bluuebunny/crossref_metadata_2025_split huggingface dataset:
Critical Role for Alpha/Beta and Gamma Interferons in Persistence of Lymphocytic Choriomeningitis Virus by Clonal Exhaustion of Cytotoxic T Cells
Authors: Ou Rong , Zhou Shenghua , Huang Lei , Moskophidis Demetrius 
Published: July 2002

ABSTRACTUnder conditions of high antigenic load during infection with invasive lymphocytic choriomeningitis virus (LCMV) strains, virus can persist by selective clonal exhaustion of antigen-specific CD8+T cells. In this work we studied the down-regulation of the virus-specific CD8+-T-cell response during a persistent infection of adult mice, with particular emphasis on the contribution of the interferon response in promoting host defense. Studies were conducted by infecting mice deficient in receptors for type I (alpha/beta interferon [IFN-α/β]), type II (IFN-γ), and both type I and II IFNs with LCMV isolates that vary in their capacity to induce T-cell exhaustion. The main conclusions of this study are as follows. (i) IFNs play a critical role in LCMV infection by reducing viral loads in the initial stages of infection and thus modifying both the extent of CD8+-T-cell exhaustion and the course of infection. The importance of IFNs in this context varies with the biological properties of the LCMV strain. (ii) An inverse correlation exists between antigen persistence and responsiveness of virus-specific CD8+T cells. This results in distinct programs of activation or tolerance (functional unresponsiveness and/or physical elimination of antigen-specific cells) during acute and chronic virus infections, respectively. (iii) A successful immune response associated with definitive viral clearance requires an appropriate balance between cellular and humoral components of the immune system. We discuss the role of IFNs in influencing virus-specific T cells that determine the outcome of persistent infections.
A Replication-Competent Feline Leukemia Virus, Subgroup A (FeLV-A), Tagged with Green Fluorescent Protein Reporter Exhibits In Vitro Biological Properties Similar to Those of the Parental FeLV-A
Authors: Chang Zongli , Pan Judong , Logg Christopher , Kasahara Noriyuki , Roy-Burman Pradip 
Published: July 2002

ABSTRACT

We previously established that lymphoid tumors could be induced in cats by intradermal injection of ecotropic feline leukemia virus (FeLV), subgroup A, plasmid DNA. In preparation for in vivo experiments to study the cell-to-cell pathway for the spread of the virus from the site of inoculation, the green fluorescent protein (GFP) transgene fused to an internal ribosome entry site (IRES) was inserted after the last nucleotide of the
env
gene in the ecotropic FeLV-A Rickard (FRA) provirus. The engineered plasmid was transfected into feline fibroblast cells for production of viruses and determination of GFP expression. The virions produced were highly infectious, and the infected cells could continue to mediate strong expression of GFP after long-term propagation in culture. Similar to parental virus, the transgene-containing ecotropic virus demonstrated recombinogenic activity with endogenous FeLV sequences in feline cells to produce polytropic recombinant FeLV subgroup B-like viruses which also contained the IRES-GFP transgene in the majority of recombinants. To date, the engineered virus has been propagated in cell culture for up to 8 months without diminished GFP expression. This is the first report of a replication-competent FeLV vector with high-level and stable expression of a transgene.
Modulation of Immunity against Herpes Simplex Virus Infection via Mucosal Genetic Transfer of Plasmid DNA Encoding Chemokines
Authors: Eo Seong Kug , Lee Sujin , Chun Sangjun , Rouse Barry T. 
Published: July 2002

ABSTRACTIn this study, we examined the effects of murine chemokine DNA, as genetic adjuvants given mucosally, on the systemic and distal mucosal immune responses to plasmid DNA encoding gB of herpes simplex virus (HSV) by using the mouse model. The CC chemokines macrophage inflammatory protein 1β (MIP-1β) and monocyte chemotactic protein 1 (MCP-1) biased the immunity to the Th2-type pattern as judged by the ratio of immunoglobulin isotypes and interleukin-4 cytokine levels produced by CD4+T cells. The CXC chemokine MIP-2 and the CC chemokine MIP-1α, however, mounted immune responses of the Th1-type pattern, and such a response rendered recipients more resistant to HSV vaginal infection. In addition, MIP-1α appeared to act via the upregulation of antigen-presenting cell (APC) function and the expression of costimulatory molecules (B7-1 and B7-2), whereas MIP-2 enhanced Th1-type CD4+T-cell-mediated adaptive immunity by increasing gamma interferon secretion from activated NK cells. Our results emphasize the value of using the mucosal route to administer DNA modulators such as chemokines that function as adjuvants by regulating the activity of innate immunity. Our findings provide new insight into the value of CXC and CC chemokines, which act on different innate cellular components as the linkage signals between innate and adaptive immunity in mucosal DNA vaccination.
A Short Sequence within Domain C of Duck Carboxypeptidase D Is Critical for Duck Hepatitis B Virus Binding and Determines Host Specificity
Authors: Spangenberg Hans Christian , Lee Hong Bock , Li Jisu , Tan Fulong , Skidgel Randal , Wands Jack R. , Tong Shuping 
Published: July 2002

