bluuebunny/crossref_metadata_2025 huggingface dataset:
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

Aminopeptidase 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

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

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

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

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

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

Cytotoxic 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

A 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

Hepatitis 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

Bovine 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

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

Gender 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.
African Swine Fever Virus Multigene Family 360 and 530 Genes Are Novel Macrophage Host Range Determinants
Authors: Zsak L. , Lu Z. , Burrage T. G. , Neilan J. G. , Kutish G. F. , Moore D. M. , Rock D. L.
Published: July 2002

Pathogenic African swine fever virus (ASFV) isolates primarily target cells of the mononuclear-phagocytic system in infected swine and replicate efficiently in primary macrophage cell cultures in vitro. ASFVs can, however, be adapted to grow in monkey cell lines. Characterization of two cell culture-adapted viruses, MS16 and BA71V, revealed that neither virus replicated in macrophage cell cultures. Cell viability experiments and ultrastructural analysis showed that infection with these viruses resulted in early macrophage cell death, which occurred prior to viral progeny production. Genomic cosmid clones from pathogenic ASFV isolate E70 were used in marker rescue experiments to identify sequences capable of restoring MS16 and BA71V growth in macrophage cell cultures. A cosmid clone representing a 38-kbp region at the left terminus of the genome completely restored the growth of both viruses. In subsequent fine-mapping experiments, an 11-kbp subclone from this region was sufficient for complete rescue of BA71V growth. Sequence analysis indicated that both MS16 and BA71V had significant deletions in the region containing members of multigene family 360 (MGF 360) and MGF530. Deletion of this same region from highly pathogenic ASFV isolate Pr4 significantly reduced viral growth in macrophage cell cultures. These findings indicate that ASFV MGF360 and MGF530 genes perform an essential macrophage host range function(s) that involves promotion of infected-cell survival.
Brome Mosaic Virus Protein 1a Recruits Viral RNA2 to RNA Replication through a 5′ Proximal RNA2 Signal
Authors: Chen Jianbo , Noueiry Amine , Ahlquist Paul
Published: July 2002

Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication factors. Membrane-associated 1a protein contains a helicase-like domain and RNA capping functions. 2a, which is targeted to membranes by 1a, contains a central polymerase-like domain. In the absence of 2a and RNA replication, 1a acts through an intergenic replication signal in BMV genomic RNA3 to stabilize RNA3 and induce RNA3 to associate with cellular membrane. Multiple results imply that 1a-induced RNA3 stabilization reflects interactions involved in recruiting RNA3 templates into replication. To determine if 1a had similar effects on another BMV RNA replication template, we constructed a plasmid expressing BMV genomic RNA2 in vivo. In vivo-expressed RNA2 templates were replicated upon expression of 1a and 2a. In the absence of 2a, 1a stabilized RNA2 and induced RNA2 to associate with membrane. Deletion analysis demonstrated that 1a-induced membrane association of RNA2 was mediated by sequences in the 5′-proximal third of RNA2. The RNA2 5′ untranslated region was sufficient to confer 1a-induced membrane association on a nonviral RNA. However, sequences in the N-terminal region of the 2a open reading frame enhanced 1a responsiveness of RNA2 and a chimeric RNA. A 5′-terminal RNA2 stem-loop important for RNA2 replication was essential for 1a-induced membrane association of RNA2 and, like the 1a-responsive RNA3 intergenic region, contained a required box B motif corresponding to the TΨC stem-loop of host tRNAs. The level of 1a-induced membrane association of various RNA2 mutants correlated well with their abilities to serve as replication templates. These results support and expand the conclusion that 1a-induced BMV RNA stabilization and membrane association reflect early, 1a-mediated steps in viral RNA replication.
Regression of a Murine Gammaherpesvirus 68-Positive B-Cell Lymphoma Mediated by CD4 T Lymphocytes
Authors: Robertson Kevin A. , Usherwood Edward J. , Nash Anthony A.
Published: July 2002

Murine gammaherpesvirus 68-infected S11 cells were injected subcutaneously into nude mice. Adoptively transferred restimulated lymphocytes consistently elicited the regression of S11 tumors. CD4 T lymphocytes were most effective in preventing tumor formation, and immunohistochemistry highlighted populations of CD4 T cells in regressing tumors.
Generation of a Highly Pathogenic Avian Influenza A Virus from an Avirulent Field Isolate by Passaging in Chickens
Authors: Ito Toshihiro , Goto Hideo , Yamamoto Eiji , Tanaka Hiroko , Takeuchi Mutsuko , Kuwayama Masaru , Kawaoka Yoshihiro , Otsuki Koichi
Published: July 2002

