bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of an Antigenic Adenovirus Epitope in a Group B Coxsackievirus
Authors: Höfling Katja , Tracy Steven , Chapman Nora , Kim Kyung-Soo , Smith Leser J.
Published: July 2002

Group B coxsackieviruses (CVB) cause human myocarditis, while human adenovirus type 2 (Ad2) is implicated as an agent of this disease. The L1 loop of the Ad2 hexon protein has been demonstrated to be antigenic in rabbits. To evaluate the feasibility of a multivalent vaccine strain against the CVB and Ad2, we cloned the sequence encoding the Ad2 hexon L1 loop, flanked by dissimilar sequences encoding the protease 2A (2Apro) recognition sites, into the genome of an attenuated strain of CVB type 3 (CVB3/0) at the junction of 2Apro and the capsid protein 1D. Progeny virus (CVB3-PL2-Ad2L1) was obtained following transfection of the construct into HeLa cells. Replication of CVB3-PL2-Ad2L1 in diverse cell cultures demonstrated that the yield of the chimeric virus was between 0.5 to 2 log units less than the parental strain. Western blot analyses of the CVB3 capsid protein 1D in CVB3-PL2-Ad2L1-infected HeLa cells demonstrated production of the expected capsid protein. Viral proteins were detected earlier and in approximately fourfold greater amounts in CVB3-PL2-Ad2L1-infected HeLa cells than in CVB3/0-infected cells. Cleavage of the CVB3-PL2-Ad2L1 polyprotein by 2Apro was slowed, accompanied by an accumulation of the fusion 1D-L1 loop protein. Reverse transcription-PCR sequence analysis of CVB3-PL2-Ad2L1 RNA demonstrated that the Ad2 hexon polypeptide coding sequence was maintained in the chimeric viral genome through at least 10 passages in HeLa cells. Mice inoculated with CVB3-PL2-Ad2L1 demonstrated a brief viremia with no replication detectable in the heart but prolonged replication of virus in the pancreas in the absence of pathologic changes in either organ. CVB3-PL2-Ad2L1 induced binding and neutralizing anti-Ad2 antibodies, in addition to antibodies against CVB3 in mice. CVB3-PL2-Ad2L1 was used to challenge mice previously inoculated with CVB3/0 and with preexisting anti-CVB3 neutralizing-antibody titers; anti-Ad2 neutralizing and binding antibodies were induced in these mice at higher levels than in mice without anti-CVB3 immunity. The data demonstrate that a CVB vector can stably express an antigenic polypeptide of Ad2 from within the CVB open reading frame that results in the induction of protective immune responses against both viruses.
Identification of a Key Target Sequence To Block Human Immunodeficiency Virus Type 1 Replication within the
gag-pol
Transframe Domain
Authors: Sei Shizuko , Yang Quan-en , O'Neill Dennis , Yoshimura Kazuhisa , Nagashima Kunio , Mitsuya Hiroaki
Published: July 2002

Although the full sequence of the human immunodeficiency virus type 1 (HIV-1) genome has been known for more than a decade, effective genetic antivirals have yet to be developed. Here we show that, of 22 regions examined, one highly conserved sequence (ACTCTTTGGCAACGA) near the 3′ end of the HIV-1
gag-pol
transframe region, encoding viral protease residues 4 to 8 and a C-terminal Vpr-binding motif of p6
Gag
protein in two different reading frames, can be successfully targeted by an antisense peptide nucleic acid oligomer named PNA
PR2
. A disrupted translation of
gag-pol
mRNA induced at the PNA
PR2
-annealing site resulted in a decreased synthesis of Pr160
Gag-Pol
polyprotein, hence the viral protease, a predominant expression of Pr55
Gag
devoid of a fully functional p6
Gag
protein, and the excessive intracellular cleavage of Gag precursor proteins, hindering the processes of virion assembly. Treatment with PNA
PR2
abolished virion production by up to 99% in chronically HIV-1-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates with the multidrug-resistant phenotype. This particular segment of the
gag-pol
transframe gene appears to offer a distinctive advantage over other regions in invading viral structural genes and restraining HIV-1 replication in infected cells and may potentially be exploited as a novel antiviral genetic target.
Octamerization Enables Soluble CD46 Receptor To Neutralize Measles Virus In Vitro and In Vivo
Authors: Christiansen Dale , Devaux Patricia , Réveil Brigitte , Evlashev Alexey , Horvat Branka , Lamy Josette , Rabourdin-Combe Chantal , Cohen Jacques H. M. , Gerlier Denis
Published: July 2002

A chimeric fusion protein encompassing the CD46 ectodomain linked to the C-terminal part of the C4b binding protein (C4bp) α chain (sCD46-C4bpα) was produced in eukaryotic cells. This protein, secreted as a disulfide-linked homo-octamer, was recognized by a panel of anti-CD46 antibodies with varying avidities. Unlike monomeric sCD46, the octameric sCD46-C4bpα protein was devoid of complement regulatory activity. However, sCD46-C4bpα was able to bind to the measles virus hemagglutinin protein expressed on murine cells with a higher avidity than soluble monomeric sCD46. Moreover, the octameric sCD46-C4bpα protein was significantly more efficient than monomeric sCD46 in inhibiting virus binding to CD46, in blocking virus induced cell-cell fusion, and in neutralizing measles virus in vitro. In addition, the octameric sCD46-C4bpα protein, but not the monomeric sCD46, fully protected CD46 transgenic mice against a lethal intracranial measles virus challenge.
Retrovirus Vectors Bearing Jaagsiekte Sheep Retrovirus Env Transduce Human Cells by Using a New Receptor Localized to Chromosome 3p21.3
Authors: Rai Sharath K. , DeMartini James C. , Miller A. Dusty
Published: July 2002

