bluuebunny/crossref_metadata_2025 huggingface dataset:
Stable High-Producer Cell Clone Expressing Virus-Like Particles of the Japanese Encephalitis Virus E Protein for a Second-Generation Subunit Vaccine
Authors: Kojima Asato , Yasuda Atsushi , Asanuma Hideki , Ishikawa Toyokazu , Takamizawa Akihisa , Yasui Kotaro , Kurata Takeshi
Published: July 2003

We produced and characterized a cell clone (J12#26 cells) that stably expresses Japanese encephalitis virus (JEV) cDNA, J12, which encodes the viral signal peptide, premembrane (prM), and envelope (E) proteins (amino acid positions 105 to 794). Rabbit kidney-derived RK13 cells were transfected with a J12 expression plasmid, selected by resistance to marker antibiotics, and cloned by two cycles of a limiting-dilution method in the presence of antibiotics, a procedure that prevents the successful generation of E-producing cell clones. J12#26 cells secreted virus-like particles containing the authentic E antigen (E-VLP) into the culture medium in a huge enzyme-linked immunosorbent assay-equivalent amount (2.5 μg per 104cells) to the internationally licensed JE vaccine JE-VAX. E-VLP production was stable after multiple cell passages and persisted over 1 year with 100% expressing cells without detectable cell fusion, apoptosis, or cell death, but was suspended when the cells grew to 100% confluency and contact inhibition occurred. Mice immunized with the purified J12#26 E-antigen without adjuvant developed high titers of neutralizing antibodies for at least 7 months and 100% protection against intraperitoneal challenge with 5 × 106PFU of JEV when examined according to the JE vaccine standardization protocol. These results suggest that the recombinant E-VLP antigen produced by the J12#26 cell clone is an effective, safe, and low-cost second-generation subunit JE vaccine.
Probing the Importance of tRNA Anticodon: Human Immunodeficiency Virus Type 1 (HIV-1) RNA Genome Complementarity with an HIV-1 That Selects tRNA
Glu
for Replication
Authors: Dupuy Lesley C. , Kelly Nathan J. , Elgavish Tricia E. , Harvey Stephen C. , Morrow Casey D.
Published: July 2003

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs at the primer binding site (PBS) that is complementary to the 3′-terminal nucleotides of tRNA
3
Lys
. Why all known strains of HIV-1 select tRNA
3
Lys
for replication is unknown. Previous studies on the effect of altering the PBS of HIV-1 on replication identified an HIV-1 with a PBS complementary to tRNA
Glu
. Since the virus was not initially designed to use tRNA
Glu
, the virus had selected tRNA
Glu
from the intracellular pool of tRNA for use in replication. Further characterization of HIV-1 that uses tRNA
Glu
may provide new insights into the preference for tRNA
3
Lys
. HIV-1 constructed with the PBS complementary to tRNA
Glu
was more stable than HIV-1 with the PBS complementary to tRNA
Met
or tRNA
His
; however, all of these viruses eventually reverted back to using tRNA
3
Lys
following growth in SupT1 cells or peripheral blood mononuclear cells (PBMCs). New HIV-1 mutants with nucleotides in U5 complementary to the anticodon of tRNA
Glu
remained stable when grown in SupT1 cells or PBMCs, although the mutants grew more slowly than the wild-type virus. Sequence analysis of the U5 region and the PBS revealed additional mutations predicted to further promote tRNA-viral genome interaction. The results support the importance of the tRNA anticodon-genome interaction in the selection of the tRNA primer and highlight the fact that unique features of tRNA
3
Lys
are exploited by HIV-1 for selection as the reverse transcription primer.
A Hepadnavirus Regulatory Element Enhances Expression of a Type 2 Bovine Viral Diarrhea Virus E2 Protein from a Bovine Herpesvirus 1 Vector
Authors: Wang Lingshu , Menon Sreekumar , Bolin Steven R. , Bello Leonard J.
Published: July 2003

Recently, the possibility of using virus vectors to immunize cattle against selected bovine viral diarrhea virus (BVDV) genes has gained widespread interest. However, when we attempted to express the E2 protein from type 2 (890 strain) BVDV in a bovine herpesvirus 1 (BHV1) vector, we observed that expression was poor. This often happens when genes from a cytoplasmic virus are expressed in the cell nucleus. To counter this effect, we attempted to enhance expression by a strategy employed by viruses. RNAs of retroviruses and hepadnaviruses containcis-acting elements that facilitate expression of RNAs that otherwise are degraded or retained within the nucleus. In Mason-Pfizer monkey virus, the required RNA sequence element is known as a constitutive transport element (CTE). A related element from woodchuck hepatitis virus is known as the woodchuck posttranscriptional regulatory element (WPRE). We tested the ability of the CTE, the WPRE, and introns to enhance expression of E2. All three elements stimulated expression of E2 from plasmids. The combination of the WPRE and an intron yielded the highest level of E2 expression in plasmids. However, when E2 was expressed from a BHV1 vector, the presence of an intron was inhibitory. In contrast, the WPRE was very efficient at stimulating E2 expression from a BHV1 vector. This result represents the first expression of a type 2 BVDV E2 protein from a mammalian virus vector and raises the possibility that the WPRE may provide a general method of enhancing foreign gene expression from BHV1 and other herpesvirus vectors.
Simian Immunodeficiency Virus Infection in Neonatal Macaques
Authors: Veazey Ronald S. , Lifson Jeffrey D. , Pandrea Ivona , Purcell Jeannette , Piatak Michael , Lackner Andrew A.
Published: July 2003

