differentiating between aberrant FI-RSV-induced immune responses and normal responses associated with RSV infection in the mouse model. Consequently, our data suggest that BBG2Na may constitute a safe RSV subunit vaccine for use in seronegative infants.
Enhanced Expression of Interferon-Regulated Genes in the Liver of Patients with Chronic Hepatitis C Virus Infection: Detection by Suppression-Subtractive Hybridization
Authors: Patzwahl René , Meier Volker , Ramadori Giuliano , Mihm Sabine 
Published: July 2002

ABSTRACTHepatitis C virus (HCV) infection causes acute and often also chronic liver disease. Worldwide, prevalence of infection is estimated to exceed that of human immunodeficiency virus infection fourfold. Because of the lack of appropriate animal models, knowledge of interactions between virus and host is still limited. Assumptions regarding pathogenesis or the activation status of innate antiviral host responses, for instance, derive mainly from clinical observations and from expression analyses of selected genes. To obtain a more objective insight into virus-host interrelationships, we used suppression-subtractive hybridization to compare gene expression in HCV-infected and non-HCV-infected liver tissues samples. Four differentially expressed genes were found: (i) the gamma interferon (IFN-γ)-inducible chemokine IP-10 gene; (ii) the IFN-α/β-inducible antiviral MxA gene; (iii) the gene encoding IFN-α/β-inducible p44, shown to be associated with ultrastructural cytoplasmic entities within hepatocytes of non-A, non-B hepatitis-infected chimpanzees; and (iv) the gene encoding IFN-α/β/γ-inducible IFI-56K, a protein recently shown to interact with the eukaryotic translation initiation factor eIF-3. Compared to hepatic gene expression in patients with liver diseases unrelated to viral infections, expression in patients with chronic HCV infection was up to 50-fold higher. While in patients with chronic HBV infection IP-10 was slightly activated as well, the IFN-α/β-regulated genes were not. Revealing a dominance of hepatic interferon-regulated processes in chronic HCV infection, data on the enhanced expression of the IFN-γ regulated IP-10 support earlier findings and may explain the composition of the hepatic cellular infiltrate. The data on enhanced expression of IFN-α/β inducible genes might be germane to therapeutic considerations.
CD5 Is Dissociated from the B-Cell Receptor in B Cells from Bovine Leukemia Virus-Infected, Persistently Lymphocytotic Cattle: Consequences to B-Cell Receptor-Mediated Apoptosis
Authors: Cantor Glenn H. , Pritchard Suzanne M. , Dequiedt Franck , Willems Luc , Kettmann Richard , Davis William C. 
Published: July 2002

ABSTRACTBovine leukemia virus (BLV), a retrovirus related to human T-cell leukemia virus types 1 and 2, can induce persistent nonneoplastic expansion of the CD5+B-cell population, termed persistent lymphocytosis (PL). As in human CD5+B cells, we report here that CD5 was physically associated with the B-cell receptor (BCR) in normal bovine CD5+B cells. In contrast, in CD5+B cells from BLV-infected PL cattle, CD5 was dissociated from the BCR. In B cells from PL cattle, apoptosis decreased when cells were stimulated with antibody to surface immunoglobulin M (sIgM), while in B cells from uninfected cattle, apoptosis increased after sIgM stimulation. The functional significance of the CD5-BCR association was suggested by experimental dissociation of the CD5-BCR interaction by cross-linking of CD5. This caused CD5+B cells from uninfected animals to decrease apoptosis when stimulated with anti-sIgM. In contrast, in CD5+B cells from PL animals, in which CD5 was already dissociated from the BCR, there was no statistically significant change in apoptosis when CD5 was cross-linked and the cells were stimulated with anti-sIgM. Disruption of CD5-BCR interactions and subsequent decreased apoptosis and increased survival in antigenically stimulated B cells may be a mechanism of BLV-induced PL.
EBNA-LP Associates with Cellular Proteins Including DNA-PK and HA95
Authors: Han Innoc , Harada Shizuko , Weaver David , Xue Yong , Lane William , Orstavik Sigurd , Skalhegg Bjorn , Kieff Elliott 
Published: July 2002

ABSTRACT
EBNA-LP-associated proteins were identified by sequencing proteins that immunoprecipitated with Flag epitope-tagged EBNA-LP (FLP) from lymphoblasts in which FLP was stably expressed. The association of EBNA-LP with Hsp70 (72/73) was confirmed, and sequences of DNA-PK catalytic subunit (DNA-PKcs), HA95, Hsp27, prolyl 4-hydroxylase α-1 subunit, α-tubulin, and β-tubulin were identified. The fraction of total cellular HA95 that associated with FLP was very high, while progressively lower fractions of the total DNA-PKcs, Hsp70, Hsp 27, α-tubulin, and β-tubulin specifically associated with EBNA-LP as determined by immunoblotting with antibodies to these proteins. EBNA-LP bound to two domains in the DNA-PKcs C terminus and DNA-PKcs associated with the EBNA-LP repeat domain. DNA-PKcs that was bound to EBNA-LP phosphorylated p53 or EBNA-LP in vitro, and the phosphorylation of EBNA-LP was inhibited by Wortmannin, a specific in vitro inhibitor of DNA-PKcs.
Gender Influences Herpes Simplex Virus Type 1 Infection in Normal and Gamma Interferon-Mutant Mice
Authors: Han Xiao , Lundberg Patric , Tanamachi Becky , Openshaw Harry , Longmate Jeff , Cantin Edouard 
Published: July 2002

ABSTRACTGender influences the incidence and severity of some bacterial and viral infections and autoimmune diseases in animal models and humans. To determine a gender-based difference, comparisons were made between male and female mice inoculated with herpes simplex virus type 1 (HSV-1) by the corneal route. Mortality was higher in the male mice of the three strains tested: 129/Sv//Ev wild type, gamma interferon (IFN-γ) knockout (GKO), and IFN-γ receptor knockout (RGKO). Similarly, in vivo HSV-1 reactivation occurred more commonly in male mice, but the male-female difference in reactivation was restricted to the two knockout strains and was not seen in the 129/Sv//Ev control. Comparison among male mice of the three strains showed a higher mortality of the RGKO mice and a higher reactivation rate of the GKO and RGKO mice than of the 129/Sv//Ev males. In contrast, female RGKO and GKO mice did not differ from female 129/Sv//Ev controls in either mortality or reactivation. HSV-1 periocular and eyelid disease was also more severe in male and dihydrotestosterone (DHT)-treated female mice than in control female mice. These results show a consistent gender difference in HSV-1 infection, with a worse outcome in male mice. In addition, the results comparing GKO and RGKO mice to controls show differences only in male mice, suggesting that some effects of IFN-γ, a key immunoregulatory molecule, are gender specific.