Highly virulent avian influenza viruses can arise from avirulent strains maintained in poultry, but evidence to support their generation from viruses in wild birds is lacking. The most likely mechanism for the acquisition of virulence by benign avian viruses is the introduction of mutations by error-prone RNA polymerase, followed by the selection of virulent viruses. To investigate whether this mechanism could apply to wild waterfowl, we studied an avirulent wild-swan virus that replicates poorly in chickens. After 24 consecutive passages by air sac inoculation, followed by five passages in chicken brain, the avirulent virus became highly pathogenic in chickens, producing a 100% mortality rate. Sequence analysis at the hemmaglutinin cleavage site of the original isolate revealed a typical avirulence type of sequence, R-E-T-R, which progressed incrementally to a typical virulence type of sequence, R-R-K-K-R, during repeated passages in chickens. These results demonstrate that avirulent viruses maintained in wild waterfowl in nature and bearing the consensus avirulence type sequence R-E-T-R have the potential to become highly pathogenic while circulating in chickens.
Neutralizing Human Fab Fragments against Measles Virus Recovered by Phage Display
Authors: de Carvalho Nicacio Cristina , Williamson R. Anthony , Parren Paul W. H. I. , Lundkvist Åke , Burton Dennis R. , Björling Ewa
Published: July 2002

Five human recombinant Fab fragments (Fabs) specific for measles virus (MV) proteins were isolated from three antibody phage display libraries generated from RNAs derived from bone marrow or splenic lymphocytes from three MV-immune individuals. All Fabs reacted in an enzyme-linked immunosorbent assay with MV antigens. In radioimmunoprecipitation assays two of the Fabs, MV12 and MT14, precipitated an ⊘80-kDa protein band corresponding to the hemagglutinin (H) protein from MV-infected Vero cell cultures, while two other Fabs, MT64 and GL29, precipitated an ⊘60-kDa protein corresponding the nucleocapsid (N) protein. In competition studies with MV fusion, H- and N protein-specific monoclonal antibodies (MAbs), the H-specific Fabs predominantly blocked the binding of H-specific MAbs, while the N-specific Fabs blocked MAbs to N. In addition, N-specific Fabs bound to denatured MV N protein in Western blotting. The specificity of the fifth Fab, MV4, could not be determined. By plaque reduction assays, three of the five Fabs, MV4, MV12, and MT14, exhibited neutralizing activity (80% cutoff) against MV (LEC-KI strain) at concentrations ranging between ≈2 and 7 μg ml
−1
. Neutralization capacity against MV strains Edmonston and Schwarz was also detected, albeit at somewhat higher Fab concentrations. In conclusion, three neutralizing Fabs were isolated, two of them reactive against the H glycoprotein of MV and another reactive against an undefined epitope. This is the first study in which MV-neutralizing human recombinant Fab antibodies have been isolated from phage display libraries.
Evolution of Porcine Reproductive and Respiratory Syndrome Virus during Sequential Passages in Pigs
Authors: Chang C.-C. , Yoon K.-J. , Zimmerman J. J. , Harmon K. M. , Dixon P. M. , Dvorak C. M. T. , Murtaugh M. P.
Published: July 2002

Porcine reproductive and respiratory syndrome (PRRS) viruses are recognized as possessing a high degree of genetic and antigenic variability. Viral diversity has led to questions regarding the association of virus mutation and persistent infection in the host and has raised concerns vis-à-vis protective immunity, the ability of diagnostic assays to detect novel variants, and the possible emergence of virulent strains. The purpose of this study was to describe ongoing changes in PRRS virus during replication in pigs under experimental conditions. Animals were inoculated with a plaque-cloned virus derived from VR-2332, the North American PRRS virus prototype. Three independent lines of in vivo replication were maintained for 367 days by pig-to-pig passage of virus at 60-day intervals. A total of 315 plaque-cloned viruses were recovered from 21 pigs over the 367-day observation period and compared to the original plaque-cloned virus by virus neutralization assay, monoclonal antibody analysis, and sequencing of open reading frames (ORFs) 1b (replicase), 5 (major envelope protein), and 7 (nucleocapsid) of the genome. Variants were detected by day 7 postinoculation, and multiple variants were present concurrently in every pig sampled over the observation period. Sequence analysis showed ORFs 1b and 7 to be highly conserved. In contrast, sequencing of ORF 5 disclosed 48 nucleotide variants which corresponded to 22 amino acid variants. Although no epitopic changes were detected under the conditions of this experiment, PRRS virus was shown to evolve continuously in infected pigs, with different genes of the viral genome undergoing various degrees of change.
Primary Immune Responses by Cord Blood CD4+T Cells and NK Cells Inhibit Epstein-Barr Virus B-Cell Transformation In Vitro
Authors: Wilson A. Douglas , Morgan Andrew J.
Published: July 2002