Children with human immunodeficiency virus infection often have higher viral loads and progress to AIDS more rapidly than adults. Since the intestinal tract is a major site of early viral replication and CD4+T-cell depletion in adults, we examined the effects of simian immunodeficiency virus (SIV) on both peripheral and intestinal lymphocytes from 13 neonatal macaques infected with SIVmac239. Normal neonates had more CD4+T cells and fewer CD8+T cells in all tissues than adults. Surprisingly, neonates had substantial percentages of CD4+T cells with an activated, memory phenotype (effector CD4+T cells) in the lamina propria of the intestine compared to peripheral lymphoid tissues, even when examined on the day of birth. Moreover, profound and selective depletion of jejunum lamina propria CD4+T cells occurred in neonatal macaques within 21 days of infection, which was preceded by large numbers of SIV-infected cells in this compartment. Furthermore, neonates with less CD4+T-cell depletion in tissues tended to have higher viral loads. The persistence of intestinal lamina propria CD4+T cells in some neonates with high viral loads suggests that increased turnover and/or resistance to CD4+T-cell loss may contribute to the higher viral loads and increased severity of disease in neonatal hosts.
Adaptation of Puumala Hantavirus to Cell Culture Is Associated with Point Mutations in the Coding Region of the L Segment and in the Noncoding Regions of the S Segment
Authors: Nemirov Kirill , Lundkvist Åke , Vaheri Antti , Plyusnin Alexander
Published: July 2003

We previously developed a model for studies on hantavirus host adaptation and initiated genetic analysis of Puumala virus variants passaged in colonized bank voles and in cultured Vero E6 cells. With the data presented in this paper, the sequence comparison of the wild-type and Vero E6-adapted variants of Puumala virus, strain Kazan, has been completed. The only amino acid substitution that distinguished the two virus variants was found in the L protein, Ser versus Phe at position 2053. Another mutation found in the L segment, the silent transition C1053U, could result from the selection of a variant with altered L RNA folding. Nucleotide substitutions observed in individual L cDNA clones, most of them A→G and U→C transitions, suggested that the population of L RNA molecules is represented by quasispecies. The mutation frequency in the L segment quasispecies appeared to be similar to the corresponding values for the S and M quasispecies. Analysis of the cDNA clones with the complete S segment sequences from passage 20 confirmed our earlier conclusion that the cell-adapted genotype of the virus is represented mostly by variants with mutated S segment noncoding regions. However, the spectrum of the S segment quasispecies appeared to be changing, suggesting that, after the initial adaptation (passages 1 to 11), the viral population is still being driven by selection for variants with higher fitness.
The Coronavirus Spike Protein Is a Class I Virus Fusion Protein: Structural and Functional Characterization of the Fusion Core Complex
Authors: Bosch Berend Jan , van der Zee Ruurd , de Haan Cornelis A. M. , Rottier Peter J. M.
Published: July 2003

Coronavirus entry is mediated by the viral spike (S) glycoprotein. The 180-kDa oligomeric S protein of the murine coronavirus mouse hepatitis virus strain A59 is posttranslationally cleaved into an S1 receptor binding unit and an S2 membrane fusion unit. The latter is thought to contain an internal fusion peptide and has two 4,3 hydrophobic (heptad) repeat regions designated HR1 and HR2. HR2 is located close to the membrane anchor, and HR1 is some 170 amino acids (aa) upstream of it. Heptad repeat (HR) regions are found in fusion proteins of many different viruses and form an important characteristic of class I viral fusion proteins. We investigated the role of these regions in coronavirus membrane fusion. Peptides HR1 (96 aa) and HR2 (39 aa), corresponding to the HR1 and HR2 regions, were produced in
Escherichia coli
. When mixed together, the two peptides were found to assemble into an extremely stable oligomeric complex. Both on their own and within the complex, the peptides were highly alpha helical. Electron microscopic analysis of the complex revealed a rod-like structure ∼14.5 nm in length. Limited proteolysis in combination with mass spectrometry indicated that HR1 and HR2 occur in the complex in an antiparallel fashion. In the native protein, such a conformation would bring the proposed fusion peptide, located in the N-terminal domain of HR1, and the transmembrane anchor into close proximity. Using biological assays, the HR2 peptide was shown to be a potent inhibitor of virus entry into the cell, as well as of cell-cell fusion. Both biochemical and functional data show that the coronavirus spike protein is a class I viral fusion protein.
Strain Variation in an Emerging Iridovirus of Warm-Water Fishes
Authors: Goldberg Tony L. , Coleman David A. , Grant Emily C. , Inendino Kate R. , Philipp David P.
Published: July 2003

Although iridoviruses vary widely within and among genera with respect to their host range and virulence, variation within iridovirus species has been less extensively characterized. This study explores the nature and extent of intraspecific variation within an emerging iridovirus of North American warm-water fishes, largemouth bass virus (LMBV). Three LMBV isolates recovered from three distinct sources differed genetically and phenotypically. Genetically, the isolates differed in the banding patterns generated from amplified fragment length polymorphism analysis but not in their DNA sequences at two loci of different degrees of evolutionary stability. In vitro, the isolates replicated at identical rates in cell culture, as determined by real-time quantitative PCR of viral particles released into suspension. In vivo, the isolates varied over fivefold in virulence, as measured by the rate at which they induced mortality in juvenile largemouth bass. This variation was reflected in the viral loads of exposed fish, measured using real-time quantitative PCR; the most virulent viral strain also replicated to the highest level in fish. Together, these results justify the designation of these isolates as different strains of LMBV. Strain variation in iridoviruses could help explain why animal populations naturally infected with iridovirus pathogens vary so extensively in their clinical responses to infection. The results of this study are especially relevant to emerging iridoviruses of aquaculture systems and wildlife.
Feline Immunodeficiency Virus Orf-A Is Required for Virus Particle Formation and Virus Infectivity
Authors: Gemeniano Malou C. , Sawai Earl T. , Leutenegger Christian M. , Sparger Ellen E.
Published: July 2003