ABSTRACT
Virus-cell surface receptor interactions are of major interest. Hepadnaviruses are a family of partially double-stranded DNA viruses with liver tropism and a narrow host range of susceptibility to infection. At least in the case of duck hepatitis B virus (DHBV), host specificity seems controlled partly at the receptor level. The middle portion in the pre-S region of the viral large envelope protein binds specifically to duck carboxypeptidase D (DCPD) but not to its human or chicken homologue. Although domain C of DCPD is implicated in ligand binding, the exact pre-S contact site remains to be determined. We prepared and tested a panel of chimeric constructs consisting of DCPD and human carboxypeptidase D (HCPD). Our results indicate that a short region at the N terminus of domain C (residues 920 to 949) is critical to DHBV binding and is a major determinant for the host specificity of DHBV infection. Replacing this region of the DCPD molecule with its human homologue abolished the DHBV interaction, whereas introducing this DCPD sequence into HCPD conferred efficient DHBV binding. Extensive analysis of site-directed mutants revealed that both conserved and nonconserved residues were important for the pre-S interaction. There were primary sequence variations and secondary structural differences that contributed to the inability of HCPD to bind the DHBV pre-S domain.
Biochemical and Genetic Studies of the Initiation of Human Rhinovirus 2 RNA Replication: Identification of a
cis
-Replicating Element in the Coding Sequence of 2A
pro
Authors: Gerber Kinga , Wimmer Eckard , Paul Aniko V. 
Published: July 2002

ABSTRACT

We have previously shown that the RNA polymerase 3D
pol
of human rhinovirus 2 (HRV2) catalyzes the covalent linkage of UMP to the terminal protein (VPg) using poly(A) as a template (K. Gerber, E. Wimmer, and A. V. Paul, J. Virol. 75:10969–10978, 2001). The products of this in vitro reaction are VPgpU, VPgpUpU, and VPg-poly(U), the 5′ end of minus-strand RNA. In the present study we used an assay system developed for poliovirus 3D
pol
(A. V. Paul, E. Rieder, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol. 74: 10359–10370, 2000) to search for a viral sequence or structure in HRV2 RNA that would provide specificity to this reaction. We now show that a small hairpin in HRV2 RNA [
cre
(2A)], located in the coding sequence of 2A
pro
, serves as the primary template for HRV2 3D
pol
in the uridylylation of HRV2 VPg, yielding VPgpU and VPgpUpU. The in vitro reaction is strongly stimulated by the addition of purified HRV2 3CD
pro
. Our analyses suggest that HRV2 3D
pol
uses a “slide-back” mechanism during synthesis of the VPg-linked precursors. The corresponding
cis-
replicating RNA elements in the 2C
ATPase
coding region of poliovirus type 1 Mahoney (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590–4600, 2000) and VP1 of HRV14 (K. L. McKnight and S. M. Lemon, RNA 4:1569–1584, 1998) can be functionally exchanged in the assay with
cre
(2A) of HRV2. Mutations of either the first or the second A in the conserved A
1
A
2
A
3
CA sequence in the loop of HRV2
cre
(2A) abolished both viral growth and the RNA's ability to serve as a template in the in vitro VPg uridylylation reaction.
Conserved, N-Linked Carbohydrates of Human Immunodeficiency Virus Type 1 gp41 Are Largely Dispensable for Viral Replication
Authors: Johnson Welkin E. , Sauvron Jennifer M. , Desrosiers Ronald C. 
Published: July 2002