Jaagsiekte sheep retrovirus (JSRV) is a type D retrovirus associated with a contagious lung tumor of sheep, ovine pulmonary carcinoma. Other than sheep, JSRV is known to infect goats, but there is no evidence of human infection. Until now it has not been possible to study the host range for JSRV because of the inability to grow this virus in culture. Here we show that the JSRV envelope protein (Env) can be used to pseudotype Moloney murine leukemia virus (MoMLV)-based retrovirus vectors and that such vectors can transduce human cells in culture. We constructed hybrid retrovirus packaging cells that express the JSRV Env and the MoMLV Gag-Pol proteins and can produce JSRV-pseudotype vectors at titers of up to 10
6
alkaline phosphatase-positive focus-forming units/ml. Using this high-titer virus, we have studied the host range for JSRV, which includes sheep, human, monkey, bovine, dog, and rabbit cells but not mouse, rat, or hamster cells. Considering the inability of the JSRV-pseudotype vector to transduce hamster cells, we used the hamster cell line-based Stanford G3 panel of whole human genome radiation hybrids to phenotypically map the JSRV receptor (
JVR
) gene within the p21.3 region of human chromosome 3. JVR is likely a new retrovirus receptor, as none of the previously identified retrovirus receptors localizes to the same position. Several chemokine receptors that have been shown to serve as coreceptors for lentivirus infection are clustered in the same region of chromosome 3; however, careful examination shows that the JSRV receptor does not colocalize with any of these genes.
Coreceptor Competition for Association with CD4 May Change the Susceptibility of Human Cells to Infection with T-Tropic and Macrophagetropic Isolates of Human Immunodeficiency Virus Type 1
Authors: Lee Shirley , Lapham Cheryl K. , Chen Hong , King Lisa , Manischewitz Jody , Romantseva Tatiana , Mostowski Howard , Stantchev Tzanko S. , Broder Christopher C. , Golding Hana
Published: July 2002

The chemokine receptors CCR5 and CXCR4 were found to function in vivo as the principal coreceptors for M-tropic and T-tropic human immunodeficiency virus (HIV) strains, respectively. Since many primary cells express multiple chemokine receptors, it was important to determine if the efficiency of virus-cell fusion is influenced not only by the presence of the appropriate coreceptor (CXCR4 or CCR5) but also by the levels of other coreceptors expressed by the same target cells. We found that in cells with low to medium surface CD4 density, coexpression of CCR5 and CXCR4 resulted in a significant reduction in the fusion with CXCR4 domain (X4) envelope-expressing cells and in their susceptibility to infection with X4 viruses. The inhibition could be reversed either by increasing the density of surface CD4 or by antibodies against the N terminus and second extracellular domains of CCR5. In addition, treatment of macrophages with a combination of anti-CCR5 antibodies or β-chemokines increased their fusion with X4 envelope-expressing cells. Conversely, overexpression of CXCR4 compared with CCR5 inhibited CCR5-dependent HIV-dependent fusion in 3T3.CD4.401 cells. Thus, coreceptor competition for association with CD4 may occur in vivo and is likely to have important implications for the course of HIV type 1 infection, as well as for the outcome of coreceptor-targeted therapies.
The Hepatitis B Virus Core Promoter Is Strongly Activated by the Liver Nuclear Receptor Fetoprotein Transcription Factor or by Ectopically Expressed Steroidogenic Factor 1
Authors: Gilbert Stéphane , Galarneau Luc , Lamontagne Alain , Roy Sylvie , Bélanger Luc
Published: July 2002

Orphan nuclear receptor fetoprotein transcription factor (FTF) was previously identified as a specific regulator of the α
1
-fetoprotein gene during early liver development and in response to hormonal signals (L. Galarneau, J.-F. Paré, D. Allard, D. Hamel, L. Lévesque, J. D. Tugwood, S. Green, and L. Bélanger, Mol. Cell. Biol. 16:3853–3865, 1996). Here we report a functional analysis of FTF interactions with the hepatitis B virus (HBV) nucleocapsid promoter. DNA-protein-binding assays show that the HBV core promoter contains two high-affinity FTF-binding sites and a third, lower-affinity site shared with other receptors. Transfections in HepG2, Hep3B, and PLC/PRF/5 hepatoma cells using chloramphenicol acetyltransferase reporter genes with the nucleocapsid promoter linked or not linked to enhancer I indicate that FTF is a potent activator of the HBV core promoter, more efficient than HNF4α, HNF3α, HNF3β, or C/EBPα. Steroidogenic factor 1, a close FTF homolog which binds to the same DNA motif and is expressed ectopically in HepG2 cells, seems to be an even stronger inducer than FTF. Point mutations of the FTF-binding sites indicate direct FTF activatory effects on the core promoter and the use of both high-affinity sites for productive interaction between the core promoter and enhancer I. Coexpression assays further indicate that FTF and HNF4α are the most efficient partners for coactivation of the pregenomic core promoter, which may largely account for the hepatic tropism and the early amplification of HBV infection. Carboxy terminus-truncated FTF behaves as a dominant negative mutant to compete all three FTF sites and strongly deactivate core promoter interactions with enhancer I; this suggests possible new ways to interfere with HBV infection.
Simian Immunodeficiency Virus Utilizes Human and Sooty Mangabey but Not Rhesus Macaque STRL33 for Efficient Entry
Authors: Pöhlmann Stefan , Lee Benhur , Meister Silke , Krumbiegel Mandy , Leslie George , Doms Robert W. , Kirchhoff Frank
Published: July 2002