The
orf-A
(
orf-2
) gene of feline immunodeficiency virus (FIV) is a small open reading frame predicted to encode a 77-amino-acid protein that contains putative domains similar to those of the ungulate lentiviral Tat protein. Orf-A is reported to be critical for efficient viral replication in vitro and in vivo. A series of FIV-pPPR-derived proviruses with in-frame deletions and point mutations within
orf-A
were constructed and tested for replication in feline lymphoid cells. Orf-A mutant proviruses were also tested for viral gene and protein expression, viral particle formation, and virion infectivity. Deletions within
orf-A
severely restricted FIV replication in feline peripheral blood mononuclear cells (PBMC) and interleukin-2-dependent T-cell lines. In addition, substitutions of alanines for leucines in the putative leucine-rich domain, for cysteines in the putative cysteine-rich domain, and for a tryptophan at position 43 in Orf-A restricted the replication of FIV mutants. Deletions and point mutations in
orf-A
imposed a small effect or no effect on FIV long-terminal-repeat-driven viral gene expression and had no effect on viral protein expression. However, release of cell-free, virion-associated viral RNA in supernatants from cells transfected with
orf-A
mutant proviruses was severely restricted but was rescued by cotransfection with a wild-type Orf-A expression vector. In addition, virions derived from
orf-A
mutant proviruses expressed reduced infectivity for feline PBMC. Our findings suggest that Orf-A functions involve multiple steps of the FIV life cycle including both virion formation and infectivity. Furthermore, these observations suggest that Orf-A represents an FIV-encoded analog more similar to the accessory gene
vpr
,
vpu
, or
nef
than to the regulatory gene
tat
encoded by the primate lentiviruses.
De Novo Synthesis of Negative-Strand RNA by Dengue Virus RNA-Dependent RNA Polymerase In Vitro: Nucleotide, Primer, and Template Parameters
Authors: Nomaguchi Masako , Ackermann Matt , Yon Changsuek , You Shihyun , Padmanbhan R.
Published: July 2003

By using a purified dengue virus RNA-dependent RNA polymerase and a subgenomic 770-nucleotide RNA template, it was shown previously that the ratio of the de novo synthesis product to hairpin product formed was inversely proportional to increments of assay temperatures (20 to 40°C). In this study, the components of the de novo preinitiation complex are defined as ATP, a high concentration of GTP (500 μM), the polymerase, and the template RNA. Even when the 3′-terminal sequence of template RNA was mutated from -GGUUCU-3′ to -GGUUUU-3′, a high GTP concentration was required for de novo initiation, suggesting that high GTP concentration plays a conformational role. Furthermore, utilization of synthetic primers by the polymerase indicated that AGAA is the optimal primer whereas AG, AGA, and AGAACC were inefficient primers. Moreover, mutational analysis of the highly conserved 3′-terminal dinucleotide CU of the template RNA indicated that change of the 3′-terminal nucleotide from U to C reduced the efficiency about fivefold. The order of preference for the 3′-terminal nucleotide, from highest to lowest, is U, A∼G, and C. However, change of the penultimate nucleotide from C to U did not affect the template activity. A model consistent with these results is that the active site of the polymerase switches from a “closed” form, catalyzing de novo initiation through synthesis of short primers, to an “open” form for elongation of a double-stranded template-primer.
Development of Recombinant Vesicular Stomatitis Viruses That Exploit Defects in Host Defense To Augment Specific Oncolytic Activity
Authors: Obuchi Masatsugu , Fernandez Marilyn , Barber Glen N.
Published: July 2003

Vesicular stomatitis virus (VSV) is a negative-stranded RNA virus normally sensitive to the antiviral actions of alpha/beta interferon (IFN-α/β). Recently, we reported that VSV replicates to high levels in many transformed cells due, in part, to susceptible cells harboring defects in the IFN system. These observations were exploited to demonstrate that VSV can be used as a viral oncolytic agent to eradicate malignant cells in vivo while leaving normal tissue relatively unaffected. To attempt to improve the specificity and efficacy of this system as a potential tool in gene therapy and against malignant disease, we have genetically engineered VSV that expresses the murine IFN-β gene. The resultant virus (VSV-IFNβ) was successfully propagated in cells not receptive to murine IFN-α/β and expressed high levels of functional heterologous IFN-β. In normal murine embryonic fibroblasts (MEFs), the growth of VSV-IFNβ was greatly reduced and diminished cytopathic effect was observed due to the production of recombinant IFN-β, which by functioning in a manner involving autocrine and paracrine mechanisms induced an antiviral effect, preventing virus growth. However, VSV-IFNβ grew to high levels and induced the rapid apoptosis of transformed cells due to defective IFN pathways being prevalent and thus unable to initiate proficient IFN-mediated host defense. Importantly, VSV expressing the human IFN-β gene (VSV-hIFNβ) behaved comparably and, while nonlytic to normal human cells, readily killed their malignant counterparts. Similar to our in vitro observations, following intravenous and intranasal inoculation in mice, recombinant VSV (rVSV)-IFNβ was also significantly attenuated compared to wild-type VSV or rVSV expressing green fluorescent protein. However, VSV-IFNβ retained propitious oncolytic activity against metastatic lung disease in immunocompetent animals and was able to generate robust antitumor T-cell responses. Our data indicate that rVSV designed to exploit defects in mechanisms of host defense can provide the basis for new generations of effective, specific, and safer viral vectors for the treatment of malignant and other disease.
Investigation of Structural and Functional Motifs within the Vaccinia Virus A14 Phosphoprotein, an Essential Component of the Virion Membrane
Authors: Mercer Jason , Traktman Paula
Published: July 2003

