bluuebunny/crossref_metadata_2025 huggingface dataset:
Chimeric Nectin1-Poliovirus Receptor Molecules Identify a Nectin1 Region Functional in Herpes Simplex Virus Entry
Authors: Cocchi Francesca , Lopez Marc , Dubreuil Patrice , Campadelli Fiume Gabriella , Menotti Laura
Published: July 2002

Human nectin1 (hNectin1), an adhesion molecule belonging to the nectin family of the immunoglobulin superfamily, mediates entry of herpes simplex virus (HSV) into cells. The hNectin1 domain that mediates virus entry into cells and also binds glycoprotein D (gD) has been localized to the first N-terminal V-type domain. The poliovirus receptor (PVR) is a structural homolog to nectins, but it cannot function as an HSV entry receptor. hNectin1-PVR chimeras were constructed to functionally locate the site on hNectin1 involved in HSV entry (HSV entry site). The epitope recognized by monoclonal antibody (MAb) R1.302, which is able to block HSV entry, was also located. The chimeric receptors were designed to preserve the overall structure of the V domain. The HSV entry activity mapped entirely to the hNectin1 portion located between residues 64 and 94 (64-94), likely to encode the C, C′, and C" β-strands and intervening loops. In turn, this site consisted of two portions: one with low-level basal activity for HSV entry (77-94), and one immediately upstream (residues 64 to 76) which greatly enhanced the HSV entry activity of the downstream region. The gD-binding site mapped substantially to the same site, whereas the MAb R1.302 epitope also required a further downstream portion (95-102). The involvement of the 64-76 portion is at difference with previous indirect mapping results that were based on competitive binding studies (C. Krummenacher et al., J. Virol. 74:10863–10872, 2000). The A, A′, B, D, E, F, and G β-strands and intervening loops did not appear to play any role in HSV entry. According to the predicted three-dimensional structure of PVR, the C C′ C" site is located peripherally in the V domain and very likely represents an accessible portion at the cell surface.
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

Under 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

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

In 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

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

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

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

Herpesvirus 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

Mouse 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

Deglycosylation 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

The 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

Replication 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

Recently, 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

To 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.
Cross-Reactive, Cell-Mediated Immunity and Protection of Chickens from Lethal H5N1 Influenza Virus Infection in Hong Kong Poultry Markets
Authors: Seo Sang Heui , Webster Robert G.
Published: July 2002

In 1997, avian H5N1 influenza virus transmitted from chickens to humans resulted in 18 confirmed infections. Despite harboring lethal H5N1 influenza viruses, most chickens in the Hong Kong poultry markets showed no disease signs. At this time, H9N2 influenza viruses were cocirculating in the markets. We investigated the role of H9N2 influenza viruses in protecting chickens from lethal H5N1 influenza virus infections. Sera from chickens infected with an H9N2 influenza virus did not cross-react with an H5N1 influenza virus in neutralization or hemagglutination inhibition assays. Most chickens primed with an H9N2 influenza virus 3 to 70 days earlier survived the lethal challenge of an H5N1 influenza virus, but infected birds shed H5N1 influenza virus in their feces. Adoptive transfer of T lymphocytes or CD8+T cells from inbred chickens (B2/B2) infected with an H9N2 influenza virus to naive inbred chickens (B2/B2) protected them from lethal H5N1 influenza virus. In vitro cytotoxicity assays showed that T lymphocytes or CD8+T cells from chickens infected with an H9N2 influenza virus recognized target cells infected with either an H5N1 or H9N2 influenza virus in a dose-dependent manner. Our findings indicate that cross-reactive cellular immunity induced by H9N2 influenza viruses protected chickens from lethal infection with H5N1 influenza viruses in the Hong Kong markets in 1997 but permitted virus shedding in the feces. Our findings are the first to suggest that cross-reactive cellular immunity can change the outcome of avian influenza virus infection in birds in live markets and create a situation for the perpetuation of H5N1 influenza viruses.
Antibody Binding and Neutralization of Primary and T-Cell Line-Adapted Isolates of Human Immunodeficiency Virus Type 1
Authors: York Joanne , Follis Kathryn E. , Trahey Meg , Nyambi Phillipe N. , Zolla-Pazner Susan , Nunberg Jack H.
Published: July 2002

