bluuebunny/crossref_metadata_2025 huggingface dataset:
Alpha/Beta Interferon Protects against Lethal West Nile Virus Infection by Restricting Cellular Tropism and Enhancing Neuronal Survival
Authors: Samuel Melanie A. , Diamond Michael S.
Published: October 2005

West Nile virus (WNV) is a mosquito-borne flavivirus that is neurotropic in humans, birds, and other animals. While adaptive immunity plays an important role in preventing WNV spread to the central nervous system (CNS), little is known about how alpha/beta interferon (IFN-α/β) protects against peripheral and CNS infection. In this study, we examine the virulence and tropism of WNV in IFN-α/β receptor-deficient (IFN- α/βR
−/−
) mice and primary neuronal cultures. IFN-α/βR
−/−
mice were acutely susceptible to WNV infection through subcutaneous inoculation, with 100% mortality and a mean time to death (MTD) of 4.6 ± 0.7 and 3.8± 0.5 days after infection with 10
0
and 10
2
PFU, respectively. In contrast, congenic wild-type 129Sv/Ev mice infected with 10
2
PFU showed 62% mortality and a MTD of 11.9 ± 1.9 days. IFN-α/βR
−/−
mice developed high viral loads by day 3 after infection in nearly all tissues assayed, including many that were not infected in wild-type mice. IFN-α/βR
−/−
mice also demonstrated altered cellular tropism, with increased infection in macrophages, B cells, and T cells in the spleen. Additionally, treatment of primary wild-type neurons in vitro with IFN-β either before or after infection increased neuronal survival independent of its effect on WNV replication. Collectively, our data suggest that IFN-α/β controls WNV infection by restricting tropism and viral burden and by preventing death of infected neurons.
Kelp Fly Virus: a Novel Group of Insect Picorna-Like Viruses as Defined by Genome Sequence Analysis and a Distinctive Virion Structure
Authors: Hartley C. J. , Greenwood D. R. , Gilbert R. J. C. , Masoumi A. , Gordon K. H. J. , Hanzlik T. N. , Fry E. E. , Stuart D. I. , Scotti P. D.
Published: October 2005

The complete genomic sequence of kelp fly virus (KFV), originally isolated from the kelp fly,
Chaetocoelopa sydneyensis
, has been determined. Analyses of its genomic and structural organization and phylogeny show that it belongs to a hitherto undescribed group within the picorna-like virus superfamily. The single-stranded genomic RNA of KFV is 11,035 nucleotides in length and contains a single large open reading frame encoding a polypeptide of 3,436 amino acids with 5′ and 3′ untranslated regions of 384 and 343 nucleotides, respectively. The predicted amino acid sequence of the polypeptide shows that it has three regions. The N-terminal region contains sequences homologous to the baculoviral inhibitor of apoptosis repeat domain, an inhibitor of apoptosis commonly found in animals and in viruses with double-stranded DNA genomes. The second region contains at least two capsid proteins. The third region has three sequence motifs characteristic of replicase proteins of many plant and animal viruses, including a helicase, a 3C chymotrypsin-like protease, and an RNA-dependent RNA polymerase. Phylogenetic analysis of the replicase motifs shows that KFV forms a distinct and distant taxon within the picorna-like virus superfamily. Cryoelectron microscopy and image reconstruction of KFV to a resolution of 15 Å reveals an icosahedral structure, with each of its 12 fivefold vertices forming a turret from the otherwise smooth surface of the 20-Å-thick capsid. The architecture of the KFV capsid is unique among the members of the picornavirus superfamily for which structures have previously been determined.
The nsp2 Replicase Proteins of Murine Hepatitis Virus and Severe Acute Respiratory Syndrome Coronavirus Are Dispensable for Viral Replication
Authors: Graham Rachel L. , Sims Amy C. , Brockway Sarah M. , Baric Ralph S. , Denison Mark R.
Published: October 2005

The positive-stranded RNA genome of the coronaviruses is translated from ORF1 to yield polyproteins that are proteolytically processed into intermediate and mature nonstructural proteins (nsps). Murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) polyproteins incorporate 16 protein domains (nsps), with nsp1 and nsp2 being the most variable among the coronaviruses and having no experimentally confirmed or predicted functions in replication. To determine if nsp2 is essential for viral replication, MHV and SARS-CoV genome RNA was generated with deletions of the nsp2 coding sequence (MHVΔnsp2 and SARSΔnsp2, respectively). Infectious MHVΔnsp2 and SARSΔnsp2 viruses recovered from electroporated cells had 0.5 to 1 log
10
reductions in peak titers in single-cycle growth assays, as well as a reduction in viral RNA synthesis that was not specific for any positive-stranded RNA species. The Δnsp2 mutant viruses lacked expression of both nsp2 and an nsp2-nsp3 precursor, but cleaved the engineered chimeric nsp1-nsp3 cleavage site as efficiently as the native nsp1-nsp2 cleavage site. Replication complexes in MHVΔnsp2-infected cells lacked nsp2 but were morphologically indistinguishable from those of wild-type MHV by immunofluorescence. nsp2 expressed in cells by stable retroviral transduction was specifically recruited to viral replication complexes upon infection with MHVΔnsp2. These results demonstrate that while nsp2 of MHV and SARS-CoV is dispensable for viral replication in cell culture, deletion of the nsp2 coding sequence attenuates viral growth and RNA synthesis. These findings also provide a system for the study of determinants of nsp targeting and function.
Liver-Directed Gamma Interferon Gene Delivery in Chronic Hepatitis C
Authors: Shin Eui-Cheol , Protzer Ulrike , Untergasser Andreas , Feinstone Stephen M. , Rice Charles M. , Hasselschwert Dana , Rehermann Barbara
Published: October 2005