We have previously reported the construction and characterization of an inducible recombinant virus in which expression of the vaccinia virus membrane protein A14 is experimentally regulated using the tetracycline operator-repressor system. Repression of A14, which results in a 1,000-fold reduction in viral yield, leads to an early block in viral morphogenesis characterized by the accumulation of large virosomes, empty “crescents” that fail to contact these virosomes, and, most strikingly, large numbers of aberrant 25-nm vesicles. Here we report the establishment of a transient-complementation system for the structure-function analysis of A14. We have constructed numerous mutant alleles of A14 designed to identify and test the importance of key structural and sequence motifs within A14, including sites of posttranslational modification, such as glycosylation, phosphorylation, and dimerization. From these studies we have determined that robust complementation ability requires an intact N terminus and two regions flanking the first membrane-spanning domain of A14. We show that A14 is modified by N-linked glycosylation both in vitro and in vivo. However, only a minority of A14 molecules are glycosylated in vivo and these are not encapsidated. In this report we also identify the sole phosphorylated serine residue of A14 as lying within the NHS
85
motif that undergoes glycosylation. Additionally, we show that the Cys
71
residue is required for intermolecular disulfide bond formation and describe the properties of a virus expressing an allele of A14 that cannot form disulfide-linked dimers.
Deletions in the Putative Cell Receptor-Binding Domain of Sindbis Virus Strain MRE16 E2 Glycoprotein Reduce Midgut Infectivity in
Aedes aegypti
Authors: Myles Kevin M. , Pierro Dennis J. , Olson Ken E.
Published: July 2003

The Sindbis virus (
Alphavirus
;
Togaviridae
) strain MRE16 efficiently infects
Aedes aegypti
mosquitoes that ingest a blood meal containing 8 to 9 log
10
PFU of virus/ml. However, a small-plaque variant of this virus, MRE16sp, poorly infects mosquitoes after oral infection with an equivalent titer. To determine the genetic differences between MRE16 and MRE16sp viruses, we have sequenced the MRE16sp structural genes and found a 90-nucleotide deletion in the E2 glycoprotein that spans the 3′ end of the coding region for the putative cell-receptor binding domain (CRBD). We examined the role of this deletion in oral infection of mosquitoes by constructing infectious clones pMRE16icΔE200-Y229 and pMRE16ic, representing MRE16 virus genomes with and without the deletion, respectively. A third infectious clone, pMRE16icΔE200-C220, was also constructed that contained a smaller deletion extending only to the 3′ terminus of the CRBD coding region. Virus derived from pMRE16ic replicated with the same efficiency as parental virus in vertebrate (BHK-21) and mosquito (C6/36) cells and orally infected
A. aegypti
. Viruses derived from pMRE16icΔE200-Y229 and pMRE16icΔE200-C220 replicated 10- to 100-fold less efficiently in C6/36 and BHK-21 cells than did MRE16ic virus. Each deletion mutant poorly infected
A. aegypti
and dramatically reduced midgut infectivity and dissemination. However, all viruses generated nearly equal titers (∼6.0 log
10
PFU/ml) in mosquitoes 4 days after infection by intrathoracic inoculation. These results suggest that the deleted portion of the E2 CRBD represents an important determinant of MRE16 virus midgut infectivity in
A. aegypti
.
Endocytosis of Hepatitis B Immune Globulin into Hepatocytes Inhibits the Secretion of Hepatitis B Virus Surface Antigen and Virions
Authors: Schilling Ralf , Ijaz Samreen , Davidoff Michail , Lee Jia Yee , Locarnini Stephen , Williams Roger , Naoumov Nikolai V.
Published: July 2003

Hepatitis B immunoglobulin is used for prophylaxis against hepatitis B virus (HBV) and is thought to act by neutralization of virions and hepatitis B virus surface antigen (HBsAg)-containing particles in circulation. Using a panel of hepatocyte-derived cell lines, the present study investigated in vitro whether HBs-specific immunoglobulin G (IgG) is internalized in hepatocytes and whether it interacts with HBsAg in the cells. By immunoelectron microscopy and immunoblotting, human IgG and FcRn receptor for IgG were demonstrated on cellular membranes and in cytoplasmic extracts, irrespective of the HBsAg status of the cells. Furthermore, HBsAg and anti-HBs were shown to be colocalized in the same cellular compartment by two-color confocal microscopy. Endocytosis of HBs-specific IgG caused intracellular accumulation of HBsAg in a dose-dependent manner and inhibited the secretion of HBsAg and HBV virions from the cells. These effects were not observed with F(ab)2fragments or nonimmune IgG as controls. The specificity of intracellular HBsAg- anti-HBs interaction was further investigated in cells transfected with HBV genomes expressing wild-type HBsAg or immune escape HBsAg (with a G145R mutation). Monoclonal anti-HBs markedly reduced the secretion of wild-type HBsAg, while the secretion of mutant HBsAg was not affected. These results suggest that HBs-specific IgG binds to hepatocytes and interacts with HBsAg within the cells. This may be relevant for the selection of surface antibody escape mutations.
Cell Cycle Arrest by Kaposi's Sarcoma-Associated Herpesvirus Replication-Associated Protein Is Mediated at both the Transcriptional and Posttranslational Levels by Binding to CCAAT/Enhancer-Binding Protein α and p21
CIP-1
Authors: Wu Frederick Y. , Wang Shizhen Emily , Tang Qi-Qun , Fujimuro Masahiro , Chiou Chuang-Jiun , Zheng Qizhi , Chen Honglin , Hayward S. Diane , Lane M. Daniel , Hayward Gary S.
Published: July 2003