ABSTRACT
The transmembrane subunit (TM) of human immunodeficiency virus type 1 (HIV-1) envelope protein contains four well-conserved sites for the attachment of N-linked carbohydrates. To study the contribution of these N-glycans to the function of TM, we systematically mutated the sites individually and in all combinations and measured the effects of each on viral replication in culture. The mutants were derived from SHIV-KB9, a simian immunodeficiency virus/HIV chimera with an envelope sequence that originated from a primary HIV-1 isolate. The attachment site mutants were generated by replacing the asparagine codon of each N-X-S/T motif with a glutamine codon. The mobilities of the variant transmembrane proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that all four sites are utilized for carbohydrate attachment. Transfection of various cell lines with the resulting panel of mutant viral constructs revealed that the N-glycan attachment sites are largely dispensable for viral replication. Fourteen of the 15 mutants were replication competent, although the kinetics of replication varied depending on the mutant and the cell type. The four single mutants (g1, g2, g3, and g4) and all six double mutants (g12, g13, g14, g23, g24, and g34) replicated in both human and rhesus monkey T-cell lines, as well as in primary rhesus peripheral blood mononuclear cells. Three of the four triple mutants (g124, g134, and g234) replicated in all cell types tested. The triple mutant g123 replicated poorly in immortalized rhesus monkey T cells (221 cells) and did not replicate detectably in CEMx174 cells. However, at 3 weeks posttransfection of 221 cells, a variant of g123 emerged with a new N-glycan attachment site which compensated for the loss of sites 1, 2, and 3 and resulted in replication kinetics similar to those of the parental virus. The quadruple mutant (g1234) did not replicate in any cell line tested, and the g1234 envelope protein was nonfunctional in a quantitative cell-cell fusion assay. The synthesis and processing of the quadruple mutant envelope protein appeared similar in transient assays to those of the parental SHIV-KB9 envelope. Given their high degree of conservation, the four N-linked carbohydrate attachment sites on the external domain of gp41 are surprisingly dispensable for viral replication. The viral variants described in this report should prove useful for investigation of the contribution of carbohydrate moieties on gp41 to recognition by antibodies, shielding from antibody-mediated neutralization, and structure-function relationships.
Gene Expression Profile of Herpesvirus-Infected T Cells Obtained Using Immunomicroarrays: Induction of Proinflammatory Mechanisms
Authors: Mayne Michael , Cheadle Chris , Soldan Samantha S. , Cermelli Claudio , Yamano Yoshihisa , Akhyani Nahid , Nagel Jim E. , Taub Dennis D. , Becker Kevin G. , Jacobson Steven 
Published: July 2002

ABSTRACTHerpesvirus infections can frequently lead to acute inflammation, yet the mechanisms regulating this event remain poorly understood. In order to determine some of the immunological mechanisms regulated by human herpesvirus infections, we studied the gene expression profile of lymphocytes infected with human herpesvirus 6 (HHV-6) by using a novel immunomicroarray. Our nylon-based immunomicroarray contained more than 1,150 immune response-related genes and was highly consistent between experiments. Experimentally, we found that independently of the HHV-6 strain used to infect T cells, multiple proinflammatory genes were increased and anti-inflammatory genes were decreased at the mRNA and protein levels. HHV-6 strains A and B increased expression of the genes for interleukin-18 (IL-18), the IL-2 receptor, members of the tumor necrosis factor alpha superfamily receptors, mitogen-activated protein kinase, and Janus kinase signaling proteins. As reported previously, CD4 protein levels were also increased significantly. Specific type 2 cytokines, including IL-10, its receptor, and IL-14, were downregulated by HHV-6 infection and, interestingly, amyloid precursor proteins and type 1 and 2 presenilins. Thus, T cells respond to HHV-6 infection by inducing a type 1 immune response that may play a significant role in the development and progression of diseases associated with HHV-6, including pediatric, hematologic, transplant, and neurologic disorders.
SJL/J Mice Are Highly Susceptible to Infection by Mouse Adenovirus Type 1
Authors: Spindler Katherine R. , Fang Lei , Moore Martin L. , Hirsch Gwen N. , Brown Corrie C. , Kajon Adriana 
Published: July 2002