The relative resistance of human immunodeficiency virus type 1 (HIV-1) primary isolates (PIs) to neutralization by a wide range of antibodies remains a theoretical and practical barrier to the development of an effective HIV vaccine. One model to account for the differential neutralization sensitivity between Pls and laboratory (or T-cell line-adapted [TCLA]) strains of HIV suggests that the envelope protein (Env) complex is made more accessible to antibody binding as a consequence of adaptation to growth in established cell lines. Here, we revisit this question using genetically related PI and TCLA viruses and molecularly clonedenvgenes. By using complementary techniques of flow cytometry and virion binding assays, we show that monoclonal antibodies targeting the V3 loop, CD4-binding site, CD4-induced determinant of gp120, or the ectodomain of gp41 bind equally well to PI and TCLA Env complexes, despite large differences in neutralization outcome. The data suggest that the differential neutralization sensitivity of PI and TCLA viruses may derive not from differences in the initial antibody binding event but rather from differences in the subsequent functioning of the PI and TCLA Envs during virus entry. An understanding of these as yet undefined differences may enhance our ability to generate broadly neutralizing HIV vaccine immunogens.
Lentivirus Vector-Mediated Hematopoietic Stem Cell Gene Transfer of Common Gamma-Chain Cytokine Receptor in Rhesus Macaques
Authors: An Dong Sung , Kung Sam K. P. , Bonifacino Aylin , Wersto Robert P. , Metzger Mark E. , Agricola Brian A. , Mao Si Hua , Chen Irvin S. Y. , Donahue Robert E.
Published: July 2002

Nonhuman primate model systems of autologous CD34
+
cell transplant are the most effective means to assess the safety and capabilities of lentivirus vectors. Toward this end, we tested the efficiency of marking, gene expression, and transplant of bone marrow and peripheral blood CD34
+
cells using a self-inactivating lentivirus vector (CS-Rh-MLV-E) bearing an internal murine leukemia virus long terminal repeat derived from a murine retrovirus adapted to replicate in rhesus macaques. In vitro cytokine stimulation was not required to achieve efficient transduction of CD34
+
cells resulting in marking and gene expression of the reporter gene encoding enhanced green fluorescent protein (EGFP) following transplant of the CD34
+
cells. Monkeys transplanted with mobilized peripheral blood CD34
+
cells resulted in EGFP expression in 1 to 10% of multilineage peripheral blood cells, including red blood cells and platelets, stable for 15 months to date. The relative level of gene expression utilizing this vector is 2- to 10-fold greater than that utilizing a non-self-inactivating lentivirus vector bearing the cytomegalovirus immediate-early promoter. In contrast, in animals transplanted with autologous bone marrow CD34
+
cells, multilineage EGFP expression was evident initially but diminished over time. We further tested our lentivirus vector system by demonstrating gene transfer of the human common gamma-chain cytokine receptor gene (γ
c
), deficient in X-linked SCID patients and recently successfully used to treat disease. Marking was 0.42 and .001 HIV-1 vector DNA copy per 100 cells in two animals. To date, all EGFP- and γ
c
-transplanted animals are healthy. This system may prove useful for expression of therapeutic genes in human hematopoietic cells.
Genetic Analysis of a Poliovirus/Hepatitis C Virus Chimera: New Structure for Domain II of the Internal Ribosomal Entry Site of Hepatitis C Virus
Authors: Zhao Wei Dong , Wimmer Eckard
Published: July 2002

