bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of a New Human Parechovirus Allows the Definition of Parechovirus Types and the Identification of RNA Structural Domains
Authors: Al-Sunaidi Mohammed , Williams Çiğdem H. , Hughes Pamela J. , Schnurr David P. , Stanway Glyn
Published: December 2006

Human
parechoviruses (HPeV), members of the
Parechovirus
genus of
Picornaviridae
, are frequent pathogens but have been
comparatively poorly studied, and little is known of their diversity,
evolution, and molecular biology. To increase the amount of information
available, we have analyzed 7 HPeV strains isolated in California
between 1973 and 1992. We found that, on the basis of VP1 sequences,
these fall into two genetic groups, one of which has not been
previously observed, bringing the number of known groups to five. While
these correlate partly with the three known serotypes, two members of
the HPeV2 serotype belong to different genetic groups. In view of the
growing importance of molecular techniques in diagnosis, we suggest
that genotype is an important criterion for identifying viruses, and we
propose that the genetic groups we have defined should be termed human
parechovirus types 1 to 5. Complete nucleotide sequence analysis of two
of the Californian isolates, representing two types, confirmed the
identification of a new genetic group and suggested a role for
recombination in parechovirus evolution. It also allowed the
identification of a putative HPeV1
cis
-acting replication
element, which is located in the VP0 coding region, as well as the
refinement of previously predicted 5′ and 3′
untranslated region structures. Thus, the results have significantly
improved our understanding of these common
pathogens.
Human Metapneumovirus Fusion Protein Vaccines That Are Immunogenic and Protective in Cotton Rats
Authors: Cseke Gabriella , Wright David W. , Tollefson Sharon J. , Johnson Joyce E. , Crowe James E. , Williams John V.
Published: December 2006

Human metapneumovirus (hMPV) is a recently described paramyxovirus that is a major cause of upper and lower respiratory infection in children and adults worldwide. A safe and effective vaccine could decrease the burden of disease associated with this novel pathogen. We previously reported the development of the cotton rat model of hMPV infection and pathogenesis (J. V. Williams et al., J. Virol. 79:10944-10951, 2005). We report here the immunogenicity of an hMPV fusion (F) protein in this model. We constructed DNA plasmids that exhibited high levels of expression of hMPV F in mammalian cells (DNA-F). These constructs were used to develop a novel strategy to produce highly pure, soluble hMPV F protein lacking the transmembrane domain (FΔTM). We then immunized cotton rats at 0 and 14 days with either control vector, DNA-F alone, DNA-F followed by FΔTM protein, or FΔTM alone. All groups were challenged intranasally at 28 days with live hMPV. All three groups that received some form of hMPV F immunization mounted neutralizing antibody responses and exhibited partial protection against virus shedding in the lungs compared to controls. The FΔTM-immunized animals showed the greatest degree of protection (>1,500-fold reduction in lung virus titer). All three immunized groups showed a modest reduction of nasal virus shedding. Neither evidence of a Th2-type response nor increased lung pathology were present in the immunized animals. We conclude that sequence-optimized hMPV F protein protects against hMPV infection when delivered as either a DNA or a protein vaccine in cotton rats.
Nuclear Import of the Preintegration Complex Is Blocked upon Infection by Human Immunodeficiency Virus Type 1 in Mouse Cells
Authors: Tsurutani Naomi , Yasuda Jiro , Yamamoto Naoki , Choi Byung-Il , Kadoki Motohiko , Iwakura Yoichiro
Published: December 2006