It has been established that many simian immunodeficiency virus (SIV) isolates utilize the orphan receptors GPR15 and STRL33 about as efficiently as the chemokine receptor CCR5 for entry into target cells. Most studies were performed, however, with coreceptors of human origin. We found that SIV from captive rhesus macaques (SIVmac) can utilize both human and simian CCR5 and GPR15 with comparable efficiencies. Strikingly, however, only human STRL33 (huSTRL33), not rhesus macaque STRL33 (rhSTRL33), functioned efficiently as an entry cofactor for a variety of isolates of SIVmac and SIV from sooty mangabeys. A single amino acid substitution of S30R in huSTRL33 impaired coreceptor activity, and the reverse change in rhSTRL33 greatly increased coreceptor activity. In comparison, species-specific sequence variations in N-terminal tyrosines in STRL33 had only moderate effects on SIV entry. These results show that a serine residue located just outside of the cellular membrane in the N terminus of STRL33 is critical for SIV coreceptor function. Interestingly, STRL33 derived from sooty mangabeys, a natural host of SIV, also contained a serine at the corresponding position and was used efficiently as an entry cofactor. These results suggest that STRL33 is not a relevant coreceptor in the SIV/macaque model but may play a role in SIV replication and transmission in naturally infected sooty mangabeys.
Pseudorabies Virus Glycoprotein K Requires the UL20 Gene Product for Processing
Authors: Dietz Petra , Klupp Barbara G. , Fuchs Walter , Köllner Bernd , Weiland Emilie , Mettenleiter Thomas C.
Published: July 2002

Glycoprotein K (gK) of pseudorabies virus (PrV) has recently been identified as a virion component which is dispensable for viral entry but required for direct cell-to-cell spread. Electron microscopic data suggested a possible function of gK in virus egress by preventing immediate fusion of released virus particles with the plasma membrane (B. G. Klupp, J. Baumeister, P. Dietz, H. Granzow, and T. C. Mettenleiter, J. Virol. 72:1949–1958, 1998). For more detailed analysis, a PrV mutant with a deletion of the UL53 (gK) open reading frame (ORF) from codons 48 to 275 was constructed, and the protein was analyzed with two monoclonal antibodies directed against PrV gK. The salient findings of this report are as follows. (i) From the PrV UL53 ORF, a functional gK is translated only from the first in-frame methionine. From the second in-frame methionine, a nonfunctional product is expressed which is not incorporated into virions. (ii) When constitutively expressed in a stable cell line without other viral proteins, gK is only incompletely processed. After superinfection with gK-deletion mutants, proper processing is restored and mature gK is incorporated into virions. (iii) The UL20 gene product is specifically required for processing of gK. gK is not correctly processed in a UL20 deletion mutant of PrV, and superinfection of gK-expressing cells with PrV-UL20
−
does not restore processing. However, all other known structural viral glycoproteins appear to be processed normally in PrV-UL20
−
-infected cells. (iv) Coexpression of gK and UL20 restored gK processing at least partially. Thus, our data show that the UL20 gene product is required for proper processing of PrV gK.
A Metastable Form of the Large Envelope Protein of Duck Hepatitis B Virus: Low-pH Release Results in a Transition to a Hydrophobic, Potentially Fusogenic Conformation
Authors: Grgacic E. V. L. , Schaller H.
Published: July 2002

We have examined the structure and fusion potential of the duck hepatitis B virus (DHBV) envelope proteins by treating subviral particles with deforming agents known to release envelope proteins of viruses from a metastable to a fusion-active state. Exposure of DHBV particles to low pH triggered a major structural change in the large envelope protein (L), resulting in exposure of trypsin sites within its S domain but without affecting the same region in the small surface protein (S) subunits. This conformational change was associated with increased hydrophobicity of the particle surface, most likely arising from surface exposure of the hydrophobic first transmembrane domain (TM1). In the hydrophobic conformation, DHBV particles were able to bind to liposomes and intact cells, while in their absence these particles aggregated, resulting in viral inactivation. These results suggests that some L molecules are in a spring-loaded metastable state which, when released, exposes a previously hidden hydrophobic domain, a transition potentially representing the fusion-active state of the envelope.
Relationship between Human Immunodeficiency Virus Type 1 Gag Multimerization and Membrane Binding
Authors: Ono Akira , Demirov Dimiter , Freed Eric O.
Published: July 2002

The human immunodeficiency virus type 1 (HIV-1) Gag precursor, Pr55
Gag
, is necessary and sufficient for the assembly and release of viruslike particles. Binding of Gag to membrane and Gag multimerization are both essential steps in virus assembly, yet the domains responsible for these events have not been fully defined. In addition, the relationship between membrane binding and Gag-Gag interaction remains to be elucidated. To investigate these issues, we analyzed, in vivo, the membrane-binding and assembly properties of a series of C-terminally truncated Gag mutants. Pr55
Gag
was truncated at the C terminus of matrix (MAstop), between the N- and C-terminal domains of capsid (CA146stop), at the C terminus of capsid (p41stop), at the C terminus of p2 (p43stop), and after the N-terminal 35 amino acids of nucleocapsid (NC35stop). The ability of these truncated Gag molecules to assemble and release viruslike particles and their capacity to copackage into particles when coexpressed with full-length Gag were determined. We demonstrate that the amount of truncated Gag incorporated into particles is incrementally increased by extension from CA146 to NC35, suggesting that multiple sites in this region are involved in Gag multimerization. Using membrane flotation centrifugation, we observe that MA shows significantly reduced membrane binding relative to full-length Gag but that CA146 displays steady-state membrane-binding properties comparable to those of Pr55
Gag
. The finding that the CA146 mutant, which contains only matrix and the N-terminal domain of capsid, exhibits levels of steady-state membrane binding equivalent to those of full-length Gag indicates that strong Gag-Gag interaction domains are not required for the efficient binding of HIV-1 Gag to membrane.
Circular Double-Stranded Forms of TT Virus DNA in the Liver
Authors: Okamoto Hiroaki , Ukita Masato , Nishizawa Tsutomu , Kishimoto Junichi , Hoshi Yuji , Mizuo Hitoshi , Tanaka Takeshi , Miyakawa Yuzo , Mayumi Makoto
Published: July 2002