ABSTRACTMouse adenovirus type 1 (MAV-1) targets endothelial and monocyte/macrophage cells throughout the mouse. Depending on the strain of mouse and dose or strain of virus, infected mice may survive, become persistently infected, or die. We surveyed inbred mouse strains and found that for the majority tested the 50% lethal doses (LD50s) were >104.4PFU. However, SJL/J mice were highly susceptible to MAV-1, with a mean LD50of 10−0.32PFU. Infected C3H/HeJ (resistant) and SJL/J (susceptible) mice showed only modest differences in histopathology. Susceptible mice had significantly higher viral loads in the brain and spleen at 8 days postinfection than resistant mice. Infection of primary macrophages or mouse embryo fibroblasts from SJL/J and C3H/HeJ mice gave equivalent yields of virus, suggesting that a receptor difference between strains is not responsible for the susceptibility difference. When C3H/HeJ mice were subjected to sublethal doses of gamma irradiation, they became susceptible to MAV-1, with an LD50like that of SJL/J mice. Antiviral immunoglobulin G (IgG) levels were measured in susceptible and resistant mice infected by an early region 1A null mutant virus that is less virulent that wild-type virus. The antiviral IgG levels were high and similar in the two strains of mice. Taken together, these results suggest that immune response differences may in part account for differences in susceptibility to MAV-1 infection.
Induction of Hepatitis C Virus E1 Envelope Protein-Specific Immune Response Can Be Enhanced by Mutation of N-Glycosylation Sites
Authors: Fournillier A. , Wychowski C. , Boucreux D. , Baumert T. F. , Meunier J.-C. , Jacobs D. , Muguet S. , Depla E. , Inchauspé G. 
Published: July 2002

ABSTRACTDeglycosylation of viral glycoproteins has been shown to influence the number of available epitopes and to modulate immune recognition of antigens. We investigated the role played by N-glycans in the immunogenicity of hepatitis C virus (HCV) E1 envelope glycoprotein, a naturally poor immunogen. Eight plasmids were engineered, encoding E1 protein mutants in which the four N-linked glycosylation sites of the protein were mutated separately or in combination. In vitro expression studies showed an influence of N-linked glycosylation on expression efficiency, instability, and/or secretion of the mutated proteins. Immunogenicity of the E1 mutants was studied in BALB/c mice following intramuscular and intraepidermal injection of the plasmids. Whereas some mutations had no or only minor effects on the antibody titers induced, mutation of the fourth glycosylation site (N4) significantly enhanced the anti-E1 humoral response in terms of both seroconversion rates and antibody titers. Moreover, antibody induced by the N4 mutant was able to recognize HCV-like particles with higher titers than those induced by the wild-type construct. Epitope mapping indicated that the E1 mutant antigens induced antibody directed at two major domains: one, located at amino acids (aa) 313 to 332, which is known to be reactive with sera from HCV patients, and a second one, located in the N-terminal domain of E1 (aa 192 to 226). Analysis of the induced immune cellular response confirmed the induction of gamma interferon-producing cells by all mutants, albeit to different levels. These results show that N-linked glycosylation can limit the antibody response to the HCV E1 protein and reveal a potential vaccine candidate with enhanced immunogenicity.
Specific Interaction of Hepatitis C Virus Protease/Helicase NS3 with the 3′-Terminal Sequences of Viral Positive- and Negative-Strand RNA
Authors: Banerjee Rajeev , Dasgupta Asim 
Published: July 2002

ABSTRACTThe hepatitis C virus (HCV)-encoded protease/helicase NS3 is likely to be involved in viral RNA replication. We have expressed and purified recombinant NS3 (protease and helicase domains) and ΔpNS3 (helicase domain only) and examined their abilities to interact with the 3′-terminal sequence of both positive and negative strands of HCV RNA. These regions of RNA were chosen because initiation of RNA synthesis is likely to occur at or near the 3′ untranslated region (UTR). The results presented here demonstrate that NS3 (and ΔpNS3) interacts efficiently and specifically with the 3′-terminal sequences of both positive- and negative-strand RNA but not with the corresponding complementary 5′-terminal RNA sequences. The interaction of NS3 with the 3′-terminal negative strand [called 3′(−) UTR127] was specific in that only homologous (and not heterologous) RNA competed efficiently in the binding reaction. A predicted stem-loop structure present at the 3′ terminus (nucleotides 5 to 20 from the 3′ end) of the negative-strand RNA appears to be important for NS3 binding to the negative-strand UTR. Deletion of the stem-loop structure almost totally impaired NS3 (and ΔpNS3) binding. Additional mutagenesis showed that three G-C pairs within the stem were critical for helicase-RNA interaction. The data presented here also suggested that both a double-stranded structure and the 3′-proximal guanosine residues in the stem were important determinants of protein binding. In contrast to the relatively stringent requirement for 3′(−) UTR binding, specific interaction of NS3 (or ΔpNS3) with the 3′-terminal sequences of the positive-strand RNA [3′(+) UTR] appears to require the entire 3′(+) UTR of HCV. Deletion of either the 98-nucleotide 3′-terminal conserved region or the 5′ half sequence containing the variable region and the poly(U) and/or poly(UC) stretch significantly impaired RNA-protein interaction. The implication of NS3 binding to the 3′-terminal sequences of viral positive- and negative-strand RNA in viral replication is discussed.
Alfalfa Mosaic Virus Replicase Proteins P1 and P2 Interact and Colocalize at the Vacuolar Membrane
Authors: Van Der Heijden Maurice W. , Carette Jan E. , Reinhoud Poula J. , Haegi Anita , Bol John F. 
Published: July 2002