Mouse cells do not support human immunodeficiency virus type 1 (HIV-1) replication because of host range barriers at steps including virus entry, transcription, RNA splicing, polyprotein processing, assembly, and release. The exact mechanisms for the suppression, however, are not completely understood. To elucidate further the barriers against HIV-1 replication in mouse cells, we analyzed the replication of the virus in lymphocytes from human CD4/CXCR4 transgenic mice. Although primary splenocytes and thymocytes allowed the entry and reverse transcription of HIV-1, the integration efficiency of the viral DNA was greatly reduced in these cells relative to human peripheral blood mononuclear cells, suggesting an additional block(s) before or at the point of host chromosome integration of the viral DNA. Preintegration processes were further analyzed using HIV-1 pseudotyped viruses. The reverse transcription step of HIV-1 pseudotyped with the envelope of murine leukemia virus or vesicular stomatitis virus glycoprotein was efficiently supported in both human and mouse cells, but nuclear import of the preintegration complex (PIC) of HIV-1 was blocked in mouse cells. We found that green fluorescent protein (GFP)-labeled HIV-1 integrase, which is known to be important in the nuclear localization of the PIC, could not be imported into the nucleus of mouse cells, in contrast to human cells. On the other hand, GFP-Vpr localized exclusively to the nuclei of both mouse and human cells. These observations suggest that, due to the dysfunction of integrase, the nuclear localization of PIC is suppressed in mouse cells.
Inhibition of Human Immunodeficiency Virus Envelope Glycoprotein- Mediated Single Cell Lysis by Low-Molecular-Weight Antagonists of Viral Entry
Authors: Madani Navid , Hubicki Amy M. , Perdigoto Ana Luisa , Springer Martin , Sodroski Joseph
Published: December 2006

The coexpression of human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins and receptors leads to the lysis of single cells by a process that is dependent upon membrane fusion. This cell lysis was inhibited by low-molecular-weight compounds that interfere with receptor binding or with receptor-induced conformational transitions in the envelope glycoproteins. A peptide, T20, potently inhibited cell-cell fusion but had no effect on single cell lysis mediated by the HIV-1 envelope glycoproteins. Thus, critical events in the lysis of single cells by the HIV-1 envelope glycoproteins occur in intracellular compartments accessible only to small inhibitory compounds.
Prolonged Adherence of Human Immunodeficiency Virus-Derived Vector Particles to Hematopoietic Target Cells Leads to Secondary Transduction In Vitro and In Vivo
Authors: Pan Yung-Wei , Scarlett Jarrad M. , Luoh Tammy T. , Kurre Peter
Published: December 2006

Human immunodeficiency virus type 1-derived lentivirus vectors bearing the vesicular stomatitis virus G (VSV-G) envelope glycoprotein demonstrate a wide host range and can stably transduce quiescent hematopoietic stem cells. In light of concerns about biosafety and potential germ line transmission, they have been used predominantly for ex vivo strategies, thought to ensure the removal of excess surface-bound particles and prevent in vivo dissemination. Studies presented here instead reveal prolonged particle adherence after ex vivo exposure, despite serial wash procedures, with subsequent transduction of secondary target cells in direct and transwell cocultures. We explored the critical parameters affecting particle retention and transfer and show that attachment to the cell surface selectively protects virus particles from serum complement-mediated inactivation. Moreover, studies with nonmyeloablated murine recipients show that transplantation of vector-exposed, washed hematopoietic cells results in systemic dissemination of functional VSV-G/lentivector particles. We demonstrate genetic marking by inadvertent transfer of vector particles and prolonged expression of transgene product in recipient tissues. Our findings have implications for biosafety, vector design, and cell biology research.
Coordinated Regulation and Widespread Cellular Expression of Interferon-Stimulated Genes (ISG) ISG-49, ISG-54, and ISG-56 in the Central Nervous System after Infection with Distinct Viruses
Authors: Wacher Christie , Müller Marcus , Hofer Markus J. , Getts Daniel R. , Zabaras Regina , Ousman Shalina S. , Terenzi Fulvia , Sen Ganes C. , King Nicholas J. C. , Campbell Iain L.
Published: December 2006