TT virus
(TTV) is an unenveloped, circular, and single-stranded DNA virus commonly infecting human beings worldwide. TTV DNAs in paired serum and liver tissues from three viremic individuals were separated by gel electrophoresis and characterized biophysically. TTV DNAs in sera migrated in sizes ranging from 2.0 to 2.5 kb. TTV DNAs in liver tissues, however, migrated at 2.0 to 2.5 kb as well as at 3.5 to 6.1 kb. Both faster- and slower-migrating forms of TTV DNAs in the liver were found to be circular and of the full genomic length of 3.8 kb. TTV DNAs migrating at 2.0 to 2.5 kb, from either serum or liver tissues, were sensitive to S1 nuclease but resistant to restriction endonucleases, and therefore, they were single-stranded. By contrast, TTV DNAs in liver tissues that migrated at 3.5 to 6.1 kb were resistant to S1 nuclease. They migrated at 3.7 to 4.0 kb after digestion with
Eco
RI, which suggests that they represent circular, double-stranded replicative intermediates of TTV. When TTV DNAs were subjected to strand-specific primer extension and then amplified by PCR with internal primers, those in serum were found to be minus-stranded DNAs while those in liver tissues were found to be a mixture of plus- and minus-stranded DNAs. These results suggest that TTV replicates in the liver via a circular double-stranded DNA.
Nonstructural Protein 5A of Hepatitis C Virus Inhibits the Function of Karyopherin β3
Authors: Chung Kyung Min , Lee Juhang , Kim Jung-Eun , Song Ok-Kyu , Cho Sungchan , Lim Jeongsim , Seedorf Matthias , Hahm Bumsuk , Jang Sung Key
Published: July 2002

It has been suggested that nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) plays a role in the incapacitation of interferon by inactivation of RNA-dependent protein kinase PKR. In order to further investigate the role of NS5A, we tried to identify cellular proteins interacting with NS5A by using the yeast two-hybrid system. The karyopherin β3 gene was isolated from a human liver cell library as a protein interacting with NS5A. The protein-protein interaction between NS5A and karyopherin β3 was confirmed by in vitro binding assay and an in vivo coimmunoprecipitation method. The effect of NS5A on the karyopherin β3 activity was investigated using a yeast cell line containing mutations in bothPSE1andKAP123, genes that are homologous to the human karyopherin β3 gene. Human karyopherin β3 complemented the loss of thePSE1andKAP123functions, supporting growth of the double mutant cells. However, expression of NS5A hampered the growth of the double mutant cells supplemented with human karyopherin β3. On the other hand, expression of NS5A by itself had no effect on the growth of the double mutant expressing wild-type yeastPSE1. This indicates that NS5A may inhibit karyopherin β3 function via protein-protein interaction. The role of NS5A in HCV replication is discussed.
Expression of Hepatitis B Virus X Protein Does Not Alter the Accumulation of Spontaneous Mutations in Transgenic Mice
Authors: Madden Charles R. , Finegold Milton J. , Slagle Betty L.
Published: July 2002

Chronic infection with hepatitis B virus (HBV) is one of the major etiological factors in the development of human hepatocellular carcinoma. Transgenic mice that express the HBV X protein (HBx) have previously been shown to be more sensitive to the effects of hepatocarcinogens, although the mechanism for this cofactor role remains unknown. The ability of HBx to inhibit DNA repair in transiently transfected cell lines suggests one possible pathway. In the present study, primary hepatocytes isolated from transgenic mice that possess the HBV
X
gene under the control of the human α-1-antitrypsin regulatory region (ATX mice) were found to be deficient in their ability to conduct unscheduled DNA synthesis in response to UV-induced DNA damage. In order to measure the impact of HBx expression on DNA repair in vivo, double-transgenic mice that express HBx and possess a bacteriophage lambda transgene were sacrificed at 30, 90, and 240 days of age. Mutation frequency was determined for high-molecular-weight liver DNA of ATX and control mice by functional analysis of the lambda transgene. Expression of HBx did not significantly increase the accumulation of spontaneous mutations. These results are consistent with previous studies of HBx transgenic mice in which no effect of HBx on liver histology was apparent. This new animal model provides a powerful system in which to investigate the in vivo cooperation between HBx expression and environmental carcinogens.
The
Autographa californica
Nuclear Polyhedrosis Virus
p143
Gene Encodes a DNA Helicase
Authors: McDougal Vivien V. , Guarino Linda A.
Published: July 2002

The P143 protein of
Autographa californica
nuclear polyhedrosis virus is essential for replication of viral DNA. To determine the function of P143, the protein was purified to near homogeneity from recombinant baculovirus-infected cells that overexpress P143. ATPase activity copurified with P143 protein during purification and also during gel filtration at a high salt concentration. The ATPase activity did not require the presence of single-stranded DNA, but was stimulated fourfold by the addition of single-stranded DNA. The ATPase activity of P143 had a

K
m

of 60 μM and a turnover of 4.5 molecules of ATP hydrolyzed/s/molecule of enzyme, indicating moderate affinity for ATP and high catalytic efficiency. P143 unwound a 40-nucleotide primer in an ATP-dependent manner, indicating that the enzyme possesses in vitro DNA helicase activity. Based on this result, it seems likely that P143 functions as a helicase in viral DNA replication.
J Domain-Independent Regulation of the Rb Family by Polyomavirus Large T Antigen
Authors: Sheng Qing , Love Tara M. , Schaffhausen Brian
Published: July 2002