Gamma interferon (IFN-γ) has been shown to inhibit replication of subgenomic and genomic hepatitis C virus (HCV) RNAs in vitro and to noncytolytically suppress hepatitis B virus (HBV) replication in vivo. IFN-γ is also known for its immunomodulatory effects and as a marker of a successful cellular immune response to HCV. Therapeutic expression of IFN-γ in the liver may therefore facilitate resolution of chronic hepatitis C, an infection that is rarely resolved spontaneously. To analyze immunomodulatory and antiviral effects of liver-specific IFN-γ expression in vivo, we intravenously injected two persistently HCV-infected chimpanzees twice with a recombinant, replication-deficient HBV vector and subsequently with a recombinant adenoviral vector. These vectors expressed human IFN-γ under control of HBV- and liver-specific promoters, respectively. Gene transfer resulted in a transient increase of intrahepatic IFN-γ mRNA, without increase in serum alanine aminotransferase levels. Ex vivo analysis of peripheral blood lymphocytes demonstrated enhanced CD16 expression on T cells and upregulation of the liver-homing marker CXCR3. Moreover, an increased frequency of HCV-specific T cells was detected ex vivo in the peripheral blood and in vitro in liver biopsy-derived, antigen-nonspecifically expanded T-cell lines. None of these immunologic effects were observed in the third chimpanzee injected with an HBV control vector. Despite these immunologic effects of the experimental vector, however, IFN-γ gene transfer did not result in a significant and long-lasting decrease of HCV titers. In conclusion, liver-directed IFN-γ gene delivery resulted in HCV-specific and nonspecific activation of cellular immune responses but did not result in effective control of HCV replication.
VP24 of Marburg Virus Influences Formation of Infectious Particles
Authors: Bamberg Sandra , Kolesnikova Larissa , Möller Peggy , Klenk Hans-Dieter , Becker Stephan
Published: October 2005

The highly pathogenic enveloped Marburg virus (MARV) is composed of seven
structural proteins and the nonsegmented negative-sense viral RNA
genome. Four proteins (NP, VP35, VP30, and L) make up the helical
nucleocapsid, which is surrounded by a matrix that is composed of the
viral proteins VP40 and VP24. VP40 is functionally homologous to the
matrix proteins of other nonsegmented negative-strand RNA viruses. As
yet, the function of VP24 remains elusive. In the present study we
found that VP24 colocalized with inclusions in MARV-infected cells that
contain preformed nucleocapsids and with nucleocapsids outside the
inclusions. Coexpression studies revealed that VP24 is recruited into
the inclusions by the presence of NP. Furthermore, VP24 displayed
membrane-binding properties and was recruited into filamentous
virus-like particles (VLPs) that are induced by VP40. The incorporation
of VP24 altered neither the morphology of VLPs nor the budding
efficiency of VLPs. When VP24 was silenced in MARV-infected cells by
small interfering RNA technology, the release of viral particles was
significantly reduced while viral transcription and replication were
unimpaired. Our data support the idea that VP24 is essential for a
process that takes place after replication and transcription and before
budding of virus progeny. It is presumed that VP24 is necessary for the
formation of transport-competent nucleocapsids and/or the interaction
between the nucleocapsids and the budding sites at the plasma
membrane.
Simian Virus 5 V Protein Acts as an Adaptor, Linking DDB1 to STAT2, To Facilitate the Ubiquitination of STAT1
Authors: Precious B. , Childs K. , Fitzpatrick-Swallow V. , Goodbourn S. , Randall R. E.
Published: October 2005

The V protein of simian virus 5 (SV5) facilitates the ubiquitination and subsequent proteasome-mediated degradation of STAT1. Here we show, by visualizing direct protein-protein interactions and by using the yeast two-hybrid system, that while the SV5 V protein fails to bind to STAT1 directly, it binds directly and independently to both DDB1 and STAT2, two cellular proteins known to be essential for SV5-mediated degradation of STAT1. We also demonstrate that STAT1 and STAT2 interact independently of SV5 V and show that SV5 V protein acts as an adaptor molecule linking DDB1 to STAT2/STAT1 heterodimers, which in the presence of additional accessory cellular proteins, including Cullin 4a, can ubiquitinate STAT1. Additionally, we show that the avidity of STAT2 for V is relatively weak but is significantly enhanced by the presence of both STAT1 and DDB1, i.e., the complex of STAT1, STAT2, DDB1, and SV5 V is more stable than a complex of STAT2 and V. From these studies we propose a dynamic model in which SV5 V acts as a bridge, bringing together a DDB1/Cullin 4a-containing ubiquitin ligase complex and STAT1/STAT2 heterodimers, which leads to the degradation of STAT1. The loss of STAT1 results in a decrease in affinity of binding of STAT2 for V such that STAT2 either dissociates from V or is displaced from V by STAT1/STAT2 complexes, thereby ensuring the cycling of the DDB1 and SV5 V containing E3 complex for continued rounds of STAT1 ubiquitination and degradation.
The Retroviral Capsid Domain Dictates Virion Size, Morphology, and Coassembly of Gag into Virus-Like Particles
Authors: Ako-Adjei Danso , Johnson Marc C. , Vogt Volker M.
Published: October 2005