Internal ribosomal entry sites (IRESs) of certain plus-strand RNA viruses direct cap-independent initiation of protein synthesis both in vitro and in vivo, as can be shown with artificial dicistronic mRNAs or with chimeric viral genomes in which IRES elements were exchanged from one virus to another. Whereas IRESs of picornaviruses can be readily analyzed in the context of their cognate genome by genetics, the IRES of hepatitis C virus (HCV), a
Hepacivirus
belonging to
Flaviviridae
, cannot as yet be subjected to such analyses because of difficulties in propagating HCV in tissue culture or in experimental animals. This enigma has been overcome by constructing a poliovirus (PV) whose translation is controled by the HCV IRES. Within the PV/HCV chimera, the HCV IRES has been subjected to systematic 5′ deletion analyses to yield a virus (P/H710-d40) whose replication kinetics match that of the parental poliovirus type 1 (Mahoney). Genetic analyses of the HCV IRES in P/H710-d40 have confirmed that the 5′ border maps to domain II, thereby supporting the validity of the experimental approach applied here. Additional genetic experiments have provided evidence for a novel structural region within domain II. Arguments that the phenotypes observed with the mutant chimera relate solely to impaired genome replication rather than deficiencies in translation have been dispelled by constructing novel dicistronic poliovirus replicons with the gene order [PV]cloverleaf-[HCV]IRES-Δcore-
R-Luc
-[PV]IRES-
F-Luc
-P2,3-3′NTR, which have allowed the measurement of HCV IRES-dependent translation independently from the replication of the replicon RNA.
Human Immunodeficiency Virus Type 1 Particles Pseudotyped with Envelope Proteins That Fuse at Low pH No Longer Require Nef for Optimal Infectivity
Authors: Chazal Nathalie , Singer Gregory , Aiken Christopher , Hammarskjöld Marie-Louise , Rekosh David
Published: July 2002

We have investigated the effects of Nef on infectivity in the context of various viral envelope proteins. These experiments were performed with a minimal vector system where Nef is the only accessory protein present. Our results support the hypothesis that the route of entry influences the ability of Nef to enhance human immunodeficiency virus (HIV) infectivity. We show that HIV particles pseudotyped with Ebola virus glycoprotein or vesicular stomatitis virus glycoprotein (VSV-G), which fuse at low pH, do not require Nef for optimal infectivity. In contrast, Nef significantly enhances the infectivity of virus particles that contain envelope proteins that fuse at neutral pH (CCR5-dependent HIV Env, CXCR4-dependent HIV Env, or amphotropic murine leukemia virus Env). In addition, our results demonstrate that virus particles containing mixed CXCR4-dependent HIV and VSV-G envelope proteins show a conditional requirement for Nef for optimal infectivity, depending on which protein is allowed to facilitate entry.
Inhibition of Host Transcription by Vesicular Stomatitis Virus Involves a Novel Mechanism That Is Independent of Phosphorylation of TATA-Binding Protein (TBP) or Association of TBP with TBP-Associated Factor Subunits
Authors: Yuan Hang , Puckett Shelby , Lyles Douglas S.
Published: July 2002

The matrix (M) protein of vesicular stomatitis virus (VSV) is a potent inhibitor in vivo of transcription by all three host RNA polymerases (RNAP). In the case of host RNA polymerase II (RNAPII), the inhibition is due to lack of activity of the TATA-binding protein (TBP), which is a subunit of the basal transcription factor TFIID. Despite the potency of M protein-induced inhibition in vivo, experiments presented here show that M protein cannot directly inactivate TFIID in vitro. Addition of M protein to nuclear extracts from uninfected cells did not inhibit transcription activity, indicating that the inhibition is indirect and is mediated through host factors. The host factors that are known to regulate TBP activity include phosphorylation by host kinases and association with different TBP-associated factor (TAF) subunits. However, TBP in VSV-infected cells was found to be assembled normally with its TAF subunits, as shown by ion exchange high-pressure liquid chromatography and sedimentation velocity analysis. A normal pattern of phosphorylation of TBP in VSV-infected cells was also observed by pH gradient gel electrophoresis. Collectively, these data indicate that M protein inactivates TBP activity in RNAPII-dependent transcription by a novel mechanism, since the known mechanisms for regulating TBP activity cannot account for the inhibition.
Association of Herpes Simplex Virus Type 1 ICP8 and ICP27 Proteins with Cellular RNA Polymerase II Holoenzyme
Authors: Zhou Changhong , Knipe David M.
Published: July 2002