The ability of polyomavirus large T antigen (LT) to promote cell cycling, to immortalize primary cells, and to block differentiation has been linked to its effects on tumor suppressors of the retinoblastoma susceptibility (Rb) gene family. Our previous studies have shown that LT requires an intact N-terminal DnaJ domain, in addition to an Rb binding site, for activation of simple E2F-containing promoters and stimulation of cell cycle progression. Here we show that some LT effects dependent on interaction with the Rb family are largely DnaJ independent. In differentiating C2C12 myoblasts, overexpression of LT caused apoptosis. Although this activity of LT completely depended on Rb binding, LTs with mutations in the J domain remained able to kill. Comparisons of Rb
−
and J
−
LTs revealed additional differences. Wild-type but not Rb
−
LT activated the cyclin A promoter under serum starvation conditions. Genetic analysis of the promoter linked the Rb requirement to an E2F site in the promoter. LTs with mutations in the J domain were still able to activate the promoter. Finally, J mutant LTs caused changes in phosphorylation of both pRb and p130. In the case of p130, Thr-986 was shown to be a site that is regulated by J mutant LT. Taken together, these observations reveal that LT regulation of Rb function can be separated into both DnaJ-dependent and DnaJ-independent pathways.
Incorporation of Tumor Vasculature Targeting Motifs into Moloney Murine Leukemia Virus Env Escort Proteins Enhances Retrovirus Binding and Transduction of Human Endothelial Cells
Authors: Liu Liqiong , Liu Ling , Anderson W. French , Beart Robert W. , Gordon Erlinda M. , Hall Frederick L.
Published: July 2002

Adhesion receptors expressed on the surfaces of tumor-activated endothelial cells provide an advantageous locus for targeting gene therapy vectors to angiogenic tissues and/or tumor vasculature. In this study, we engineered a series of Asn-Gly-Arg (NGR)-containing congeners of the presumptive cell binding motif contained within the ninth type III repeat of fibronectin and displayed these tumor vasculature targeting motifs (TVTMs) within the context of Moloney murine leukemia envelope “escort” proteins. Comparative studies of envelope incorporation into viral particles and evaluation of the cell binding properties of the targeted vectors revealed critical structural features, thus identifying a subset of optimal TVTMs. Utilizing a modified ELISA to evaluate viral binding to target cells, we observed a significant down-regulation of TVTM-virion binding to human endothelial cells following sustained (48-h) exposure to VEGF. Normalized for equivalent titers (10
6
CFU/ml), as assayed on NIH 3T3 cells, vectors displaying TVTM escort proteins significantly enhanced the transduction efficiency from 12.2 to 37.4% in human KSY-1 endothelial cell cultures (
P
< 0.001) and from 0.4 to 4.1% in human umbilical vein endothelial cell (HUVEC) cultures (
P
< 0.001). In summary, these studies utilized an engineering approach to identify a subset of TVTMs that are stably incorporated as envelope “escort” proteins into retroviral vectors and that, by functioning to improve the binding efficiency and transduction of both HUVEC and KSY1 endothelial cells, may have therapeutic potential for targeting gene delivery to the tumor-associated vasculature.
Activation of Divergent Neuronal Cell Death Pathways in Different Target Cell Populations during Neuroadapted Sindbis Virus Infection of Mice
Authors: Havert Michael B. , Schofield Brian , Griffin Diane E. , Irani David N.
Published: July 2002

Infection of adult mice with neuroadapted Sindbis virus (NSV) results in a severe encephalomyelitis accompanied by prominent hindlimb paralysis. We find that the onset of paralysis parallels morphologic changes in motor neuron cell bodies in the lumbar spinal cord and in motor neuron axons in ventral nerve roots, many of which are eventually lost over time. However, unlike NSV-induced neuronal cell death found in the brain of infected animals, the loss of motor neurons does not appear to be apoptotic, as judged by morphologic and biochemical criteria. This may be explained in part by the lack of detectable caspase-3 expression in these cells.
Major Histocompatibility Complex Class I Gene Controls the Generation of Gamma Interferon-Producing CD4+and CD8+T Cells Important for Recovery from Friend Retrovirus-Induced Leukemia
Authors: Peterson Karin E. , Iwashiro Michihiro , Hasenkrug Kim J. , Chesebro Bruce
Published: July 2002

Recovery from leukemia induced by Friend virus complex (FV) requires strong CD4+helper, CD8+cytotoxic T-lymphocyte, and B-cell responses. The development of these immune responses is dependent on the major histocompatibility complex (MHC) (H-2) genotype of the mouse. InH-2b/bmice, which spontaneously recover from FV-induced erythroleukemia, neutralization of gamma interferon (IFN-γ) in vivo inhibited recovery, which indicated that IFN-γ was a necessary component of the immune response to FV. Furthermore, inH-2b/bmice, high numbers of IFN-γ-producing cells were detected after FV infection, whereas inH-2a/bmice, which have a low-recovery phenotype, only low numbers of IFN-γ-producing cells were detected. Similarly,H-2bm14/bmice, which cannot recover from FV infection due to a point mutation in one allele of theH-2Dbgene, also had low numbers of IFN-γ-producing T cells. Surprisingly, this effect was observed for both CD8+and CD4+T cells. These findings reveal a novel influence of MHC class I genes on CD4+T-cell responses to viral infection. Furthermore, the influence of MHC class I genotype on the generation of both IFN-γ-producing CD4+and CD8+T cells helps explain the major impact of theH-2Dgene on recovery from FV disease.
Structure-Function Studies of the Self-Assembly Domain of the Human Immunodeficiency Virus Type 1 Transmembrane Protein gp41
Authors: Weng Yongkai , Yang Zhongning , Weiss Carol D.
Published: July 2002