Epstein-Barr virus (EBV) transformation of B cells from fetal cord blood in vitro varies depending on the individual sample. When a single preparation of EBV was simultaneously used to transform fetal cord blood samples from six different individuals, the virus transformation titer varied from less than zero to 105.9. We show that this variation in EBV transformation is associated with a marked primary immune response in cord blood samples predominately involving CD4+T cells and CD16+CD56+NK cells. After virus challenge both CD4+T cells and NK cells in fetal cord blood cultures expressed the lymphocyte activation marker CD69. The cytotoxic response against autologous EBV-infected lymphoblastoid cell line (LCL) targets correlated with the number of CD16+CD69+cells and was inversely correlated with the virus transformation titer. Although NK activity was detected in fresh cord blood and increased following activation by the virus, killing of autologous LCLs was detected only following activation by exposure to the virus. Both activated CD4+T cells and CD16+NK cells were independently able to kill autologous LCLs. Both interleukin-2 and gamma interferon were produced by CD4+T cells after virus challenge. The titer of EBV was lower when purified B cells were used than when whole cord blood was used. Addition of monocytes restored the virus titer, while addition of resting T cells or EBV-activated CD4+T-cell blasts reduced the virus titer. We conclude that there are primary NK-cell and Th1-type CD4+T-cell responses to EBV in fetal cord blood that limit the expansion of EBV-infected cells and in some cases eliminate virus infection in vitro.
Expression of a Membrane-Anchored Glycoprotein, the Influenza Virus Hemagglutinin, by Dicistronic Replicons Derived from the Poliovirus Genome
Authors: Vignuzzi Marco , Gerbaud Sylvie , van der Werf Sylvie , Escriou Nicolas
Published: July 2002

Mono- and dicistronic poliovirus replicons were constructed to express the influenza virus hemagglutinin, retaining its signal peptide and transmembrane region. Picornavirus genomes do not normally encode glycoproteins, and only the dicistronic replicon, in which the foreign and poliovirus sequences were separated by the encephalomyocarditis virus internal ribosomal entry site, replicated and expressed glycosylated hemagglutinin.
Bone Marrow Chimeras Reveal Non-H-2Hematopoietic Control of Susceptibility to Theiler's Virus Persistent Infection
Authors: Aubagnac Stéphanie , Brahic Michel , Bureau Jean-François
Published: July 2002

The DA strain of Theiler's murine encephalomyelitis virus persists in the white matter of the spinal cords of susceptible mice. Previous results showed that the difference in susceptibility to viral persistence between the susceptible SJL/J strain and the resistant B10.S strain was due to multiple non-H-2loci. The respective roles of hematopoietic and nonhematopoietic cells in this difference have been evaluated with bone marrow chimeras. The results show that non-H-2loci with a major effect on susceptibility are expressed in hematopoietic cells. However, the study of the SJL.B10-D10Mit180-D10Mit74congenic line suggests that other loci expressed in nonhematopoietic cells also play a role.
Identification of Active-Site Amino Acid Residues in the Chiba Virus 3C-Like Protease
Authors: Someya Yuichi , Takeda Naokazu , Miyamura Tatsuo
Published: July 2002