Herpes simplex virus 1 (HSV-1) infection causes the shutoff of host gene transcription and the induction of a transcriptional program of viral gene expression. Cellular RNA polymerase II is responsible for transcription of all the viral genes, but several viral proteins stimulate viral gene transcription. ICP4 is required for all delayed-early and late gene transcription, ICP0 stimulates transcription of viral genes, and ICP27 stimulates expression of some early genes and transcription of at least some late viral genes. The early DNA-binding protein, ICP8, also stimulates late gene transcription. We therefore investigated which HSV proteins interact with RNA polymerase II. Using immunoprecipitation and Western blotting methods, we observed the coprecipitation of ICP27 and ICP8 with RNA polymerase II holoenzyme. The association of ICP27 with RNA polymerase II was detectable as early as 3 h postinfection, while ICP8 association became evident by 5 h postinfection, and the association of both was independent of viral DNA synthesis. Infections with
ICP27
gene mutant viruses revealed that ICP27 is required for the association of ICP8 with RNA polymerase II, while studies with
ICP8
gene deletion mutants showed no apparent role for ICP8 in the association of ICP27 with RNA polymerase II. The association of ICP27 and ICP8 with RNA polymerase II holoenzyme appeared to be independent of nucleic acids. We hypothesize that the interaction of ICP27 with RNA polymerase II holoenzyme reflects its role in stimulating early and late gene expression and/or its role in inhibiting host transcription and that the interaction of ICP8 with RNA polymerase II holoenzyme reflects its role in stimulating late gene transcription.
A Variable Region 3 (V3) Mutation Determines a Global Neutralization Phenotype and CD4-Independent Infectivity of a Human Immunodeficiency Virus Type 1 Envelope Associated with a Broadly Cross-Reactive, Primary Virus-Neutralizing Antibody Response
Authors: Zhang Peng Fei , Bouma Peter , Park Eun Ju , Margolick Joseph B. , Robinson James E. , Zolla-Pazner Susan , Flora Michael N. , Quinnan Gerald V.
Published: July 2002

The human serum human immunodeficiency virus type 1 (HIV-1)-neutralizing serum 2 (HNS2) neutralizes many primary isolates of different clades of HIV-1, and virus expressing envelope from the same donor, clone R2, is neutralized cross-reactively by HIV-immune human sera. The basis for this cross-reactivity was investigated. It was found that a rare mutation in the proximal limb of variable region 3 (V3), 313-4 PM, caused virus pseudotyped with the R2 envelope to be highly sensitive to neutralization by monoclonal antibodies (MAbs) directed against conformation-sensitive epitopes at the tip of the V3 loop, such as 19b, and moderately sensitive to MAbs against CD4 binding site (CD4bs) and CD4-induced (CD4i) epitopes, soluble CD4 (sCD4), and HNS2. In addition, introduction of this sequence by mutagenesis caused enhanced sensitivity to neutralization by 19b, anti-CD4i MAb, and HNS2 in three other primary HIV-1 envelopes and by anti-CD4bs MAb and sCD4 in one of the three. The 313-4 PM sequence also conferred increased infectivity for CD4+CCR5+cells and the ability to infect CCR5+cells upon all of these four and two of these four HIV-1 envelopes, respectively. Neutralization of R2 by HNS2 was substantially inhibited by the cyclized R2 V3 35-mer synthetic peptide. Similarly, the peptide also had some lesser efficacy in blocking neutralization of R2 by other sera or of neutralization of other primary viruses by HNS2. Together, these results indicate that the unusual V3 mutation in the R2 clone accounts for its uncommon neutralization sensitivity phenotype and its capacity to mediate CD4-independent infection, both of which could relate to immunogenicity and the neutralizing activity of HNS2. This is also the first primary HIV-1 isolate envelope glycoprotein found to be competent for CD4-independent infection.
RelB Nuclear Translocation Mediated by C-Terminal Activator Regions of Epstein-Barr Virus-Encoded Latent Membrane Protein 1 and Its Effect on Antigen-Presenting Function in B Cells
Authors: Pai Saparna , O'Sullivan Brendan J. , Cooper Leanne , Thomas Ranjeny , Khanna Rajiv
Published: July 2002