The coiled-coil region of the human immunodeficiency virus type 1 transmembrane protein (gp41) makes up the interior core of the six-helix bundle structure of the gp41 self-assembly domain. We extended our previous study of this domain (Y. Weng and C. D. Weiss, J. Virol. 72:9676–9682, 1998) by analyzing 23 additional mutants at positions that lie at the interface of the interior core and outer helices. We found nine new functional mutants. For most mutants, the activity could be explained by the ability of the modeled mutants to stabilize the six-helix bundle structure. The present study provides insights into the envelope glycoprotein fusion mechanism and information for rational drug and vaccine design.
Probing the Structure of Rotavirus NSP4: a Short Sequence at the Extreme C Terminus Mediates Binding to the Inner Capsid Particle
Authors: O'Brien Judith A. , Taylor John A. , Bellamy A. R.
Published: July 2002

The rotavirus nonstructural glycoprotein NSP4 functions as the receptor for the inner capsid particle (ICP) which buds into the lumen of the endoplasmic reticulum during virus maturation. The structure of the cytoplasmic domain of NSP4 from rotavirus strain SA11 has been investigated by using limited proteolysis and mass spectrometry. Digestion with trypsin and V8 protease reveals a C-terminal protease-sensitive region that is 28 amino acids long. The minimal sequence requirements for receptor function have been defined by constructing fusions with glutathione
S
-transferase and assessing their ability to bind ICPs. These experiments demonstrate that 17 to 20 amino acids from the extreme C terminus are necessary and sufficient for ICP binding and that this binding is cooperative. These observations are consistent with a model for the structure of the NSP4 cytoplasmic region in which four flexible regions of 28 amino acids are presented by a protease-resistant coiled-coil tetramerization domain, with only the last ∼20 amino acids of each peptide interacting with the surface binding sites on the ICP.
Gamma Interferon Is Not Required for Mucosal Cytotoxic T-Lymphocyte Responses or Heterosubtypic Immunity to Influenza A Virus Infection in Mice
Authors: Nguyen Huan H. , van Ginkel Frederik W. , Vu Huong L. , Novak Miroslav J. , McGhee Jerry R. , Mestecky Jiri
Published: July 2002

Heterosubtypic immunity (HSI) is defined as cross-protection against influenza virus of a different serotype than the virus initially encountered and is thought to be mediated by influenza virus-specific cytotoxic T lymphocytes (CTL). Since gamma interferon (IFN-γ) stimulates cytotoxic cells, including antigen-specific CTL which may control virus replication by secretion of antiviral cytokines such as tumor necrosis factor alpha and IFN-γ, we have investigated the mechanism of HSI by analyzing the role of IFN-γ for HSI in IFN-γ gene-deleted (IFN-γ−/−) mice. It has been reported that IFN-γ is not required for recovery from primary infection with influenza virus but is important for HSI. Here, we conclusively show that IFN-γ is not required for induction of secondary influenza virus-specific CTL responses in mediastinal lymph nodes and HSI to lethal influenza A virus infection. Although T helper 2 (Th2)-type cytokines were upregulated in the lungs of IFN-γ−/−mice after virus challenge, either Th1- or Th2-biased responses could provide heterosubtypic protection. Furthermore, titers of serum-neutralizing and cross-reactive antibodies to conserved nucleoprotein in IFN-γ−/−mice did not differ significantly from those in immunocompetent mice. These results indicate that lack of IFN-γ does not impair cross-reactive virus-specific immune responses and HSI to lethal infection with influenza virus. Our findings provide new insight for the mechanisms of HSI and should be valuable in the development of protective mucosal vaccines against variant virus strains, such as influenza and human immunodeficiency virus.
Rubella Virus Capsid Associates with Host Cell Protein p32 and Localizes to Mitochondria
Authors: Beatch Martin D. , Hobman Tom C.
Published: July 2002

Togavirus nucleocapsids have a characteristic icosahedral structure and are composed of multiple copies of a capsid protein complexed with genomic RNA. The assembly of rubella virus nucleocapsids is unique among togaviruses in that the process occurs late in virus assembly and in association with intracellular membranes. The goal of this study was to identify host cell proteins which may be involved in regulating rubella virus nucleocapsid assembly through their interactions with the capsid protein. Capsid was used as bait to screen a CV1 cDNA library using the yeast two-hybrid system. One protein that interacted strongly with capsid was p32, a cellular protein which is known to interact with other viral proteins. The interaction between capsid and p32 was confirmed using a number of different in vitro and in vivo methods, and the site of interaction between these two proteins was shown to be at the mitochondria. Interestingly, overexpression of the rubella virus structural proteins resulted in clustering of the mitochondria in the perinuclear region. The p32-binding site in capsid is a potentially phosphorylated region that overlaps the viral RNA-binding domain of capsid. Our results are consistent with the possibility that the interaction of p32 with capsid plays a role in the regulation of nucleocapsid assembly and/or virus-host interactions.
Application of the Intracellular Gamma Interferon Assay To Recalculate the Potency of CD8+T-Cell Responses to Herpes Simplex Virus
Authors: Kumaraguru Udayasankar , Rouse Barry T.
Published: July 2002

Enumeration and characterization of herpes simplex virus (HSV)-specific CD8+T lymphocytes are tedious and indirect. We quantitated antigen-specific CD8+T cells during acute and secondary stages of HSV infection using intracellular gamma interferon production upon stimulation with virus or immunodominant peptide. Results show a substantial increase in the number of CD8+T cells which was otherwise underestimated with the conventional limiting dilution analysis.
Role of NH
2
- and COOH-Terminal Domains of the P Protein of Human Parainfluenza Virus Type 3 in Transcription and Replication
Authors: De Bishnu P. , Hoffman Michael A. , Choudhary Suresh , Huntley Clayton C. , Banerjee Amiya K.
Published: July 2002