The 3C-like protease of the Chiba virus, a Norwalk-like virus, is one of the chymotrypsin-like proteases. To identify active-site amino acid residues in this protease, 37 charged amino acid residues and a putative nucleophile, Cys139, within the GDCG sequence were individually replaced with Ala in the 3BC precursor, followed by expression in
Escherichia coli
, where the active 3C-like protease would cleave 3BC into 3B (VPg) and 3C (protease). Among 38 Ala mutants, 7 mutants (R8A, H30A, K88A, R89A, D138A, C139A, and H157A) completely or nearly completely lost the proteolytic activity. Cys139 was replaceable only with Ser, suggesting that an SH or OH group in the less bulky side chain was required for the side chain of the residue at position 139. His30, Arg89, and Asp138 could not be replaced with any other amino acids. Although Arg8 was also not replaceable for the 3B/3C cleavage and the 3C/3D cleavage, the N-terminal truncated mutant devoid of Arg8 significantly cleaved 3CD into 3C and 3D (polymerase), indicating that Arg8 itself was not directly involved in the proteolytic cleavage. As for position 88, a positively charged residue was required because the Arg mutant showed significant activity. As deduced by the X-ray structure of the hepatitis A virus 3C protease, Arg8, Lys88, and Arg89 are far away from the active site, and the side chain of Asp138 is directed away from the active site. Therefore, these are not catalytic residues. On the other hand, all of the mutants of His157 in the S1 specificity pocket tended to retain very slight activity, suggesting a decreased level of substrate recognition. These results, together with a sequence alignment with the picornavirus 3C proteases, indicate that His30 and Cys139 are active-site residues, forming a catalytic dyad without a carboxylate directly participating in the proteolysis.
A Tyrosine-Based Motif in the Cytoplasmic Tail of Pseudorabies Virus Glycoprotein B Is Important for both Antibody-Induced Internalization of Viral Glycoproteins and Efficient Cell-to-Cell Spread
Authors: Favoreel Herman W. , Van Minnebruggen Geert , Nauwynck Hans J. , Enquist Lynn W. , Pensaert Maurice B.
Published: July 2002

Pseudorabies virus (PRV), a swine alphaherpesvirus, is capable of causing viremia in vaccinated animals. Two mechanisms that may help PRV avoid recognition by the host immune system during this viremia are direct cell-to-cell spread in tissue and antibody-induced internalization of viral cell surface glycoproteins in PRV-infected blood monocytes, the carrier cells of the virus in the blood. PRV glycoprotein B (gB) is crucial during both processes. Here we show that mutating a tyrosine residue located in a YXXΦ motif in the gB cytoplasmic tail results in decreased efficiency of cell-to-cell spread and a strong reduction in antibody-induced internalization of viral cell surface glycoproteins. Mutating the dileucine motif in the gB tail led to an increased cell-to-cell spread of the virus and the formation of large syncytia.
K15 Protein of Kaposi’s Sarcoma-Associated Herpesvirus Is Latently Expressed and Binds to HAX-1, a Protein with Antiapoptotic Function
Authors: Sharp Tyson V. , Wang Hsei-Wei , Koumi Andrew , Hollyman Daniel , Endo Yoshio , Ye Hongtao , Du Ming-Qing , Boshoff Chris
Published: July 2002

The Kaposi’s sarcoma-associated herpesvirus (KSHV) (or human herpesvirus 8) open reading frame (ORF) K15 encodes a putative integral transmembrane protein in the same genomic location as latent membrane protein 2A of Epstein-Barr virus. Ectopic expression of K15 in cell lines revealed the presence of several different forms ranging in size from full length, ∼50 kDa, to 17 kDa. Of these different species the 35- and 23-kDa forms were predominant. Mutational analysis of the initiator AUG indicated that translation initiation from this first AUG is required for K15 expression. Computational analysis indicates that the different forms detected may arise due to proteolytic cleavage at internal signal peptide sites. We show that K15 is latently expressed in KSHV-positive primary effusion lymphoma cell lines and in multicentric Castleman’s disease. Using a yeast two-hybrid screen we identified HAX-1 (HS1 associated protein X-1) as a binding partner to the C terminus of K15 and show that K15 interacts with cellular HAX-1 in vitro and in vivo. Furthermore, HAX-1 colocalizes with K15 in the endoplasmic reticulum and mitochondria. The function of HAX-1 is unknown, although the similarity of its sequence to those of Nip3 and Bcl-2 infers a role in the regulation of apoptosis. We show here that HAX-1 can form homodimers in vivo and is a potent inhibitor of apoptosis and therefore represents a new apoptosis regulatory protein. The putative functions of K15 with respect to its interaction with HAX-1 are discussed.
Herpes Simplex Virus Type 1 Immediate-Early Protein ICP0 and Its Isolated RING Finger Domain Act as Ubiquitin E3 Ligases In Vitro
Authors: Boutell Chris , Sadis Seth , Everett Roger D.
Published: July 2002