Lytic-cycle replication of Kaposi's sarcoma-associated herpesvirus (KSHV) in PEL cells causes G
1
cell cycle arrest mediated by the virus-encoded replication-associated protein (RAP) (or K8 protein), which induces high-level expression of the cellular C/EBPα and p21 proteins. Here we have examined the mechanism of this induction at both the transcriptional and posttranslational levels. RAP proved to bind very efficiently to both C/EBPα and p21 and stabilized them by up to 10-fold from proteasome-mediated degradation in vitro. Cross-linking revealed that RAP itself forms stable dimers and tetramers in solution and forms higher-order complexes but not heterodimers with C/EBPα. Cotransfection of RAP with C/EBPα cooperatively stimulated both the C/EBPα and p21 promoters in luciferase reporter gene assays. Only the basic/leucine zipper region of RAP was needed for interaction with and stabilization of C/EBPα, but both the N-terminal and C-terminal domains were required for transcriptional augmentation. In vitro-translated RAP interfered with DNA binding by C/EBPα in electrophonetic mobility shift assay (EMSA) experiments but did not itself bind to the target C/EBPα sites or form supershifted bands. However, in endogenous chromatin immunoprecipitation (ChIP) assays with tetradecanoyl phorbol acetate-induced PEL cells, RAP proved to specifically associate with the C/EBPα promoter in vivo, but only in a C/EBPα-dependent manner, implying an in vivo piggyback interaction with DNA-bound C/EBPα. Expression of exogenous RAP (Ad-RAP) caused G
1
/S cell cycle arrest in human dermal microvascular endothelial cells and also induced both the C/EBPα and p21 proteins, which formed punctate nuclear patterns that colocalized with RAP in PML nuclear bodies. In the presence of RAP, C/EBPα was also efficiently recruited into viral DNA replication compartments in both infected and cotransfected cells. In support of a direct role for this interaction in viral DNA replication, three C/EBPα binding sites were identified by in vitro EMSA experiments within a 220-bp core segment of the duplicated KSHV Ori-Lyt region, and although RAP did not bind to Ori-Lyt DNA directly in vitro, both endogenous RAP and C/EBPα were found to be associated with the Ori-Lyt region by ChIP assays in lytically induced PEL cells. Finally, we found that the KSHV lytic cycle could not be triggered by either synchronizing KSHV latently infected PEL cells in G
1
phase or inducing p21 in a C/EBPα-independent process.
Residues in the Apical Domain of the Feline and Canine Transferrin Receptors Control Host-Specific Binding and Cell Infection of Canine and Feline Parvoviruses
Authors: Palermo Laura M. , Hueffer Karsten , Parrish Colin R.
Published: July 2003

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) capsids bind to the transferrin receptors (TfRs) of their hosts and use these receptors to infect cells. The binding is partially host specific, as FPV binds only to the feline TfR, while CPV binds to both the canine and feline TfRs. The host-specific binding is controlled by a combination of residues within a raised region of the capsid. To define the TfR structures that interact with the virus, we altered the apical domain of the feline or canine TfR or prepared chimeras of these receptors and tested the altered receptors for binding to FPV or CPV capsids. Most changes in the apical domain of the feline TfR did not affect binding, but replacing Leu221 with Ser or Asp prevented receptor binding to either FPV or CPV capsids, while replacing Leu221 with Lys resulted in a receptor that bound only to CPV but not to FPV. Analysis of recombinants of the feline and canine TfRs showed that sequences controlling CPV-specific binding were within the apical domain and that more than one difference between these receptors determined the CPV-specific binding of the canine TfR. Single changes within the canine TfR which removed a single amino acid insertion or which eliminated a glycosylation site gave that receptor the expanded ability to bind to FPV and CPV. In some cases, binding of capsids to mutant receptors did not result in infection, suggesting a structural role for the receptor in cell infection by the viruses.
Incorporation of Tick-Borne Encephalitis Virus Replicons into Virus-Like Particles by a Packaging Cell Line
Authors: Gehrke Rainer , Ecker Michael , Aberle Stephan W. , Allison Steven L. , Heinz Franz X. , Mandl Christian W.
Published: July 2003

RNA replicons derived from flavivirus genomes show considerable potential as gene transfer and immunization vectors. A convenient and efficient encapsidation system is an important prerequisite for the practical application of such vectors. In this work, tick-borne encephalitis (TBE) virus replicons and an appropriate packaging cell line were constructed and characterized. A stable CHO cell line constitutively expressing the two surface proteins prM/M and E (named CHO-ME cells) was generated and shown to efficiently export mature recombinant subviral particles (RSPs). When replicon NdΔME lacking the prM/M and E genes was introduced into CHO-ME cells, virus-like particles (VLPs) capable of initiating a single round of infection were released, yielding titers of up to 5 × 107/ml in the supernatant of these cells. Another replicon (NdΔCME) lacking the region encoding most of the capsid protein C in addition to proteins prM/M and E was not packaged by CHO-ME cells. As observed with other flavivirus replicons, both TBE virus replicons appeared to exert no cytopathic effect on their host cells. Sedimentation analysis revealed that the NdΔME-containing VLPs were physically distinct from RSPs and similar to infectious virions. VLPs could be repeatedly passaged in CHO-ME cells but maintained the property of being able to initiate only a single round of infection in other cells during these passages. CHO-ME cells can thus be used both as a source for mature TBE virus RSPs and as a safe and convenient replicon packaging cell line, providing the TBE virus surface proteins prM/M and E intrans.
Reovirus-Induced Alteration in Expression of Apoptosis and DNA Repair Genes with Potential Roles in Viral Pathogenesis
Authors: DeBiasi Roberta L. , Clarke Penny , Meintzer Suzanne , Jotte Robert , Kleinschmidt-Demasters B. K. , Johnson Gary L. , Tyler Kenneth L.
Published: July 2003