ABSTRACTReplication ofAlfalfa mosaic virus(AMV) RNAs depends on the virus-encoded proteins P1 and P2. P1 contains methyltransferase- and helicase-like domains, and P2 contains a polymerase-like domain. Coimmunoprecipitation experiments revealed an interaction between in vitro translated-P1 and P2 and showed that these proteins are present together in fractions with RNA-dependent RNA polymerase activity. A deletion analysis in the yeast two-hybrid system showed that in P1 the C-terminal sequence of 509 amino acids with the helicase domain was necessary for the interaction. In P2, the sequence of the N-terminal 241 aa was required for the interaction. In infected protoplasts, P1 and P2 colocalized at a membrane structure that was identified as the tonoplast (i.e., the membrane that surrounds the vacuoles) by using a tonoplast intrinsic protein as a marker in immunofluorescence studies. While P1 was exclusively localized on the tonoplast, P2 was found both at the tonoplast and at other locations in the cell. AsBrome mosaic virusreplication complexes have been found to be associated with the endoplasmic reticulum (M. A. Restrepo-Hartwig and P. Ahlquist, J. Virol. 70:8908–8916, 1996), viruses in the familyBromoviridaeapparently select different cellular membranes for the assembly of their replication complexes.
Enhancement of Sindbis Virus Self-Replicating RNA Vaccine Potency by Linkage of Herpes Simplex Virus Type 1 VP22 Protein to Antigen
Authors: Cheng Wen-Fang , Hung Chien-Fu , Chai Chee-Yin , Hsu Keng-Fu , He Liangmai , Ling Morris , Wu T.-C. 
Published: July 2002

ABSTRACTRecently, self-replicating and self-limiting RNA vaccines (RNA replicons) have emerged as an important form of nucleic acid vaccines. Self-replicating RNA eventually causes lysis of transfected cells and does not raise the concern associated with naked DNA vaccines of integration into the host genome. This is particularly important for development of vaccines targeting proteins that are potentially oncogenic. However, the potency of RNA replicons is significantly limited by their lack of intrinsic ability to spread in vivo. The herpes simplex virus type 1 protein VP22 has demonstrated the remarkable property of intercellular transport and provides the opportunity to enhance RNA replicon vaccine potency. We therefore created a novel fusion of VP22 with a model tumor antigen, human papillomavirus type 16 E7, in a Sindbis virus RNA replicon vector. The linkage of VP22 with E7 resulted in a significant enhancement of E7-specific CD8+T-cell activities in vaccinated mice and converted a less effective RNA replicon vaccine into one with significant potency against E7-expressing tumors. These results indicate that fusion of VP22 to an antigen gene may greatly enhance the potency of RNA replicon vaccines.
Infrequent Occurrence of Natural Mutations in the pp65495–503Epitope Sequence Presented by the HLA A∗0201 Allele among Human Cytomegalovirus Isolates
Authors: Zaia John A. , Gallez-Hawkins Ghislaine , Li Xiuli , Yao Zhi-Qiang , Lomeli Norma , Molinder Karen , La Rosa Corinna , Diamond Don J. 
Published: July 2002

ABSTRACTTo determine if mutations of an immunodominant HLA-restricted cytomegalovirus (CMV) peptide sequence occur in nature, the sequence corresponding to the HLA A∗0201-specific peptide CMVpp65495–503was determined in 50 human CMV isolates. Rare mutations were detected; 6 of 50 were silent mutations at the amino terminus of the peptide, while 3 of 50 were mutations of the native methionine residue to isoleucine (M499I). The observed M499I mutation in three isolates decreased cytolytic targeting.