The interferon (IFN)-stimulated genes (ISGs) ISG-49, ISG-54, and ISG-56 are highly responsive to viral infection, yet the regulation and function of these genes in vivo are unknown. We examined the simultaneous regulation of these ISGs in the brains of mice during infection with either lymphocytic choriomeningitis virus (LCMV) or West Nile virus (WNV). Expression of the ISG-49 and ISG-56 genes increased significantly during LCMV infection, being widespread and localized predominantly to common as well as distinct neuronal populations. Expression of the ISG-54 gene also increased but to lower levels and with a more restricted distribution. Although expression of the ISG-49, ISG-54, and ISG-56 genes was increased in the brains of LCMV-infected STAT1 and STAT2 knockout (KO) mice, this was blunted, delayed, and restricted to the choroid plexus, meninges, and endothelium. ISG-56 protein was regulated in parallel with the corresponding RNA transcript in the brain during LCMV infection in wild-type and STAT KO mice. Similar changes in ISG-49, ISG-54, and ISG-56 RNA levels and ISG-56 protein levels were observed in the brains of wild-type mice following infection with WNV. Thus, the ISG-49, ISG-54, and ISG-56 genes are coordinately upregulated in the brain during LCMV and WNV infection; this upregulation, in the case of LCMV, was totally (neurons) or partially (non-neurons) dependent on the IFN-signaling molecules STAT1 and STAT2. These findings suggest a dominant role for the ISG-49, ISG-54, and ISG-56 genes in the host response to different viruses in the central nervous system, where, particularly in neurons, these genes may have nonredundant functions.
Cell-Type-Specific Growth Restriction of Vesicular Stomatitis Virus polR Mutants Is Linked to Defective Viral Polymerase Function
Authors: Ostertag Derek , Hoblitzell-Ostertag Traci M. , Perrault Jacques
Published: December 2006

Vesicular stomatitis virus polR mutants synthesize defective RNA replication products in vitro and display growth restriction in some cultured cells (J. L. Chuang, R. L. Jackson, and J. Perrault, Virology 229:57-67, 1997). We show here that a recombinant virus carrying the polR N protein mutation (R179H) yielded ∼100-fold- and ∼40-fold-lower amounts of infectious virus than the wild type in mouse L-929 and rat 3Y1 cells, respectively, but only ∼3-fold less in hamster BHK cells. Virus genome accumulation was inhibited 6- to 10-fold in restricting cells, but transcription was not affected. No defect in encapsidation of replication products was detected, but virus protein accumulation was reduced two- to threefold in both restricting and nonrestricting cells. polR virus particles released from the latter were 5- to 10-fold less infectious than the wild type but showed no difference in protein composition. Phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF-2α) was enhanced ∼3-fold in polR versus wild-type virus-infected L-929 cells, but neither inhibition of host gene transcription nor inhibition of double-stranded RNA (dsRNA)-activated protein kinase showed significant effects on restriction. Conditioned medium studies revealed no evidence for secretion of antiviral factors from restricting cells. We conclude that the block in polR growth is due to the combined effect of reduced genome replication and lower infectivity of released virus particles and may be due to overproduction of dsRNA. An accompanying paper (D. Ostertag, T. M. Hoblitzell-Ostertag, and J. Perrault, J. Virol. 81:503-513, 2007) provides compelling evidence for the role of dsRNA in this unique restriction phenomenon.
Overproduction of Double-Stranded RNA in Vesicular Stomatitis Virus-Infected Cells Activates a Constitutive Cell-Type-Specific Antiviral Response
Authors: Ostertag Derek , Hoblitzell-Ostertag Traci M. , Perrault Jacques
Published: December 2006

In a companion paper (D. Ostertag, T. M. Hoblitzell-Ostertag, and J. Perrault, J. Virol. 81:492-502, 2007), we provided indirect evidence that cell-type-specific growth restriction of the vesicular stomatitis virus (VSV) polR mutants may be due to enhanced production of double-stranded RNA (dsRNA). We show here that polR growth in mouse L-929 cells was rescued by vaccinia virus coinfection and that sole expression of the vaccinia virus dsRNA-binding E3L protein, via coinfection with an engineered VSV minigenome, also restored polR growth. Expression of dsRNA-binding protein NS1A or NS1B from influenza virus, but not C protein from Sendai virus, which does not bind dsRNA, likewise effected polR rescue. The N-terminal dsRNA-binding domain of NS1A, only 73 amino acids in length, but not a full-size mutant NS1A lacking dsRNA-binding activity, restored polR growth. Both key aspects of polR growth restriction, namely inhibition of genome replication and release of low-infectivity virus particles, were countered by expression of the dsRNA-binding proteins. We tested the effects of overproducing dsRNA in wild-type VSV infections by coinfecting cells with a VSV recombinant expressing the sense strand of the enhanced green fluorescent protein gene (VSV-GFP) and one expressing the antisense strand (VSV-PFG). These coinfections mimicked all aspects of polR restriction, including host range, lack of effect on transcription, reduced virus particle infectivity, and insensitivity to inhibition of host gene transcription or dsRNA-activated protein kinase activity. We conclude that, for some cell types, overproduction of dsRNA during VSV infection triggers an immediate and constitutive host cell antiviral effector response independent of interferon induction or signaling.
Visualization of the Interaction between the Precursors of VPg, the Viral Protein Linked to the Genome ofTurnip Mosaic Virus, and the Translation Eukaryotic Initiation Factor iso 4E In Planta
Authors: Beauchemin Chantal , Boutet Nathalie , Laliberté Jean-François
Published: December 2006