Reoviruses are a leading model for understanding cellular mechanisms of virus-induced apoptosis. Reoviruses induce apoptosis in multiple cell lines in vitro, and apoptosis plays a key role in virus-induced tissue injury of the heart and brain in vivo. The activation of transcription factors NF-κB and c-Jun are key events in reovirus-induced apoptosis, indicating that new gene expression is critical to this process. We used high-density oligonucleotide microarrays to analyze cellular transcriptional alterations in HEK293 cells after infection with reovirus strain T3A (i.e., apoptosis inducing) compared to infection with reovirus strain T1L (i.e., minimally apoptosis inducing) and uninfected cells. These strains also differ dramatically in their potential to induce apoptotic injury in hearts of infected mice in vivo—T3A is myocarditic, whereas T1L is not. Using high-throughput microarray analysis of over 12,000 genes, we identified differential expression of a defined subset of genes involved in apoptosis and DNA repair after reovirus infection. This provides the first comparative analysis of altered gene expression after infection with viruses of differing apoptotic phenotypes and provides insight into pathogenic mechanisms of virus-induced disease.
Local Delivery of CpG Oligodeoxynucleotides Induces Rapid Changes in the Genital Mucosa and Inhibits Replication, but Not Entry, of Herpes Simplex Virus Type 2
Authors: Ashkar Ali A. , Bauer Stefan , Mitchell William J. , Vieira Jeff , Rosenthal Kenneth L.
Published: July 2003

Mucosal surfaces are the entry sites for the vast majority of infectious pathogens and provide the first line of defense against infection. In addition to the epithelial barrier, the innate immune system plays a key role in recognizing and rapidly responding to invading pathogens via innate receptors, such as Toll-like receptors (TLR). Bacterial CpG DNA, a potent activator of innate immunity, is recognized by TLR9. Here, we confirm that local mucosal, but not systemic, delivery of CpG oligodeoxynucleotides (ODN) to the genital tract protects mice from a subsequent lethal vaginal herpes simplex virus type 2 (HSV-2) challenge. Since these effects were so local in action, we examined the genital mucosa. Local delivery of CpG ODN induced rapid proliferation and thickening of the genital epithelium and caused significant recruitment of inflammatory cells to the submucosa. Local CpG ODN treatment also resulted in inhibition of HSV-2 replication but had no effect on HSV-2 entry into the genital mucosa. CpG ODN-induced protection against HSV-2 was not associated with early increases in gamma interferon (IFN-γ) secretion in the genital tract, and CpG ODN-treated IFN-γ−/−mice were protected from subsequent challenge with a lethal dose of HSV-2. Treatment of human HEK-293 cells transfected with murine TLR9 showed that the antiviral activity of CpG ODN was mediated through TLR9. These studies suggest that local induction of mucosal innate immunity can provide protection against sexually transmitted infections, such as HSV-2 or possibly human immunodeficiency virus, at the mucosal surfaces.
Conditional Suppression of Cellular Genes: Lentivirus Vector-Mediated Drug-Inducible RNA Interference
Authors: Wiznerowicz Maciej , Trono Didier
Published: July 2003

RNA interference has emerged as a powerful technique to downregulate the expression of specific genes in cells and in animals, thus opening new perspectives in fields ranging from developmental genetics to molecular therapeutics. Here, we describe a method that significantly expands the potential of RNA interference by permitting the conditional suppression of genes in mammalian cells. Within a lentivirus vector background, we subjected the polymerase III promoter-dependent production of small interfering RNAs to doxycycline-controllable transcriptional repression. The resulting system can achieve the highly efficient and completely drug-inducible knockdown of cellular genes. As lentivirus vectors can stably transduce a wide variety of targets both in vitro and in vivo and can be used to generate transgenic animals, the present system should have broad applications.
Function of Herpes Simplex Virus Type 1 gD Mutants with Different Receptor-Binding Affinities in Virus Entry and Fusion
Authors: Milne Richard S. B. , Hanna Sheri L. , Rux Ann H. , Willis Sharon H. , Cohen Gary H. , Eisenberg Roselyn J.
Published: July 2003

We have studied the receptor-specific function of four linker-insertion mutants of herpes simplex virus type 1 glycoprotein D (gD) representing each of the functional regions of gD. We used biosensor analysis to measure binding of the gD mutants to the receptors HVEM (HveA) and nectin-1 (HveC). One of the mutants, gD(∇34t), failed to bind HVEMt but showed essentially wild-type (WT) affinity for nectin-1t. The receptor-binding kinetics and affinities of the other three gD mutants varied over a 1,000-fold range, but each mutant had the same affinity for both receptors. All of the mutants were functionally impaired in virus entry and cell fusion, and the levels of activity were strikingly similar in these two assays. gD(∇34)-containing virus was defective on HVEM-expressing cells but did enter nectin-1-expressing cells to about 60% of WT levels. This showed that the defect of this form of gD on HVEM-expressing cells was primarily one of binding and that this was separable from its later function in virus entry. gD(∇243t) showed WT binding affinity for both receptors, but virus containing this form of gD had a markedly reduced rate of entry, suggesting that gD(∇243) is impaired in a postbinding step in the entry process. There was no correlation between gD mutant activity in fusion or virus entry and receptor-binding affinity. We conclude that gD functions in virus entry and cell fusion regardless of its receptor-binding kinetics and that as long as binding to a functional receptor occurs, entry will progress.
Complete Genomic Sequencing Shows that Polioviruses and Members of Human Enterovirus Species C Are Closely Related in the Noncapsid Coding Region
Authors: Brown Betty , Oberste M. Steven , Maher Kaija , Pallansch Mark A.
Published: July 2003