The phosphoproteins (P proteins) of paramyxoviruses play a central role in transcription and replication of the viruses by forming the RNA polymerase complex L-P and encapsidation complex (N-P) with nucleocapsid protein (N) and binding to N protein-encapsidated genome RNA template (N-RNA template). We have analyzed the human parainfluenza virus type 3 (HPIV3) P protein and deletion mutants thereof in an in vitro transcription and in vivo replication system. The in vitro system utilizes purified N-RNA template and cell extract containing L and P proteins coexpressed via plasmids using a recombinant vaccinia virus expression system. The in vivo system takes advantage of minigenome replication, which measures luciferase reporter gene expression from HPIV3 minigenomes by viral proteins in a recombinant vaccinia virus expression system. These studies revealed that the C-terminal 20-amino-acid region of P is absolutely required for transcription in vitro and luciferase expression in vivo, suggesting its critical role in viral RNA synthesis. The N-terminal 40-amino-acid region, on the other hand, is essential for luciferase expression but dispensable for transcription in vitro. Consistent with these findings, the C-terminal domain is required for binding of P protein to the N-RNA template involved in both transcription and replication, whereas the N-terminal domain is required for the formation of soluble N-P complex involved in encapsidation of nascent RNA chains during replication. Coimmunoprecipitation analysis showed that the P protein forms a stable homooligomer (perhaps a trimer) that is present in L-P and N-P complexes in the higher oligomeric forms (at least a pentamer). Interestingly, coexpression of a large excess of N- or C-terminally deleted P with wild-type P had no effect on minigenome replication in vivo, notwithstanding the formation of heterooligomeric complexes. These data indicate that P protein with a deleted terminal domain can function normally within the P heterooligomeric complex to carry out transcription and replication in vivo.
Interleukin-12 (IL-12) Enhancement of the Cellular Immune Response against Human Immunodeficiency Virus Type 1 Env Antigen in a DNA Prime/Vaccinia Virus Boost Vaccine Regimen Is Time and Dose Dependent: Suppressive Effects of IL-12 Boost Are Mediated by Nitric Oxide
Authors: Gherardi M. Magdalena , Ramírez Juan C. , Esteban Mariano
Published: July 2002

We previously demonstrated that codelivery of interleukin-12 (IL-12) with the human immunodeficiency virus type 1 (HIV-1) Env antigen from a recombinant vaccinia virus (rVV) can enhance the specific anti-Env cell-mediated immune (CMI) response. In the present study, we have investigated the effects of IL-12 in mice when it is expressed in a DNA prime/VV boost vaccine regimen. The delivery of IL-12 and Env product during priming with a DNA vector, followed by a booster with VV expressing the Env gene (rVVenv), was found to trigger the optimal CMI response compared with other immunization schedules studied. Significantly, if IL-12 is also delivered as a booster from the viral vector, an impairment of the effects of IL-12 was observed involving nitric oxide (NO), since it was overcome by specific inhibitors of inducible NO synthase. NO caused transient immunosuppression rather than impairment of viral replication. Moreover, at certain viral doses, coadministration of the NO inhibitor during the booster resulted in IL-12-mediated enhancement of the specific CD8+T-cell response. In addition, the dose of the IL-12-encoding plasmid (pIL-12) and the route of administration of both vectors were relevant factors for optimal CMI responses. Maximal numbers of Env-specific CD8+gamma interferon-secreting cells were obtained when 50 μg of pIL-12 was administered intramuscularly at priming, followed by an intravenous rVVenv boost. Our results demonstrate, in a murine model, critical parameters affecting the success of vaccination schedules based on a combination of DNA and VV vectors in conjunction with immunomodulators.
DNA Vaccines Encoding Viral Glycoproteins Induce Nonspecific Immunity and Mx Protein Synthesis in Fish
Authors: Kim Carol H. , Johnson Marc C. , Drennan John D. , Simon Benjamin E. , Thomann Estela , Leong Jo-Ann C.
Published: July 2002

Protective immunity by vaccination with plasmid DNA encoding a viral glycoprotein (G) has long been assumed to result from the induction of a specific immune response. We report here that the initial protection may be due to the induction of alpha/beta interferon, with long-term protection due to a specific response to the encoded viral G. DNA vaccines encoding the Gs of three serologically unrelated fish rhabdoviruses were used to vaccinate rainbow trout against a lethal challenge with infectious hematopoietic necrosis virus (IHNV). All three vaccines, each encoding the G gene of either IHNV (IHNV-G), snakehead rhabdovirus (SHRV) (SHRV-G), or spring viremia of carp virus (SVCV) (SVCV-G), elicited protective immunity against IHNV. Vaccinated fish were challenged at 30 or 70 days postvaccination with lethal doses of IHNV. At 30 days postvaccination, only 5% of fish that had received any of the G vaccines died, whereas more than 50% of the control fish succumbed to virus challenge. When fish were vaccinated and challenged at 70 days postvaccination, only 12% of the IHNV-G-vaccinated fish died compared to 68% for the SHRV-G- and 76% for the SVCV-G-vaccinated fish. Assays for trout Mx protein, an indicator of alpha/beta interferon induction, showed that only fish vaccinated with a G-containing plasmid produced high levels of Mx protein in the kidneys and liver. Interestingly, at day 7 after virus challenge, all of the fish vaccinated with the IHNV-G plasmid were negative for Mx, but the SHRV-G- and SVCV-G-vaccinated fish still showed detectable levels of Mx. These results suggest that DNA vaccines in fish induce an early, nonspecific antiviral protection mediated by an alpha/beta interferon and, later, a specific immune response.
Use of a Transient Assay for Studying the Genetic Determinants of
Fv1
Restriction
Authors: Bock Michael , Bishop Kate N. , Towers Greg , Stoye Jonathan P.
Published: July 2002