The retroviral structural protein, Gag, is capable of independently assembling into virus-like particles (VLPs) in living cells and in vitro. Immature VLPs of human immunodeficiency virus type 1 (HIV-1) and of Rous sarcoma virus (RSV) are morphologically distinct when viewed by transmission electron microscopy (TEM). To better understand the nature of the Gag-Gag interactions leading to these distinctions, we constructed vectors encoding several RSV/HIV-1 chimeric Gag proteins for expression in either insect cells or vertebrate cells. We used TEM, confocal fluorescence microscopy, and a novel correlative scanning EM (SEM)-confocal microscopy technique to study the assembly properties of these proteins. Most chimeric proteins assembled into regular VLPs, with the capsid (CA) domain being the primary determinant of overall particle diameter and morphology. The presence of domains between matrix and CA also influenced particle morphology by increasing the spacing between the inner electron-dense ring and the VLP membrane. Fluorescently tagged versions of wild-type RSV, HIV-1, or murine leukemia virus Gag did not colocalize in cells. However, wild-type Gag proteins colocalized extensively with chimeric Gag proteins bearing the same CA domain, implying that Gag interactions are mediated by CA. A dramatic example of this phenomenon was provided by a nuclear export-deficient chimera of RSV Gag carrying the HIV-1 CA domain, which by itself localized to the nucleus but relocalized to the cytoplasm in the presence of wild type HIV-1 Gag. Wild-type and chimeric Gag proteins were capable of coassembly into a single VLP as viewed by correlative fluorescence SEM if, and only if, the CA domain was derived from the same virus. These results imply that the primary selectivity of Gag-Gag interactions is determined by the CA domain.
CD8 T Cells Require Gamma Interferon To Clear Borna Disease Virus from the Brain and Prevent Immune System-Mediated Neuronal Damage
Authors: Hausmann Jürgen , Pagenstecher Axel , Baur Karen , Richter Kirsten , Rziha Hanns-Joachim , Staeheli Peter
Published: October 2005

Borna disease virus (BDV) frequently causes meningoencephalitis and fatal neurological disease in young but not old mice of strain MRL. Disease does not result from the virus-induced destruction of infected neurons. Rather, it is mediated byH-2k-restricted antiviral CD8 T cells that recognize a peptide derived from the BDV nucleoprotein N. Persistent BDV infection in mice is not spontaneously cleared. We report here that N-specific vaccination can protect wild-type MRL mice but not mutant MRL mice lacking gamma interferon (IFN-γ) from persistent infection with BDV. Furthermore, we observed a significant degree of resistance of old MRL mice to persistent BDV infection that depended on the presence of CD8 T cells. We found that virus initially infected hippocampal neurons around 2 weeks after intracerebral infection but was eventually cleared in most wild-type MRL mice. Unexpectedly, young as well as old IFN-γ-deficient MRL mice were completely susceptible to infection with BDV. Moreover, neurons in the CA1 region of the hippocampus were severely damaged in most diseased IFN-γ-deficient mice but not in wild-type mice. Furthermore, large numbers of eosinophils were present in the inflamed brains of IFN-γ-deficient mice but not in those of wild-type mice, presumably because of increased intracerebral synthesis of interleukin-13 and the chemokines CCL1 and CCL11, which can attract eosinophils. These results demonstrate that IFN-γ plays a central role in host resistance against infection of the central nervous system with BDV and in clearance of BDV from neurons. They further indicate that IFN-γ may function as a neuroprotective factor that can limit the loss of neurons in the course of antiviral immune responses in the brain.
Nonrandom Packaging of Host RNAs in Moloney Murine Leukemia Virus
Authors: Onafuwa-Nuga Adewunmi A. , King Steven R. , Telesnitsky Alice
Published: October 2005

Moloney murine leukemia virus (MLV) particles contain both viral genomic RNA and an assortment of host cell RNAs. Packaging of virus-encoded RNA is selective, with virions virtually devoid of spliced
env
mRNA and highly enriched for unspliced genome. Except for primer tRNA, it is unclear whether packaged host RNAs are randomly sampled from the cell or specifically encapsidated. To address possible biases in host RNA sampling, the relative abundances of several host RNAs in MLV particles and in producer cells were compared. Using 7SL RNA as a standard, some cellular RNAs, such as those of the Ro RNP, were found to be enriched in MLV particles in that their ratios relative to 7SL differed little, if at all, from their ratios in cells. Some RNAs were underrepresented, with ratios relative to 7SL several orders of magnitude lower in virions than in cells, while others displayed intermediate values. At least some enriched RNAs were encapsidated by genome-defective nucleocapsid mutants. Virion RNAs were not a random sample of the cytosol as a whole, since some cytoplasmic RNAs like tRNA
Met
were vastly underrepresented, while U6 spliceosomal RNA, which functions in the nucleus, was enriched. Real-time PCR demonstrated that
env
mRNA, although several orders of magnitude less abundant than unspliced viral RNA, was slightly enriched relative to actin mRNA in virions. These data demonstrate that certain host RNAs are nearly as enriched in virions as genomic RNA and suggest that Ψ
−
mRNAs and some other host RNAs may be specifically excluded from assembly sites.
β-Adrenoreceptors Reactivate Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication via PKA-Dependent Control of Viral RTA
Authors: Chang Margaret , Brown Helen J. , Collado-Hidalgo Alicia , Arevalo Jesusa M. , Galic Zoran , Symensma Tonia L. , Tanaka Lena , Deng Hongyu , Zack Jerome A. , Sun Ren , Cole Steve W.
Published: October 2005

Reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication is mediated by the viral RTA transcription factor, but little is known about the physiological processes controlling its expression or activity. Links between autonomic nervous system activity and AIDS-associated Kaposi's sarcoma led us to examine the potential influence of catecholamine neurotransmitters. Physiological concentrations of epinephrine and norepinephrine efficiently reactivated lytic replication of KSHV in latently infected primary effusion lymphoma cells via β-adrenergic activation of the cellular cyclic AMP/protein kinase A (PKA) signaling pathway. Effects were blocked by PKA antagonists and mimicked by pharmacological and physiological PKA activators (prostaglandin E2and histamine) or overexpression of the PKA catalytic subunit. PKA up-regulatedRTAgene expression, enhanced activity of theRTApromoter, and posttranslationally enhanced RTA'strans-activating capacity for its own promoter and heterologous lytic promoters (e.g., the viralPANgene). Mutation of predicted phosphorylation targets at RTA serines 525 and 526 inhibited PKA-mediated enhancement of RTAtrans-activating capacity. Given the high catecholamine levels at sites of KSHV latency such as the vasculature and lymphoid organs, these data suggest that β-adrenergic control of RTA might constitute a significant physiological regulator of KSHV lytic replication. These findings also suggest novel therapeutic strategies for controlling the activity of this oncogenic gammaherpesvirus in vivo.
Double-Stranded RNA Induces Sequence-Specific Antiviral Silencing in Addition to Nonspecific Immunity in a Marine Shrimp: Convergence of RNAInterference and Innate Immunity in the Invertebrate Antiviral Response?
Authors: Robalino Javier , Bartlett Thomas , Shepard Eleanor , Prior Sarah , Jaramillo Guillermo , Scura Edward , Chapman Robert W. , Gross Paul S. , Browdy Craig L. , Warr Gregory W.
Published: October 2005

Double-stranded
RNA (dsRNA) is a common by-product of viral infections and a potent
inducer of innate antiviral immune responses in vertebrates. In the
marine shrimp
Litopenaeus vannamei
, innate antiviral immunity
is also induced by dsRNA in a sequence-independent manner. In this
study, the hypothesis that dsRNA can evoke not only innate antiviral
immunity but also a sequence-specific antiviral response in shrimp was
tested. It was found that viral sequence-specific dsRNA affords potent
antiviral immunity in vivo, implying the involvement of RNA
interference (RNAi)-like mechanisms in the antiviral response of the
shrimp. Consistent with the activation of RNAi by virus-specific dsRNA,
endogenous shrimp genes could be silenced in a systemic fashion by the
administration of cognate long dsRNA. While innate antiviral immunity,
sequence-dependent antiviral protection, and gene silencing could all
be induced by injection of long dsRNA molecules, injection of short
interfering RNAs failed to induce similar responses, suggesting a size
requirement for extracellular dsRNA to engage antiviral mechanisms and
gene silencing. We propose a model of antiviral immunity in shrimp by
which viral dsRNA engages not only innate immune pathways but also an
RNAi-like mechanism to induce potent antiviral responses in
vivo.
Stochastic Interplay between Mutation and Recombination during the Acquisition of Drug Resistance Mutations in Human Immunodeficiency Virus Type 1
Authors: Althaus Christian L. , Bonhoeffer Sebastian
Published: October 2005

The emergence of drug resistance mutations in human immunodeficiency virus (HIV) has been a major setback in the treatment of infected patients. Besides the high mutation rate, recombination has been conjectured to have an important impact on the emergence of drug resistance. Population genetic theory suggests that in populations limited in size recombination may facilitate the acquisition of beneficial mutations. The viral population in an infected patient may indeed represent such a population limited in size, since current estimates of the effective population size range from 500 to 105. To address the effects of limited population size, we therefore expand a previously described deterministic population genetic model of HIV replication by incorporating the stochastic processes that occur in finite populations of infected cells. Using parameter estimates from the literature, we simulate the evolution of drug-resistant viral strains. The simulations show that recombination has only a minor effect on the rate of acquisition of drug resistance mutations in populations with effective population sizes as small as 1,000, since in these populations, viral strains typically fix beneficial mutations sequentially. However, for intermediate effective population sizes (104to 105), recombination can accelerate the evolution of drug resistance by up to 25%. Furthermore, a reduction in population size caused by drug therapy can be overcome by a higher viral mutation rate, leading to a faster evolution of drug resistance.
A Single Amino Acid Position in the Helper Component of Cauliflower Mosaic Virus Can Change the Spectrum of Transmitting Vector Species
Authors: Moreno Aranzazu , Hébrard Eugénie , Uzest Marilyne , Blanc Stéphane , Fereres Alberto
Published: October 2005

Viruses frequently use insect vectors to effect rapid spread through host populations. In plant viruses, vector transmission is the major mode of transmission, used by nearly 80% of species described to date. Despite the importance of this phenomenon in epidemiology, the specificity of the virus-vector relationship is poorly understood at both the molecular and the evolutionary level, and very limited data are available on the precise viral protein motifs that control specificity. Here, using the aphid-transmitted
Cauliflower mosaic virus
(CaMV) as a biological model, we confirm that the “noncirculative” mode of transmission dominant in plant viruses (designated “mechanical vector transmission” in animal viruses) involves extremely specific virus-vector recognition, and we identify an amino acid position in the “helper component” (HC) protein of CaMV involved in such recognition. Site-directed mutagenesis revealed that changing the residue at this position can differentially affect transmission rates obtained with various aphid species, thus modifying the spectrum of vector species for CaMV. Most interestingly, in a virus line transmitted by a single vector species, we observed the rapid appearance of a spontaneous mutant specifically losing its transmissibility by another aphid species. Hence, in addition to the first identification of an HC motif directly involved in specific vector recognition, we demonstrate that change of a virus to a different vector species requires only a single mutation and can occur rapidly and spontaneously.
Brd2/RING3 Interacts with a Chromatin-Binding Domain in the Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen 1 (LANA-1) That Is Required for Multiple Functions of LANA-1
Authors: Viejo-Borbolla Abel , Ottinger Matthias , Brüning Eva , Bürger Antje , König Regina , Kati Emrah , Sheldon Julie A. , Schulz Thomas F.
Published: October 2005