The RNA genome ofTurnip mosaic virusis covalently linked at its 5′ end to a viral protein known as VPg. This protein binds to the translation eukaryotic initiation factor iso 4E [eIF(iso)4E]. This interaction has been shown to be important for virus infection, although its exact biological function(s) has not been elucidated. In this study, we investigated the subcellular site of the VPg-eIF(iso)4E interaction using bimolecular fluorescence complementation (BiFC). As a first step, eIF(iso)4E, 6K-VPg-Pro, and VPg-Pro were expressed as full-length green fluorescent protein (GFP) fusions inNicotiana benthamiana, and their subcellular localizations were visualized by confocal microscopy. eIF(iso)4E was predominantly associated with the endoplasmic reticulum (ER), and VPg-Pro was observed in the nucleus and possibly the nucleolus, while 6K-VPg-Pro-GFP induced the formation of cytoplasmic vesicles budding from the ER. In BiFC experiments, reconstituted green fluorescence was observed throughout the nucleus, with a preferential accumulation in subnuclear structures when the GFP split fragments were fused to VPg-Pro and eIF(iso)4E. On the other hand, the interaction of 6K-VPg-Pro with eIF(iso)4E was observed in cytoplasmic vesicles embedded in the ER. These data suggest that the association of VPg with the translation factor might be needed for two different functions, depending of the VPg precursor involved in the interaction. VPg-Pro interaction with eIF(iso)4E may be involved in perturbing normal cellular functions, while 6K-VPg-Pro interaction with the translation factor may be needed for viral RNA translation and/or replication.
Structure of the 5′ Nontranslated Region of the Coxsackievirus B3 Genome:Chemical Modification and Comparative Sequence Analysis
Authors: Bailey Jennifer M. , Tapprich William E.
Published: December 2006

Coxsackievirus B3 (CVB3) is a picornavirus which causes myocarditis and pancreatitis
and may play a role in type I diabetes. The viral genome is a single
7,400-nucleotide polyadenylated RNA encoding 11 proteins in a single
open reading frame. The 5′ end of the viral genome contains a
highly structured nontranslated region (5′NTR) which folds to
form an internal ribosome entry site (IRES) as well as structures
responsible for genome replication, both of which are critical for
virulence. A structural model of the CVB3 5′NTR, generated
primarily by comparative sequence analysis and energy minimization,
shows seven domains (I to VII). While this model provides a preliminary
basis for structural analysis, the model lacks comprehensive
experimental validation. Here we provide experimental evidence from
chemical modification analysis to determine the structure of the CVB3
5′NTR. Chemical probing results show that the theoretical model
for the CVB3 5′NTR is largely, but not completely, supported
experimentally. In combination with our chemical probing data, we have
used the RNASTRUCTURE algorithm and sequence comparison of 105
enterovirus sequences to provide evidence for novel secondary and
tertiary interactions. A comprehensive examination of secondary
structure is discussed, along with new evidence for tertiary
interactions. These include a loop E motif in domain III and a
long-range pairing interaction that links domain II to domain V. The
results of our work provide mechanistic insight into key functional
elements in the cloverleaf and IRES, thereby establishing a base of
structural information from which to interpret experiments with CVB3
and other
picornaviruses.
Borna Disease Virus Matrix Protein Is an Integral Component of the Viral Ribonucleoprotein Complex That Does Not Interfere with Polymerase Activity
Authors: Chase Geoffrey , Mayer Daniel , Hildebrand Antonia , Frank Ronald , Hayashi Yohei , Tomonaga Keizo , Schwemmle Martin
Published: December 2006