Previous studies have shown that Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) is uniquely able to up-regulate the expression of the peptide transporters (referred to as TAP-1 and TAP-2) and major histocompatibility complex (MHC) class I in Burkitt's lymphoma (BL) cell lines. This up-regulation is often accompanied by a restoration of antigen-presenting function as measured by the ability of these cells to present endogenously expressed viral antigen to cytotoxic T lymphocytes. Here we show that the expression of LMP1 resulted in up-regulation and nuclear translocation of RelB that were coincident with increased expression of MHC class I in BL cells. Deletion of the C-terminal activator regions (CTARs) of LMP1 significantly impaired the abilities of LMP1 to translocate RelB into the nucleus and to up-regulate the expression of antigen-processing genes. Further analysis with single-point mutations within the CTARs confirmed that the residues critical for NF-κB activation directly contribute to antigen-processing function regulation in BL cells. This LMP1-mediated effect was blocked following expression of either dominant negative IκBα S32/36A, an NF-κB inhibitor, or antisense RelB. These observations indicate that upregulation of antigen-presenting function in B cells mediated by LMP1 is signaled through the NF-κB subunit RelB. The data provide a mechanism by which LMP1 modulates immunogenicity of Epstein-Barr virus-infected normal and malignant cells.
Expression of Herpes Simplex Virus ICP0 Inhibits the Induction of Interferon-Stimulated Genes by Viral Infection
Authors: Eidson Kasey M. , Hobbs William E. , Manning Brian J. , Carlson Paul , DeLuca Neal A.
Published: July 2002

The herpes simplex virus type 1 (HSV-1) mutantd109 does not express any of the immediate-early (IE) proteins and persists in cells for a prolonged length of time. As has been shown by Nicholl et al. (J. Gen. Virol. 81:2215-2218, 2000) and Mossman et al. (J. Virol. 75:750-758, 2001) using other mutants defective for IE gene expression, infection withd109 induced the expression of a number of interferon-stimulated genes. Induction of these genes was significantly greater at multiplicities of infection (MOI) of 10 PFU/cell or greater, and the resulting antiviral effect was only seen at MOIs greater than 10 PFU/cell. Using mutants defective for sets of IE genes established that the lack of ICP0 expression was necessary for high levels of interferon-stimulated gene expression in HEL cells. The induction of interferon-stimulated genes byd109 could also be inhibited by infection with an E1−:E3−:E4− adenovirus expressing levels of ICP0 that are comparable to those expressed within the first hour of wild-type virus infection. Lastly, the addition of the proteasome inhibitor MG132 to cells infected with a mutant that expresses ICP0,d106, also resulted in the induction of interferon-stimulated genes. Thus, ICP0 may function through the proteasome very early in HSV infection to inhibit a cellular antiviral response induced by the virion.
Distinct Roles of Adenovirus Vector-Transduced Dendritic Cells, Myoblasts, and Endothelial Cells in Mediating an Immune Response against a Transgene Product
Authors: Mercier Stéphanie , Gahéry-Segard Hanne , Monteil Martine , Lengagne Renée , Guillet Jean-Gérard , Eloit Marc , Denesvre Caroline
Published: July 2002

Adenovirus-mediated gene delivery via the intramuscular route efficiently promotes an immune response against the transgene product. In this study, a recombinant adenovirus vector encoding β-galactosidase (AdβGal) was used to transduce dendritic cells (DC), which are antigen-presenting cells, as well as myoblasts and endothelial cells (EC), neither of which present antigens. C57BL/6 mice received a single intramuscular injection of AdβGal-transduced DC, EC, or myoblasts and were then monitored for anti-β-galactosidase (anti-β-Gal) antibody production, induction of gamma interferon-secreting CD8+T cells, and protection against melanoma tumor cells expressing β-Gal. While all transduced cell types were able to elicit an antibody response against the transgene product, the specific isotypes were distinct, with exclusive production of immunoglobulin G2a (IgG2a) antibodies following injection of transduced DC and EC versus equivalent IgG1 and IgG2a responses in mice inoculated with transduced myoblasts. Transduced DC induced a strong ex vivo CD8+T-cell response at a level of 50% of the specific response obtained with the AdβGal control. In contrast, this response was 6- to 10-fold-lower in animals injected with transduced myoblasts and EC. Accordingly, only animals injected with transduced DC were protected against a β-Gal tumor challenge. Thus, in order to induce a strong and protective immune response to an adenovirus-encoded transgene product, it is necessary to transduce cells of dendritic lineage. Importantly, it will be advantageous to block the transduction of DC for adenovirus-based gene therapy strategies.
Photocolouration of 2,4,4,6-Tetraaryl-4H-thiopyrans: A Semiempirical Quantum Chemical Study
Authors: Böhm Stanislav , Šebek Pavel , Nešpůrek Stanislav , Kuthan Josef
Published: July 2002