To probe the genetic determinants controlling the interaction between the retroviral restriction gene
Fv1
and its murine leukemia virus target, we set out to develop rapid, transient assays for
Fv1
function. Cells were transfected or transduced with
Fv1
expression plasmids which can produce green fluorescent protein via an internal ribosome entry site positioned between the
Fv1
and green fluorescent protein coding sequences.
Fv1
function was then assessed by comparing virus replication in green fluorescent protein-positive and -negative cells, using retroviral vectors encoding a second fluorescent marker, yellow fluorescent protein, or β-galactosidase. Using this assay, we could show that
Fv1
specificities were not as absolute as previously thought, since the

Fv1
b

allele was capable of interacting with “nonrestricted” B- and NB-tropic viruses and by shuffling the n- and b-alleles of
Fv1
, it was possible to generate a Fv1 molecule capable of restricting N-, B-, and NB-tropic viruses equally efficiently. Further, we could show that the presence of nonrestricting Fv1 in the same cell as restrictive Fv1 abrogates restriction, implying competition for binding to the retroviral target.
Analysis of the Pore Structure of the Influenza A Virus M
2
Ion Channel by the Substituted-Cysteine Accessibility Method
Authors: Shuck Kevin , Lamb Robert A. , Pinto Lawrence H.
Published: July 2002

The M
2
ion channel of influenza A virus is a small integral membrane protein whose active form is a homotetramer with each polypeptide chain containing 96-amino-acid residues. To identify residues of the transmembrane (TM) domain that line the presumed central ion-conducting pore, a set of mutants was generated in which each residue of the TM domain (residues 25 to 44) was replaced by cysteine. The accessibility of the cysteine mutants to modification by the sulfhydryl-specific reagents methane thiosulfonate ethylammonium (MTSEA) and MTS tetraethylammonium (MTSET) was tested. Extracellular application of MTSEA evoked decreases in the conductances measured from two mutants, M
2
-A30C and M
2
-G34C. The changes observed were not reversible on washout, indicative of a covalent modification. Inhibition by MTSEA, or by the larger reagent MTSET, was not detected for residues closer to the extracellular end of the channel than Ala-30, indicating the pore may be wider near the extracellular opening. To investigate the accessibility of the cysteine mutants to reagents applied intracellularly, oocytes were microinjected directly with reagents during recordings. The conductance of the M
2
-W41C mutant was decreased by intracellular injection of a concentrated MTSET solution. However, intracellular application of MTSET caused no change in the conductance of the M
2
-G34C mutant, a result in contrast to that obtained when the reagent was applied extracellularly. These data suggest that a constriction in the pore exists between residues 34 and 41 which prevents passage of the MTS reagent. These findings are consistent with the proposed role for His-37 as the selectivity filter. Taken together, these data confirm our earlier model that Ala-30, Gly-34, His-37, and Trp-41 line the channel pore (L. H. Pinto, G. R. Dieckmann, C. S. Gandhi, C. G. Papworth, J. Braman, M. A. Shaughnessy, J. D. Lear, R. A. Lamb, and W. F. DeGrado, Proc. Natl. Acad. Sci. USA 94:11301–11306, 1997).
Poxvirus-Induced Immunostimulating Effects on Porcine Leukocytes
Authors: Fachinger Vicky , Schlapp Tobias , Strube Walter , Schmeer Norbert , Saalmüller Armin
Published: July 2002

The prophylactic application of inactivated parapox ovis viruses (Baypamun; Bayer AG, Leverkusen, Germany) has been shown to reduce efficiently the outbreak of stress-mediated diseases in different species. However, little is known about the basic mechanism behind this observed stimulatory property. We therefore tested eight inactivated poxvirus strains belonging to three different genera (Orthopoxvirus,Avipoxvirus, andParapoxvirus) for their capacity to activate cells of the porcine innate and specific immune systems in vitro. The results indicated that poxviruses failed to induce increased phagocytosis, oxidative burst, or natural killer cell activity in swine. In contrast, enhanced release of interleukin-2, alpha interferon, and gamma interferon, as well as strong proliferation, could be measured. Flow cytometric analyses and cell sorting experiments identified T-helper cells as the main target responding to inactivated poxviruses: the activated cells had a CD4highCD25+major histocompatibility complex type II-positive phenotype and were the major source of secreted cytokines. Together, the results demonstrated that all tested poxviruses possessed immunostimulating capacity. These in vitro poxvirus-induced effects may be responsible at least in part for the in vivo immunostimulating capacity of inactivated poxviruses.
Response of Foot-and-Mouth Disease Virus to Increased Mutagenesis: Influence of Viral Load and Fitness in Loss of Infectivity
Authors: Sierra Saleta , Dávila Mercedes , Lowenstein Pedro R. , Domingo Esteban
Published: July 2002

Passage of foot-and-mouth disease virus (FMDV) in cell culture in the presence of the mutagenic base analog 5-fluorouracil or 5-azacytidine resulted in decreases of infectivity and occasional extinction of the virus. Low viral loads and low viral fitness enhanced the frequency of extinction events; this finding was shown with a number of closely related FMDV clones and populations differing by up to 10
6
-fold in relative fitness in infections involving either single or multiple passages in the absence or presence of the chemical mutagens. The mutagenic treatments resulted in increases of 2- to 6.4-fold in mutation frequency and up to 3-fold in mutant spectrum complexity. The largest increase observed corresponded to the 3D (polymerase)-coding region, which is highly conserved in nonmutagenized FMDV populations. As a result, nucleotide sequence heterogeneity for the 3D-coding region became very similar to that for the variable VP1-coding region in FMDVs multiply passaged in the presence of chemical mutagens. The results suggest that strategies to combine reductions of viral load and viral fitness could be effectively associated with extinction mutagenesis as a potential new antiviral strategy.