The 65 human enterovirus serotypes are currently classified into five species:
Poliovirus
(3 serotypes),
Human enterovirus A
(HEV-A) (12 serotypes), HEV-B (37 serotypes), HEV-C (11 serotypes), and HEV-D (2 serotypes). Coxsackie A virus (CAV) serotypes 1, 11, 13, 15, 17, 18, 19, 20, 21, 22, and 24 constitute HEV-C. We have determined the complete genome sequences for the remaining nine HEV-C serotypes and compared them with the complete sequences of CAV21, CAV24, and the polioviruses. The viruses were most diverse in the capsid region (4 to 36% amino acid difference). A high degree of capsid sequence conservation (96% amino acid identity) suggests that CAV15 and CAV18 should be classified as strains of CAV11 and CAV13, respectively. In the 3CD region, CAV1, CAV19, and CAV22 differed from one another by only 1.2 to 1.4% and CAV11, CAV13, CAV17, CAV20, CAV21, CAV24, and the polioviruses differed from one another by only 1.2 to 3.6%. The two groups, however, differed from one another by 14.6 to 16.2%. The polioviruses as a group were monophyletic only in the capsid region. Only one group of serotypes (CAV1, CAV19, and CAV22) was consistently monophyletic in multiple genome regions. Incongruities among phylogenetic trees based on different genome regions strongly suggest that recombination has occurred between the polioviruses, CAV11, CAV13, CAV17, and CAV20. The close relationship among the polioviruses and CAV11, CAV13, CAV17, CAV20, CAV21, and CAV24 and the uniqueness of CAV1, CAV19, and CAV22 suggest that revisions should be made to the classification of these viruses.
Cellular Localization of Nectin-1 and Glycoprotein D during Herpes Simplex Virus Infection
Authors: Krummenacher Claude , Baribaud Isabelle , Eisenberg Roselyn J. , Cohen Gary H.
Published: July 2003

During viral entry, herpes simplex virus (HSV) glycoprotein D (gD) interacts with a specific cellular receptor such as nectin-1 (PRR1/HveC/CD111) or the herpesvirus entry mediator A (HVEM/HveA). Nectin-1 is involved in cell-to-cell adhesion. It is located at adherens junctions, where it bridges cells through homophilic or heterophilic interactions with other nectins. Binding of HSV gD prevents nectin-1-mediated cell aggregation. Since HSV gD affects the natural function of nectin-1, we further investigated the effects of gD expression on nectin-1 during HSV infection or in transfected cells. We also studied the importance of the interaction between nectin-1 and the cytoplasmic protein afadin for HSV entry and spread as well as the effects of infection on this interaction. In these investigations, we used a panel of cells expressing nectin-1 or nectin-1-green fluorescent protein fusions as the only mediators of HSV entry. During HSV infection, nectin-1 localization at adherens junction was dramatically altered in a manner dependent on gD expression. Nectin-1 and gD colocalized at cell contact areas between infected and noninfected cells and at the edges of plaques. This specific accumulation of gD at junctions was driven by expression of nectin-1 in
trans
on the surface of adjacent cells. Reciprocally, nectin-1 was maintained at junctions by the
trans
expression of gD in the absence of a cellular natural ligand. Our observations indicate that newly synthesized gD substitutes for nectin-1 of infected cells at junctions with noninfected cells. We propose that gD attracts and maintains the receptor at junctions where it can be used for virus spread.
Identification of an Insulator in AAVS1, a Preferred Region for Integration of Adeno-Associated Virus DNA
Authors: Ogata Toshihiko , Kozuka Takuyo , Kanda Tadahito
Published: July 2003

In latent adeno-associated virus (AAV) infection, the viral genome is integrated preferentially into the human chromosome 19 q arm at a specific region designated AAVS1, which has an open chromatin conformation as indicated by the presence of a DNase I-hypersensitive site (DHS-S1). We examined whether an insulator, which defines the domain of gene expression by directionally blocking the action of enhancers and by preventing the spread of heterochomatin, is present near the DHS-S1 in the middle of a 2.6-kbp AAVS1-related DNA fragment used in this study. The fragment, cloned into an Epstein-Barr virus (EBV)-based eukaryotic episomal plasmid, was introduced into HEK293 cells. The DHS-S1 on the plasmid replicating in the nuclei was hypersensitive to DNase I digestion, and thus, the EBV plasmid system was used in an enhancer-blocking assay with the 2.6-kbp DNA and two shortened DNAs, of 1.6 kbp and 336 bp, containing DHS-S1. The three DNA fragments, when inserted in the proper direction between the cytomegalovirus immediate-early enhancer and minimal promoter, repressed the expression of a reporter gene. Thus, the enhancer-blocking activity was located within the 336-bp DNA containing the entire region (300 bp) of DHS-S1. To investigate the prevention of repression caused by heterochromatin, a transgene-expressing cassette flanked by the two 336-bp DNAs placed in the enhancer-blocking direction was introduced into HEK293 and HeLa cells. All the cell clones examined with the cassette integrated into cell DNA continued to express the transgene, which indicates that the pair of 336-bp DNA apparently prevented the spread of heterochromatin. The results show that an insulator lies between nucleotides 17 and 354 near the DHS-S1 in AAVS1. In a gel shift test, the 336-bp DNA did not bind an in vitro-prepared CCCTC-binding factor that binds to the chicken β-globin insulator, suggesting that the AAVS1 insulator requires an as yet unidentified binding protein. The newly identified AAVS1 insulator is likely to contribute to the maintenance of an open chromatin conformation that affects the life cycle of AAV.
Intracellular Trafficking of a Palmitoylated Membrane-Associated Protein Component of Enveloped Vaccinia Virus
Authors: Husain Matloob , Moss Bernard
Published: July 2003