Latency-associated nuclear antigen 1 (LANA-1) of Kaposi's sarcoma-associated herpesvirus (KSHV) mediates the episomal replication of the KSHV genome, as well as transcriptional regulation, in latently infected cells. Interaction of LANA-1 with cellular chromatin is required for both these functions. An N-terminal heterochromatin-binding site in LANA-1 is essential for the replication and maintenance of latent episomes, as well as transcriptional regulation. We have recently described a C-terminal domain in LANA-1 that modulates the interaction with cellular interphase chromatin or elements of the nuclear matrix. Here, we used a series of LANA-1 deletion mutants to investigate the relationship between the different functions of LANA-1 and its interaction with the host chromatin-binding protein Brd2/RING3. Our findings suggest that the C-terminal chromatin-binding domain in LANA-1 is required for multiple LANA-1 functions, including the ability to bind to and replicate viral episomal DNA, to modulate transcription, and to interact with Brd2/RING3. Similar to the recently described tethering of bovine papillomavirus E2 protein to host chromatin via Brd4/MCAP, Brd2/RING3, another member of the Brd family of chromatin-binding proteins, therefore interacts with a chromatin-binding region of another viral latent nuclear protein and could play a role in its multiple functions.
Comparative Antigenicity and Immunogenicity of Hepadnavirus Core Proteins
Authors: Billaud Jean-Noel , Peterson Darrell , Schödel Florian , Chen Antony , Sallberg Matti , Garduno Fermin , Goldstein Phillip , McDowell Wendy , Hughes Janice , Jones Joyce , Milich David
Published: October 2005

The hepatitis B virus core protein (HBcAg) is a uniquely immunogenic particulate antigen and as such has been used as a vaccine carrier platform. The use of other hepadnavirus core proteins as vaccine carriers has not been explored. To determine whether the rodent hepadnavirus core proteins derived from the woodchuck (WHcAg), ground squirrel (GScAg), and arctic squirrel (AScAg) viruses possess immunogen characteristics similar to those of HBcAg, comparative antigenicity and immunogenicity studies were performed. The results indicate that (i) the rodent core proteins are equal in immunogenicity to or more immunogenic than HBcAg at the B-cell and T-cell levels; (ii) major histocompatibility complex (MHC) genes influence the immune response to the rodent core proteins (however, nonresponder haplotypes were not identified); (iii) WHcAg can behave as a T-cell-independent antigen in athymic mice; (iv) the rodent core proteins are not significantly cross-reactive with the HBcAg at the antibody level (however, the nonparticulate “eAgs” do appear to be cross-reactive); (v) the rodent core proteins are only partially cross-reactive with HBcAg at the CD4+T-cell level, depending on MHC haplotype; and (vi) the rodent core proteins are competent to function as vaccine carrier platforms for heterologous, B-cell epitopes. These results have implications for the selection of an optimal hepadnavirus core protein for vaccine design, especially in view of the “preexisting” immunity problem that is inherent in the use of HBcAg for human vaccine development.
Combinatorial Approach to Hepadnavirus-Like Particle Vaccine Design
Authors: Billaud Jean-Noel , Peterson Darrell , Barr Margaret , Chen Antony , Sallberg Matti , Garduno Fermin , Goldstein Phillip , McDowell Wendy , Hughes Janice , Jones Joyce , Milich David
Published: October 2005

The particulate hepatitis core protein (HBcAg) represents an efficient carrier platform with many of the characteristics uniquely required for the delivery of weak immunogens to the immune system. Although the HBcAg is highly immunogenic, the existing HBcAg-based platform technology has a number of theoretical and practical limitations, most notably the “preexisting immunity” and “assembly” problems. To address the assembly problem, we have developed the core protein from the woodchuck hepadnavirus (WHcAg) as a new particulate carrier platform system. WHcAg appears to tolerate insertions of foreign epitopes at a greater number of positions than HBcAg. For example, both within the external loop region and outside the loop region a total of 17 insertion sites were identified on WHcAg. Importantly, the identification of an expanded number of insertion sites was dependent on additional modifications to the C terminus that appear to stabilize the various internal insertions. Indeed, 21 separate C-terminal modifications have been generated that can be used in combination with the 17 insertion sites to ensure efficient hybrid WHcAg particle assembly. This combinatorial technology is also dependent on the sequence of the heterologous insert. Therefore, the three variables of insert position, C terminus, and epitope sequence are relevant in the design of hybrid WHcAg particles for vaccine purposes.
A Norovirus Protease Structure Provides Insights into Active and Substrate Binding Site Integrity
Authors: Nakamura Kentaro , Someya Yuichi , Kumasaka Takashi , Ueno Go , Yamamoto Masaki , Sato Takao , Takeda Naokazu , Miyamura Tatsuo , Tanaka Nobuo
Published: October 2005