We have recently shown that the matrix protein M of Borna disease virus (BDV) copurifies with the affinity-purified nucleoprotein (N) from BDV-infected cells, suggesting that M is an integral component of the viral ribonucleoprotein complex (RNP). However, further studies were hampered by the lack of appropriate tools. Here we generated an M-specific rabbit polyclonal antiserum to investigate the intracellular distribution of M as well as its colocalization with other viral proteins in BDV-infected cells. Immunofluorescence analysis revealed that M is located both in the cytoplasm and in nuclear punctate structures typical for BDV infection. Colocalization studies indicated an association of M with nucleocapsid proteins in these nuclear punctate structures. In situ hybridization analysis revealed that M also colocalizes with the viral genome, implying that M associates directly with viral RNPs. Biochemical studies demonstrated that M binds specifically to the phosphoprotein P but not to N. Binding of M to P involves the N terminus of P and is independent of the ability of P to oligomerize. Surprisingly, despite P-M complex formation, BDV polymerase activity was not inhibited but rather slightly elevated by M, as revealed in a minireplicon assay. Thus, unlike M proteins of other negative-strand RNA viruses, BDV-M seems to be an integral component of the RNPs without interfering with the viral polymerase activity. We propose that this unique feature of BDV-M is a prerequisite for the establishment of BDV persistence.
Analysis of CD127 and KLRG1 Expression on Hepatitis C Virus-Specific CD8
+
T Cells Reveals the Existence of Different Memory T-Cell Subsets in the Peripheral Blood and Liver
Authors: Bengsch Bertram , Spangenberg Hans Christian , Kersting Nadine , Neumann-Haefelin Christoph , Panther Elisabeth , von Weizsäcker Fritz , Blum Hubert E. , Pircher Hanspeter , Thimme Robert
Published: December 2006

The differentiation and functional status of virus-specific CD8
+
T cells is significantly influenced by specific and ongoing antigen recognition. Importantly, the expression profiles of the interleukin-7 receptor alpha chain (CD127) and the killer cell lectin-like receptor G1 (KLRG1) have been shown to be differentially influenced by repetitive T-cell receptor interactions. Indeed, antigen-specific CD8
+
T cells targeting persistent viruses (e.g., human immunodeficiency virus and Epstein-Barr virus) have been shown to have low CD127 and high KLRG1 expressions, while CD8
+
T cells targeting resolved viral antigens (e.g., FLU) typically display high CD127 and low KLRG1 expressions. Here, we analyzed the surface phenotype and function of hepatitis C virus (HCV)-specific CD8
+
T cells. Surprisingly, despite viral persistence, we found that a large fraction of peripheral HCV-specific CD8
+
T cells were CD127
+
and KLRG1
−
and had good proliferative capacities, thus resembling memory cells that usually develop following acute resolving infection. Intrahepatic virus-specific CD8
+
T cells displayed significantly reduced levels of CD127 expression but similar levels of KLRG1 expression compared to the peripheral blood. These results extend previous studies that demonstrated central memory (CCR7
+
) and early-differentiated phenotypes of HCV-specific CD8
+
T cells and suggest that insufficient stimulation of virus-specific CD8
+
T cells by viral antigen may be responsible for this alteration in HCV-specific CD8
+
T-cell differentiation during chronic HCV infection.
Sheep-Passaged Bovine Spongiform Encephalopathy Agent Exhibits Altered Pathobiological Properties in Bovine-PrP Transgenic Mice
Authors: Espinosa Juan Carlos , Andréoletti Olivier , Castilla Joaquín , Herva María Eugenia , Morales Mónica , Alamillo Elia , San-Segundo Fayna Díaz , Lacroux Caroline , Lugan Séverine , Salguero Francisco Javier , Langeveld Jan , Torres Juan María
Published: December 2006