Molecular geometries of 4H-thiopyrans Ia, Ib and their possible photoisomers IVa - VIIa and IVb - VIIb were optimized by the AM1 and PM3 method and used for the calculation of electronic absorption spectra by the CNDO/S-CI procedure. Comparison of the theoretical data with experimental UV-VIS absorption spectra made it possible to postulate two probable mechanisms of the photocolouration of Ia, Ib caused by sunlight or UV-illumination.
Amperometric Enzyme Electrodes for Substrates of Immobilized Pyranose Oxidase
Authors: Petřivalský Marek , Skládal Petr , Macholán Lumír , Volc Jindřich
Published: July 2002

Two kinds of biosensors for the determination of pyranose oxidase substrates were developed, based on the detection of evolving hydrogen peroxide on a platinum or platinized graphite electrode at +650 or +400 mV, respectively. The membranes consisted of enzyme immobilized by covalent bonds on nylon net and were stable for 8 months of dry storage at 4 °C. In addition to D-glucose, low concentrations of D-xylose, D-galactose and L-sorbose can also be measured with the biosensor. The shift of the optimum pH of the immobilized enzyme to the alkaline region (8.0 - 9.5) is convenient for the borate buffer medium which extends the linear concentration region of the biosensor to 15 mmol l-1 for D-galactose, 30 mmol l-1 for D-xylose and 30 mmol l-1 for maltose. L-Sorbose provides no response up to a concentration of 10 mmol l-1 in 0.05 M borate and up to a concentration of 30 mmol l-1 in 0.2 M borate at pH 9.2. Interfering D-glucose was eliminated up to 2.5 mmol l-1 by means of an enzyme pre-membrane with immobilized hexokinase. The effect of ascorbate was eliminated, up to 75 mmol l-1, by using a cellulose acetate electrostatic barrier. D-Galactose, however, decreases the sensor response to D-glucose.
Continuous Precipitation of Lead Iodide
Authors: Žáček Stanislav , Nývlt Jaroslav
Published: July 2002

Lead iodide was precipitated from aqueous solutions of 0.015 - 0.1 M Pb(NO3)2 and 0.03 - 0.2 M KI in the equimolar ratio using a laboratory model of a stirred continuous crystallizer at 22 °C. After reaching the steady state, the PbI2 crystal size distribution was measured sedimentometrically and the crystallization kinetics was evaluated based on the mean particle size. Both the linear crystal growth rate and the nucleation rate depend on the specific output of the crystallizer. The system crystallization constant either points to a significant effect of secondary nucleation by the mechanism of contact of the crystals with the stirrer blade, or depends on the concentrations of the components added due to the micromixing mechanism.
Determination of 2-Nitrophenol, 4-Nitrophenol, 2-Methoxy-5-nitrophenol, and 2,4-Dinitrophenol by Differential Pulse Voltammetry and Adsorptive Stripping Voltammetry
Authors: Barek Jiří , Ebertová Hana , Mejstřík Viktor , Zima Jiří
Published: July 2002

The behaviour of the title substances, which are components of the commercial plant growth stimulator Sviton, in differential pulse voltammetry at a hanging mercury drop electrode was studied and the optimum conditions were established for their quantitation over the concentration region of 1 .10-7 to 1 .10-5 mol l-1. Additional decrease in the limit of determination was achieved in adsorptive stripping voltammetry, by which 4-nitrophenol and 2-nitrophenol can be determined within the concentration regions of 2 .10-10 to 1 .10-7 and 2 .10-8 to 1. 10-7 mol l-1, respectively, and 2,4-dinitrophenol and 2-methoxy-5-nitrophenol within the region of 2 .10-9 to 1 .10-7 mol l-1.