Proteasome-dependent degradation of ubiquitinated proteins plays a key role in many important cellular processes. Ubiquitination requires the E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme, and frequently a substrate-specific ubiquitin protein ligase (E3). One class of E3 ubiquitin ligases has been shown to contain a common zinc-binding RING finger motif. We have previously shown that herpes simplex virus type 1 ICP0, itself a RING finger protein, induces the proteasome-dependent degradation of several cellular proteins and induces the accumulation of colocalizing conjugated ubiquitin in vivo. We now report that both full-length ICP0 and its isolated RING finger domain induce the accumulation of polyubiquitin chains in vitro in the presence of E1 and the E2 enzymes UbcH5a and UbcH6. Mutations within the RING finger region that abolish the in vitro ubiquitination activity also cause severe reductions in ICP0 activity in other assays. We conclude that ICP0 has the potential to act as an E3 ubiquitin ligase during viral infection and to target specific cellular proteins for destruction by the 26S proteasome.
Functional Replacement and Positional Dependence of Homologous and Heterologous L Domains in Equine Infectious Anemia Virus Replication
Authors: Li Feng , Chen Chaoping , Puffer Bridget A. , Montelaro Ronald C.
Published: July 2002

We have previously demonstrated by Gag polyprotein budding assays that the Gag p9 protein of equine infectious anemia virus (EIAV) utilizes a unique YPDL motif as a late assembly domain (L domain) to facilitate release of the budding virus particle from the host cell plasma membrane (B. A. Puffer, L. J. Parent, J. W. Wills, and R. C. Montelaro, J. Virol. 71:6541-6546, 1997). To characterize in more detail the role of the YPDL L domain in the EIAV life cycle, we have examined the replication properties of a series of EIAV proviral mutants in which the parental YPDL L domain was replaced by a human immunodeficiency virus type 1 (HIV-1) PTAP or Rous sarcoma virus (RSV) PPPY L domain in the p9 protein or by proviruses in which the parental YPDL or HIV-1 PTAP L domain was inserted in the viral matrix protein. The replication properties of these L-domain variants were examined with respect to Gag protein expression and processing, virus particle production, and virus infectivity. The data from these experiments indicate that (i) the YPDL L domain of p9 is required for replication competence (assembly and infectivity) in equine cell cultures, including the natural target equine macrophages; (ii) all of the functions of the YPDL L domain in the EIAV life cycle can be replaced by replacement of the parental YPDL sequence in p9 with the PTAP L-domain segment of HIV-1 p6 or the PPPY L domain of RSV p2b; and (iii) the assembly, but not infectivity, functions of the EIAV proviral YPDL substitution mutants can be partially rescued by inclusions of YPDL and PTAP L-domain sequences in the C-terminal region of the EIAV MA protein. Taken together, these data demonstrate that the EIAV YPDL L domain mediates distinct functions in viral budding and infectivity and that the HIV-1 PTAP and RSV PPPY L domains can effectively facilitate these dual replication functions in the context of the p9 protein. In light of the fact that YPDL, PTAP, and PPPY domains evidently have distinct characteristic binding specificities, these observations may indicate different portals into common cellular processes that mediate EIAV budding and infectivity, respectively.
PrP Expression and Replication by Schwann Cells: Implications in Prion Spreading
Authors: Follet Jérôme , Lemaire-Vieille Catherine , Blanquet-Grossard Françoise , Podevin-Dimster Valérie , Lehmann Sylvain , Chauvin Jean-Paul , Decavel Jean-Pierre , Varea Ruth , Grassi Jacques , Fontès Michel , Cesbron Jean-Yves
Published: July 2002