Norovirus 3C-like proteases are crucial to proteolytic processing of norovirus polyproteins. We determined the crystal structure of the 3C-like protease from Chiba virus, a norovirus, at 2.8-Å resolution. An active site including Cys139 and His30 is present, as is a hydrogen bond network that stabilizes the active site conformation. In the oxyanion hole backbone, a structural difference was observed probably upon substrate binding. A peptide substrate/enzyme model shows that several interactions between the two components are critical for substrate binding and that the S1 and S2 sites appropriately accommodate the substrate P1 and P2 residues, respectively. Knowledge of the structure and a previous mutagenesis study allow us to correlate proteolysis and structure.
Prophylactic Alpha Interferon Treatment Increases the Therapeutic Index of Oncolytic Vesicular Stomatitis Virus Virotherapy for Advanced Hepatocellular Carcinoma in Immune-Competent Rats
Authors: Shinozaki Katsunori , Ebert Oliver , Suriawinata Arief , Thung Swan N. , Woo Savio L. C.
Published: October 2005

Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with intrinsic oncolytic specificity due to substantially attenuated antiviral responses in many tumors. We have recently reported that recombinant VSV vector can be used as an effective oncolytic agent to safely treat multifocal hepatocellular carcinoma (HCC) in the livers of immune-competent rats via hepatic artery infusion. When administered at doses above the maximum tolerated dose (MTD), however, the animals suffered from neurotoxicity and/or acute lethal hepatotoxicity. Since VSV is extremely sensitive to the antiviral actions of alpha/beta interferon (IFN-α/β) in normal cells, we tested if prophylactic treatment with rat IFN-α would enhance VSV safety without compromising treatment efficacy in tumor-bearing rats. We found that VSV retained its replication potential in human and rat HCC cells after preincubation with relatively high doses of rat and human IFN-α in vitro, and its MTD in tumor-bearing rats treated systemically with rat IFN-α at 66 IU/g body weight (BW), equivalent to a human IFN-α dose that is currently prescribed for patients with viral hepatitis, was elevated by more than 1/2 log unit. Furthermore, we demonstrate that intratumoral replication of VSV was not attenuated by administration of 66 IU/g BW rat IFN-α, as tumor response and survival advantage in VSV-treated rats in the presence or absence of rat IFN-α were equivalent. The results suggest that prophylactic rat IFN-α treatment elevates the therapeutic index of hepatic arterial VSV therapy for multifocal HCC in rats. Since human IFN-α is currently in clinical use, its prophylactic application should be considered in future clinical translational protocols for VSV-mediated oncolytic virotherapy as a novel therapeutic modality in patients with advanced HCC, as well as other types of cancer.
LFA-1 Is a Key Determinant for Preferential Infection of Memory CD4+T Cells by Human Immunodeficiency Virus Type 1
Authors: Tardif Mélanie R. , Tremblay Michel J.
Published: October 2005

Memory CD4+T cells are considered a stable latent reservoir for human immunodeficiency virus type 1 (HIV-1) and a barrier to eradication of this retroviral infection in patients under therapy. It has been shown that memory CD4+T cells are preferentially infected with HIV-1, but the exact mechanism(s) responsible for this higher susceptibility remains obscure. Previous findings indicate that incorporation of host-derived intercellular adhesion molecule 1 (ICAM-1) in HIV-1 increases virus infectivity. To measure the putative involvement of virus-anchored ICAM-1 in the preferential infection of memory cells by HIV-1, quiescent and activated naive and memory T-cell subsets were exposed to isogenic virions either lacking or bearing ICAM-1. Memory CD4+T cells were found to be more susceptible than naive CD4+T cells to infection with ICAM-1-bearing virions, as exemplified by a more important virus replication, an increase in integrated viral DNA copies, and a more efficient entry process. Interactions between virus-associated host ICAM-1 and cell surface LFA-1 under a cluster formation seem to be responsible for the preferential HIV-1 infection of the memory cell subset. Altogether, these data shed light on a potential mechanism by which HIV-1 preferentially targets long-lived memory CD4+T cells.
Human Immunodeficiency Virus Type 1 Vpr Interacts with Antiapoptotic Mitochondrial Protein HAX-1
Authors: Yedavalli Venkat S. R. K. , Shih Hsiu-Ming , Chiang Yu-Ping , Lu Chun-Yi , Chang Luan-Yin , Chen Mao-Yuan , Chuang Che-Yen , Dayton Andrew I. , Jeang Kuan-Teh , Huang Li-Min
Published: October 2005

Human immunodeficiency virus type 1 viral protein R (Vpr) is required for viral pathogenesis and has been implicated in T-cell apoptosis through its activation of caspase 3 and caspase 9 and perturbation of mitochondrial membrane potential. To understand better Vpr-mitochondria interaction, we report here the identification of antiapoptotic mitochondrial protein HAX-1 as a novel Vpr target. We show that Vpr and HAX-1 physically associate with each other. Overexpression of Vpr in cells dislocates HAX-1 from its normal residence in mitochondria and creates mitochondrion instability and cell death. Conversely, overexpression of HAX-1 suppressed the proapoptotic activity of Vpr.
Brome Mosaic Virus 1a Nucleoside Triphosphatase/Helicase Domain Plays Crucial Roles in Recruiting RNA Replication Templates
Authors: Wang Xiaofeng , Lee Wai-Ming , Watanabe Tokiko , Schwartz Michael , Janda Michael , Ahlquist Paul
Published: October 2005