Sheep can be experimentally infected with bovine spongiform encephalopathy (BSE), and the ensuing disease is similar to scrapie in terms of pathogenesis and clinical signs. BSE infection in sheep is an animal and human health concern. In this study, the transmission in BoPrP-Tg110 mice of prions from BSE-infected sheep was examined and compared to the transmission of original cattle BSE in cattle and sheep scrapie prions. Our results indicate no transmission barrier for sheep BSE prions to infect BoPrP-Tg110 mice, but the course of the disease is accelerated compared to the effects of the original BSE isolate. The shortened incubation period of sheep BSE in the model was conserved in subsequent passage in BoPrP-Tg110 mice, indicating that it is not related to infectious titer differences. Biochemical signature, lesion profile, and PrP
Sc
deposition pattern of both cattle and sheep BSE were similar. In contrast, all three sheep scrapie isolates tested showed an evident transmission barrier and further adaptation in subsequent passage. Taken together, those data indicate that BSE agent can be altered by crossing a species barrier, raising concerns about the virulence of this new prion towards other species, including humans. The BoPrP-Tg110 mouse bioassay should be considered as a valuable tool for discriminating scrapie and BSE in sheep.
Viral Interactions in Human Lymphoid Tissue: Human Herpesvirus 7 Suppresses the Replication of CCR5-Tropic Human Immunodeficiency Virus Type 1 via CD4 Modulation
Authors: Lisco Andrea , Grivel Jean-Charles , Biancotto Angélique , Vanpouille Christophe , Origgi Francesco , Malnati Mauro S. , Schols Dominique , Lusso Paolo , Margolis Leonid B.
Published: December 2006

Human immunodeficiency virus (HIV) infection is often accompanied by infection with other pathogens that affect the clinical course of HIV disease. Here, we identified another virus, human herpesvirus 7 (HHV-7) that interferes with HIV type 1 (HIV-1) replication in human lymphoid tissue, where critical events of HIV disease occur. Like the closely related HHV-6, HHV-7 suppresses the replication of CCR5-tropic (R5) HIV-1 in coinfected blocks of human lymphoid tissue. Unlike HHV-6, which affects HIV-1 by upregulating RANTES, HHV-7 did not upregulate any CCR5-binding chemokine. Rather, the inhibition of R5 HIV-1 by HHV-7 was associated with a marked downregulation of CD4, the cellular receptor shared by HHV-7 and HIV-1. HHV-7-induced CD4 downregulation was sufficient for HIV-1 inhibition, since comparable downregulation of CD4 with cyclotriazadisulfonamide, a synthetic macrocycle that specifically modulates expression of CD4, resulted in the suppression of HIV infection similar to that seen in HHV-7-infected tissues. In contrast to R5 HIV-1, CXCR4-tropic (X4) HIV-1 was only minimally suppressed by HHV-7 coinfection. This selectivity in suppression of R5 and X4 HIV-1 is explained by a suppression of HHV-7 replication in X4- but not in R5-coinfected tissues. These results suggest that HIV-1 and HHV-7 may interfere in lymphoid tissue in vivo, thus potentially affecting the progression of HIV-1 disease. Knowledge of the mechanisms of interaction of HIV-1 with HHV-7, as well as with other pathogens that modulate HIV-1 replication, may provide new insights into HIV pathogenesis and lead to the development of new anti-HIV therapeutic strategies.
Epstein-Barr Virus BZLF1 Gene, a Switch from Latency to Lytic Infection, Is Expressed as an Immediate-Early Gene after Primary Infection of B Lymphocytes
Authors: Wen Wangrong , Iwakiri Dai , Yamamoto Koji , Maruo Seiji , Kanda Teru , Takada Kenzo
Published: December 2006

We demonstrate here that the Epstein-Barr virus (EBV) BZLF1 gene, a switch from latent infection to lytic infection, is expressed as early as 1.5 h after EBV infection in Burkitt's lymphoma-derived, EBV-negative Akata and Daudi cells and primary B lymphocytes. Since BZLF1 mRNA is expressed even when the cells are infected with EBV in the presence of anisomycin, an inhibitor of protein synthesis, its expression does not require prerequisite protein synthesis, indicating that BZLF1 is expressed as an immediate-early gene following primary EBV infection of B lymphocytes.
Mouse Hepatitis Virus Does Not Induce Beta Interferon Synthesis and Does Not Inhibit Its Induction by Double-Stranded RNA
Authors: Zhou Haixia , Perlman Stanley
Published: December 2006

Mouse hepatitis virus (MHV) does not induce interferon (IFN) production in fibroblasts or bone marrow-derived dendritic cells. In this report, we show that the essential IFN-β transcription factors NF-κB and IFN regulatory factor 3 are not activated for nuclear translocation and gene induction during infection. However, MHV was unable to inhibit the activation of these factors and subsequent IFN-β production induced by poly(I:C). Further, MHV infection did not inhibit IFN-β production mediated by known host pattern recognition receptors (PRRs) (RIG-I, Mda-5, and TLR3). These results are consistent with the notion that double-stranded RNA, produced during MHV infection, is not accessible to cellular PRRs.
Inefficient Human Immunodeficiency Virus Replication in Mobile Lymphocytes
Authors: Sourisseau Marion , Sol-Foulon Nathalie , Porrot Françoise , Blanchet Fabien , Schwartz Olivier
Published: December 2006