Prion infection relies on a continuous chain of PrP
c
-expressing tissues to spread from peripheral sites to the central nervous system (CNS). Direct neuroinvasion via peripheral nerves has long been considered likely. However, the speed of axonal flow is incompatible with the lengthy delay prior to the detection of PrP
Sc
in the brain. We hypothesized that Schwann cells could be the candidate implicated in this mechanism; for that, it has to express PrP
c
and to allow PrP
Sc
conversion. We investigated in vivo localization of PrP
c
in sciatic nerve samples from different strains of mice. We demonstrated that PrP
c
is mainly localized at the cell membrane of the Schwann cell. We also studied in vitro expression of PrP
c
in the Schwann cell line MSC-80 and demonstrated that it expresses PrP
c
at the same location. More specifically, we demonstrated that this glial cell line, when infected in vitro with the mouse Chandler prion strain, both produces the PrP
Sc
till after 18 passages and is able to transmit disease to mice, which then develop the typical signs of prion diseases. It is the first time that infection and replication of PrP
Sc
are shown in a peripheral glial cell line.
Recombinant Vaccinia Virus-Induced T-Cell Immunity: Quantitation of the Response to the Virus Vector and the Foreign Epitope
Authors: Harrington Laurie E. , Most Robbert van der , Whitton J. Lindsay , Ahmed Rafi
Published: July 2002

Recombinant vaccinia viruses (rVV) have been extensively used as vaccines, but there is little information about the total magnitude of the VV-specific T-cell response and how this compares to the immune response to the foreign gene(s) expressed by the rVV. To address this issue, we quantitated the T-cell responses to both the viral vector and the insert following the infection of mice with VV expressing a cytotoxic T lymphocyte (CTL) epitope (NP118-126) from lymphocytic choriomeningitis virus (LCMV). The LCMV epitope-specific response was quantitated by intracellular cytokine staining after stimulation with the specific peptide. To analyze the total VV-specific response, we developed a simple intracellular cytokine staining assay using VV-infected major histocompatibility complex class I and II matched cells as stimulators. Using this approach, we made the following determinations. (i) VV-NP118 induced potent and long-lasting CD8 and CD4 T-cell responses to the vector; at the peak of the response (∼1 week), there were ∼107VV-specific CD8 T cells (25% of the CD8 T cells) and ∼106VV-specific CD4 T cells (∼5% of the CD4 T cells) in the spleen. These numbers decreased to ∼5 × 105CD8 T cells (∼5% frequency) and ∼105CD4 T cells (∼0.5% frequency), respectively, by day 30 and were then stably maintained at these levels for >300 days. The size of this VV-specific T-cell response was comparable to that of the T-cell response induced following an acute LCMV infection. (ii) VV-specific CD8 and CD4 T cells were capable of producing gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-2; all cells were able to make IFN-γ, a subset produced both IFN-γ and TNF-α, and another subset produced all three cytokines. (iii) The CD8 T-cell response to the foreign gene (LCMV NP118-126 epitope) was coordinately regulated with the response to the vector during all three phases (expansion, contraction, and memory) of the T-cell response. The total number of CD8 T cells responding to NP118-126 were ∼20- to 30-fold lower than the number responding to the VV vector (∼1% at the peak and 0.2% in memory). This study provides a better understanding of T-cell immunity induced by VV-based vaccines, and in addition, the technique described in the study can be readily extended to other viral vectors to determine the ratio of the T-cell response to the insert versus the vector. This information will be useful in optimizing prime-boost regimens for vaccination.
Replication of Bovine Papillomavirus Type 1 (BPV-1) DNA in
Saccharomyces cerevisiae
following Infection with BPV-1 Virions
Authors: Zhao Kong-Nan , Frazer Ian H.
Published: July 2002

Saccharomyces cerevisiae
protoplasts exposed to bovine papillomavirus type 1 (BPV-1) virions demonstrated uptake of virions on electron microscopy.
S. cerevisiae
cells looked larger after exposure to BPV-1 virions, and cell wall regeneration was delayed. Southern blot hybridization of Hirt DNA from cells exposed to BPV-1 virions demonstrated BPV-1 DNA, which could be detected over 80 days of culture and at least 13 rounds of division. Two-dimensional gel analysis of Hirt DNA showed replicative intermediates, confirming that the BPV-1 genome was replicating within
S. cerevisiae
. Nicked circle, linear, and supercoiled BPV-1 DNA species were observed in Hirt DNA preparations from
S. cerevisiae
cells infected for over 50 days, and restriction digestion showed fragments hybridizing to BPV-1 in accord with the predicted restriction map for circular BPV-1 episomes. These data suggest that BPV-1 can infect
S. cerevisiae
and that BPV-1 episomes can replicate in the infected
S. cerevisiae
cells.