Positive-strand RNA virus RNA replication is invariably membrane associated and frequently involves viral proteins with nucleoside triphosphatase (NTPase)/helicase motifs or activities. Brome mosaic virus (BMV) encodes two RNA replication factors: 1a has a C-terminal NTPase/helicase-like domain, and 2a
pol
has a central polymerase domain. 1a accumulates on endoplasmic reticulum membranes, recruits 2a
pol
, and induces 50- to 70-nm membrane invaginations (spherules) serving as RNA replication compartments. 1a also recruits BMV replication templates such as genomic RNA3. In the absence of 2a
pol
, 1a dramatically stabilizes RNA3 by transferring RNA3 to a membrane-associated, nuclease-resistant state that appears to correspond to the interior of the 1a-induced spherules. Prior results show that the 1a NTPase/helicase-like domain contributes to RNA recruitment. Here, we tested mutations in the conserved helicase motifs of 1a to further define the roles of this domain in RNA template recruitment. All 1a helicase mutations tested showed normal 1a accumulation, localization to perinuclear endoplasmic reticulum membranes, and recruitment of 2a
pol
. Most 1a helicase mutants also supported normal spherule formation. Nevertheless, these mutations severely inhibited RNA replication and 1a-induced stabilization of RNA3 in vivo. For such 1a mutants, the membrane-associated RNA3 pool was both reduced and highly susceptible to added nuclease. Thus, 1a recruitment of viral RNA templates to a membrane-associated, nuclease-resistant state requires additional functions beyond forming spherules and recruiting RNA to membranes, and these functions depend on the 1a helicase motifs. The possibility that, similar to some double-stranded RNA viruses, the 1a NTPase/helicase-like domain may be involved in importing viral RNAs into a preformed replication compartment is discussed.
Activation of Kaposi's Sarcoma-Associated Herpesvirus Lytic Gene Expression during Epithelial Differentiation
Authors: Johnson Andrew S. , Maronian Nicole , Vieira Jeffrey
Published: October 2005

The oral cavity has been identified as the major site for the shedding of infectious Kaposi's sarcoma-associated herpesvirus (KSHV). While KSHV DNA is frequently detected in the saliva of KSHV seropositive persons, it does not appear to replicate in salivary glands. Some viruses employ the process of epithelial differentiation for productive viral replication. To test if KSHV utilizes the differentiation of oral epithelium as a mechanism for the activation of lytic replication and virus production, we developed an organotypic raft culture model of epithelium using keratinocytes from human tonsils. This system produced a nonkeratinized stratified squamous oral epithelium in vitro, as demonstrated by the presence of nucleated cells at the apical surface; the expression of involucrin and keratins 6, 13, 14, and 19; and the absence of keratin 1. The activation of KSHV lytic-gene expression was examined in this system using rKSHV.219, a recombinant virus that expresses the green fluorescent protein during latency from the cellular EF-1α promoter and the red fluorescent protein (RFP) during lytic replication from the viral early PAN promoter. Infection of keratinocytes with rKSHV.219 resulted in latent infection; however, when these keratinocytes differentiated into a multilayered epithelium, lytic cycle activation of rKSHV.219 occurred, as evidenced by RFP expression, the expression of the late virion protein open reading frame K8.1, and the production of infectious rKSHV.219 at the epithelial surface. These findings demonstrate that KSHV lytic activation occurs as keratinocytes differentiate into a mature epithelium, and it may be responsible for the presence of infectious KSHV in saliva.
Interspecies Transmission of Chronic Wasting Disease Prions to Squirrel Monkeys (
Saimiri sciureus
)
Authors: Marsh Richard F. , Kincaid Anthony E. , Bessen Richard A. , Bartz Jason C.
Published: October 2005

Chronic wasting disease (CWD) is an emerging prion disease of deer and elk. The risk of CWD transmission to humans following exposure to CWD-infected tissues is unknown. To assess the susceptibility of nonhuman primates to CWD, two squirrel monkeys were inoculated with brain tissue from a CWD-infected mule deer. The CWD-inoculated squirrel monkeys developed a progressive neurodegenerative disease and were euthanized at 31 and 34 months postinfection. Brain tissue from the CWD-infected squirrel monkeys contained the abnormal isoform of the prion protein, PrP-res, and displayed spongiform degeneration. This is the first reported transmission of CWD to primates.
Dengue Fever in Humanized NOD/SCID Mice
Authors: Bente Dennis A. , Melkus Michael W. , Garcia J. Victor , Rico-Hesse Rebeca
Published: October 2005

The increased transmission and geographic spread of dengue fever (DF) and its more severe presentation, dengue hemorrhagic fever (DHF), make it the most important mosquito-borne viral disease of humans (50 to 100 million infections/year) (World Health Organization, Fact sheet 117, 2002). There are no vaccines or treatment for DF or DHF because there are no animal or other models of human disease; even higher primates do not show symptoms after infection (W. F. Scherer, P. K. Russell, L. Rosen, J. Casals, and R. W. Dickerman, Am. J. Trop. Med. Hyg.
27:
590-599, 1978). We demonstrate that nonobese diabetic/severely compromised immunodeficient (NOD/SCID) mice xenografted with human CD34
+
cells develop clinical signs of DF as in humans (fever, rash, and thrombocytopenia), when infected in a manner mimicking mosquito transmission (dose and mode). These results suggest this is a valuable model with which to study pathogenesis and test antidengue products.
An Aptamer That Neutralizes R5 Strains of Human Immunodeficiency Virus Type 1 Blocks gp120-CCR5 Interaction
Authors: Dey Antu K. , Khati Makobetsa , Tang Min , Wyatt Richard , Lea Susan M. , James William
Published: October 2005