Cell-to-cell viral transfer facilitates the spread of lymphotropic retroviruses such as human immunodeficiency virus (HIV) and human T-cell leukemia virus (HTLV), likely through the formation of “virological synapses” between donor and target cells. Regarding HIV replication, the importance of cell contacts has been demonstrated, but this phenomenon remains only partly characterized. In order to alter cell-to-cell HIV transmission, we have maintained cultures under continuous gentle shaking and followed viral replication in this experimental system. In lymphoid cell lines, as well as in primary lymphocytes, viral replication was dramatically reduced in shaken cultures. To document this phenomenon, we have developed an assay to assess the relative contributions of free and cell-associated virions in HIV propagation. Acutely infected donor cells were mixed with carboxyfluorescein diacetate succinimidyl ester-labeled lymphocytes as targets, and viral production was followed by measuring HIV Gag expression at different time points by flow cytometry. We report that cellular contacts drastically enhance productive viral transfer compared to what is seen with infection with free virus. Productive cell-to-cell viral transmission required fusogenic viral envelope glycoproteins on donor cells and adequate receptors on targets. Only a few syncytia were observed in this coculture system. Virus release from donor cells was unaffected when cultures were gently shaken, whereas virus transfer to recipient cells was severely impaired. Altogether, these results indicate that cell-to-cell transfer is the predominant mode of HIV spread and help to explain why this virus replicates so efficiently in lymphoid organs.
Assembly of Two Independent Populations of Flock House Virus Particles with Distinct RNA Packaging Characteristics in the Same Cell
Authors: Venter P. Arno , Schneemann Anette
Published: December 2006

Flock House virus
(FHV;
Nodaviridae
) is a positive-strand RNA virus that encapsidates a bipartite genome consisting of RNA1 and RNA2. We recently showed that specific recognition of these RNAs for packaging into progeny particles requires coat protein translated from replicating viral RNA. In the present study, we investigated whether the entire assembly pathway, i.e., the formation of the initial nucleating complex and the subsequent completion of the capsid, is restricted to the same pool of coat protein subunits. To test this, coat proteins carrying either FLAG or hemagglutinin epitopes were synthesized from replicating or nonreplicating RNA in the same cell, and the resulting particle population and its RNA packaging phenotype were analyzed. Results from immunoprecipitation analysis and ion-exchange chromatography showed that the differentially tagged proteins segregated into two distinct populations of virus particles with distinct RNA packaging phenotypes. Particles assembled from coat protein that was translated from replicating RNA contained the FHV genome, whereas particles assembled from coat protein that was translated from nonreplicating mRNA contained random cellular RNA. These data demonstrate that only coat proteins synthesized from replicating RNA partake in the assembly of virions that package the viral genome and that RNA replication, coat protein translation, and virion assembly are processes that are tightly coupled during the life cycle of FHV.
Suppression of Coronavirus Replication by Inhibition of the MEK Signaling Pathway
Authors: Cai Yingyun , Liu Yin , Zhang Xuming
Published: December 2006