The F13L protein of vaccinia virus, an essential and abundant palmitoylated peripheral membrane component of intra- and extracellular enveloped virions, associates with Golgi, endosomal, and plasma membranes in the presence or absence of other viral proteins. In the present study, the trafficking of a fully functional F13L-green fluorescent protein (GFP) chimera in transfected and productively infected cells was analyzed using specific markers and inhibitors. We found that Sar1H79G, atrans-dominant-negative protein inhibitor of cargo transport from the endoplasmic reticulum, had no apparent effect on the intracellular distribution of F13L-GFP, suggesting that the initial membrane localization occurs at a downstream compartment of the secretory pathway. Recycling of F13L-GFP from the plasma membrane was demonstrated by partial colocalization with FM4-64, a fluorescent membrane marker of endocytosis. Punctate F13L-GFP fluorescence overlapped with clathrin and Texas red-conjugated transferrin, suggesting that endocytosis occurred via clathrin-coated pits. The inhibitory effects of chlorpromazine andtrans-dominant-negative forms of dynamin and Eps15 protein on the recycling of F13L-GFP provided further evidence for clathrin-mediated endocytosis. In addition, the F13L protein was specifically coimmunoprecipitated with α-adaptin, a component of the AP-2 complex that interacts with Eps15. Nocodazole and wortmannin perturbed the intracellular trafficking of F13L-GFP, consistent with its entry into late and early endosomes through the secretory and endocytic pathways, respectively. The recycling pathway described here provides a mechanism for the reutilization of the F13L protein following its deposition in the plasma membrane during the exocytosis of enveloped virions.
Identification of a C-Terminal Regulatory Motif in Hepatitis C Virus RNA-Dependent RNA Polymerase: Structural and Biochemical Analysis
Authors: Lévêque Vincent J.-P. , Johnson Robert B. , Parsons Stephen , Ren Jianxin , Xie Congping , Zhang Faming , Wang Q. May
Published: July 2003

The NS5B RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) is a key component of the viral replicase. Reported here is the three-dimensional structure of HCV NS5B polymerase, with the highlight on its C-terminal folding, determined by X-ray crystallography at 2.1-Å resolution. Structural analysis revealed that a stretch of C-terminal residues of HCV NS5B inserted into the putative RNA binding cleft, where they formed a hydrophobic pocket and interacted with several important structural elements. This region was found to be conserved and unique to the RNA polymerases encoded by HCV and related viruses. Through biochemical analyses, we confirmed that this region interfered with the binding of HCV NS5B to RNA. Deletion of this fragment from HCV NS5B enhanced the RNA synthesis rate up to ∼50-fold. These results provide not only direct experimental insights into the role of the C-terminal tail of HCV NS5B polymerase but also a working model for the RNA synthesis mechanism employed by HCV and related viruses.
Major Histocompatibility Complex Class I Alleles Associated with Slow Simian Immunodeficiency Virus Disease Progression Bind Epitopes Recognized by Dominant Acute-Phase Cytotoxic-T-Lymphocyte Responses
Authors: O'Connor David H. , Mothe Bianca R. , Weinfurter Jason T. , Fuenger Sarah , Rehrauer William M. , Jing Peicheng , Rudersdorf Richard R. , Liebl Max E. , Krebs Kendall , Vasquez Joshua , Dodds Elizabeth , Loffredo John , Martin Sarah , McDermott Adrian B. , Allen Todd M. , Wang Chenxi , Doxiadis G. G. , Montefiori David C. , Hughes Austin , Burton Dennis R. , Allison David B. , Wolinsky Steven M. , Bontrop Ronald , Picker Louis J. , Watkins David I.
Published: July 2003

Certain major histocompatibility complex class I (MHC-I) alleles are associated with delayed disease progression in individuals infected with human immunodeficiency virus (HIV) and in macaques infected with simian immunodeficiency virus (SIV). However, little is known about the influence of these MHC alleles on acute-phase cellular immune responses. Here we follow 51 animals infected with SIVmac239 and demonstrate a dramatic association between Mamu-A*01 and -B*17 expression and slowed disease progression. We show that the dominant acute-phase cytotoxic T lymphocyte (CTL) responses in animals expressing these alleles are largely directed against two epitopes restricted by Mamu-A*01 and one epitope restricted by Mamu-B*17. One Mamu-A*01-restricted response (Tat28-35SL8) and the Mamu-B*17-restricted response (Nef165-173IW9) typically select for viral escape variants in early SIVmac239 infection. Interestingly, animals expressing Mamu-A*1 and -B*17 have less variation in the Tat28-35SL8 epitope during chronic infection than animals that express only Mamu-A*01. Our results show that MHC-I alleles that are associated with slow progression to AIDS bind epitopes recognized by dominant CTL responses during acute infection and underscore the importance of understanding CTL responses during primary HIV infection.