We recently described the isolation and structural characterization of 2′-fluoropyrimidine-substituted RNA aptamers that bind to gp120 of R5 strains of human immunodeficiency virus type 1 and thereby potently neutralize the infectivity of phylogenetically diverse R5 strains. Here we investigate the physical basis of their antiviral action. We show that both N-linked oligosaccharides and the variable loops V1/V2 and V3 are not required for binding of one aptamer, B40, to gp120. Using surface plasmon resonance binding analyses, we show that the aptamer binds to the CCR5-binding site on gp120 in a relatively CD4-independent manner, providing a mechanistic explanation for its neutralizing potency.
Sequences Intervening between the Core Packaging Determinants Are Dispensable for Maintaining the Packaging Potential and Propagation of Feline Immunodeficiency Virus Transfer Vector RNAs
Authors: Mustafa Farah , Ghazawi Akela , Jayanth Preethi , Phillip Pretty Susan , Ali Jahabar , Rizvi Tahir A.
Published: October 2005

The packaging determinants of feline immunodeficiency virus (FIV) consist of two discontinuous core regions, extending from R to ∼150 bp of the 5′ untranslated region and the first ∼100 bp of
gag
. However, the role of sequences intervening between the core regions in packaging has not been clear. A mutational analysis was conducted to determine whether the intervening sequences played a role in FIV RNA packaging, using an in vivo packaging assay complemented with semiquantitative reverse transcriptase PCR. Our analyses reveal that the intervening sequences are dispensable not only for vector RNA packaging but also for propagation, confirming the discontinuous nature of the FIV packaging signal.
Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Induces a Strong Bend on Binding to Terminal Repeat DNA
Authors: Wong Lai-Yee , Wilson Angus C.
Published: October 2005

During latency, the Kaposi's sarcoma-associated herpesvirus genome is maintained as a circular episome, replicating in synchrony with host chromosomes. Replication requires the latency-associated nuclear antigen (LANA) and an origin of latent DNA replication located in the viral terminal repeats, consisting of two LANA binding sites (LBSs) and a GC-rich sequence. Here, we show that the recruitment of a LANA dimer to high-affinity site LBS-1 bends DNA by 57° and towards the major groove. The cooccupancy of LBS-1 and lower-affinity LBS-2 induces a symmetrical bend of 110°. By changing the origin architecture, LANA may help to assemble a specific nucleoprotein structure important for the initiation of DNA replication.
Eosinophils alter colonic epithelial barrier function: role for major basic protein
Authors: Furuta Glenn T. , Nieuwenhuis Edward E. S. , Karhausen Jorn , Gleich Gerald , Blumberg Richard S. , Lee James J. , Ackerman Steven J.
Published: October 2005

Mucosal eosinophils increase in a number of gastrointestinal diseases that are often associated with altered epithelial barrier function, including food allergic enteropathies and inflammatory bowel diseases. Although eosinophils are known to secrete biologically active mediators including granule proteins, their role in gastrointestinal diseases is uncertain. The aim of this study was to determine the impact of eosinophils on intestinal barrier function. Epithelial barrier function was determined in a coculture of eosinophils and T84 epithelial cells and in a murine model of T helper (Th) type 2-mediated colitis. Coculture conditions resulted in decreased transepithelial resistance (TER) and increased transepithelial flux. Cell-free coculture supernatants contained a ≥5-kDa soluble factor that also diminished TER; these supernatants contained the eosinophil-granule proteins major basic protein (MBP) and eosinophil-derived neurotoxin (EDN). T84 barrier function decreased significantly when basolateral surfaces were exposed to native human MBP but not EDN. Additional studies identified downregulation of the tight junctional molecule occludin as at least one mechanism for MBP action. MBP-null mice were protected from inflammation associated with oxazolone colitis compared with wild-type mice. In conclusion, MBP decreases epithelial barrier function and in this manner contributes to the pathogenesis of inflammatory bowel diseases.
Volume-activated chloride currents in interstitial cells of Cajal
Authors: Park Sung Jin , Mckay Catherine M. , Zhu Yaohui , Huizinga Jan D.
Published: October 2005

Interstitial cells of Cajal (ICC) undergo marked morphological changes on contraction of the musculature, making it essential to understand properties of mechanosensitive ion channels. The whole cell patch-clamp technique was used to identify and to characterize volume-activated Cl− currents in ICC cultured through the explant technique. Hypotonic solutions (≈210 mosM) activated an outwardly rectifying current, which reversed near the equilibrium potential for Cl−. Time-dependent inactivation occurred only at pulse potentials of +80 mV, with a time constant of 478 ± 182 ms. The degree of outward rectification was calculated using a rectification index, the ratio between the slope conductances of +65 and −55 mV, which was 13.9 ± 1.5 at 76 mM initial extracellular Cl− concentration. The sequence of relative anion permeability of the outwardly rectifying Cl− channel was I− > Cl− > aspartate−. The chloride channel blockers, DIDS and 5-nitro-2-(3-phenlypropl-amino)benzoic acid, caused a voltage-dependent block of the outwardly rectifying Cl− current, inhibition occurring primarily at depolarized potentials. On exposure to hypotonic solution, the slope conductance significantly increased at the resting membrane potential (−70 mV) from 1.2 ± 0.2 to 2.0 ± 0.4 nS and at the slow-wave plateau potential (−35 mV) from 2.1 ± 0.3 to 5.0 ± 1.0 nS. The current was constitutively active in ICC and contributed to the resting membrane potential and excitability at the slow-wave plateau. In conclusion, swelling or volume change will depolarize ICC through activation of outwardly rectifying chloride channels, thereby increasing cell excitability.