We previously demonstrated that infection of cultured cells with murine coronavirus mouse hepatitis virus (MHV) resulted in activation of the mitogen-activated protein kinase (Raf/MEK/ERK) signal transduction pathway (Y. Cai et al., Virology 355:152-163, 2006). Here we show that inhibition of the Raf/MEK/ERK signaling pathway by the MEK inhibitor UO126 significantly impaired MHV progeny production (a reduction of 95 to 99% in virus titer), which correlated with the phosphorylation status of ERK1/2. Moreover, knockdown of MEK1/2 and ERK1/2 by small interfering RNAs suppressed MHV replication. The inhibitory effect of UO126 on MHV production appeared to be a general phenomenon since the effect was consistently observed in all six different MHV strains and in three different cell types tested; it was likely exerted at the postentry steps of the virus life cycle because the virus titers were similarly inhibited from infected cells treated at 1 h prior to, during, or after infection. Furthermore, the treatment did not affect the virus entry, as revealed by the virus internalization assay. Metabolic labeling and reporter gene assays demonstrated that translation of cellular and viral mRNAs appeared unaffected by UO126 treatment. However, synthesis of viral genomic and subgenomic RNAs was severely suppressed by UO126 treatment, as demonstrated by a reduced incorporation of [
3
H]uridine and a decrease in chloramphenicol acetyltransferase (CAT) activity in a defective-interfering RNA-CAT reporter assay. These findings indicate that the Raf/MEK/ERK signaling pathway is involved in MHV RNA synthesis.
Adenovirus Ubiquitin-Protein Ligase Stimulates Viral Late mRNA NuclearExport
Authors: Woo Jennifer L. , Berk Arnold J.
Published: December 2006

Theadenovirus type 5 (Ad5) E1B-55K and E4orf6 proteins are required
together to stimulate viral late nuclear mRNA export to the cytoplasm
and to restrict host cell nuclear mRNA export during the late phase of
infection. Previous studies have shown that these two viral proteins
interact with the cellular proteins elongins B and C,
cullin 5, RBX1, and additional cellular proteins to form an E3
ubiquitin-protein ligase that polyubiquitinates p53 and probably one or
more subunits of the MRE11-RAD50-NBS1 (MRN) complex, directing their
proteasomal degradation. The MRN complex is required for cellular DNA
double-strand break repair and induction of the DNA damage response by
adenovirus infection. To determine if the ability of E1B-55K and E4orf6
to stimulate viral late mRNA nuclear export requires the
ubiquitin-protein ligase activity of this viral ubiquitin-protein
ligase complex, we designed and expressed a dominant-negative mutant
form of cullin 5 in HeLa cells before infection with wild-type Ad5 or
the E1B-55K null mutant
dl
1520. The dominant-negative cullin 5
protein stabilized p53 and the MRN complex, indicating that it
inhibited the viral ubiquitin-protein ligase but had no effect on viral
early mRNA synthesis, early protein synthesis, or viral DNA
replication. However, expression of the dominant-negative cullin 5
protein caused a decrease in viral late protein synthesis and viral
nuclear mRNA export similar to the phenotype produced by mutations in
E1B-55K. We conclude that the stimulation of adenovirus late mRNA
nuclear export by E1B-55K and E4orf6 results from the ubiquitin-protein
ligase activity of the adenovirus ubiquitin-protein ligase
complex.
Mechanisms of Gastrointestinal CD4+T-Cell Depletion during Acuteand Early Human Immunodeficiency Virus Type 1 Infection
Authors: Mehandru Saurabh , Poles Michael A. , Tenner-Racz Klara , Manuelli Victoria , Jean-Pierre Patrick , Lopez Peter , Shet Anita , Low Andrea , Mohri Hiroshi , Boden Daniel , Racz Paul , Markowitz Martin
Published: December 2006

During acute and early human immunodeficiency virus type 1 (HIV-1) infection (AEI) more than 50% of CD4+T cells are preferentially depleted from the gastrointestinal (GI) lamina propria. To better understand the underlying mechanisms, we studied virological and immunological events within the peripheral blood (PB) and GI tract during AEI. A total of 32 AEI subjects and 18 uninfected controls underwent colonic biopsy. HIV-1 viral DNA and RNA levels were quantified in CD4+T cells derived from the GI tract and PB by using real-time PCR. The phenotype of infected cells was characterized by using combinations of immunohistochemistry and in situ hybridization. Markers of immunological memory, activation, and proliferation were examined by flow cytometry and immunohistochemistry, and the host-derived cytotoxic cellular response was examined by using immunohistochemistry. GI CD4+T cells harbored, on average, 13-fold higher HIV-1 viral DNA levels and 10-fold higher HIV-1 RNA levels than PB CD4+T cells during AEI. HIV-1 RNA was detected in both “activated” and“ nonactivated” mucosal CD4+T cells. A significantly higher number of activated and proliferating T cells were detected in the GI tract compared to the PB, and a robust cytotoxic response (HIV-1 specificity not determined) was detected in the GI tract as early as 18 days postinfection. Mucosal CD4+T-cell depletion is multifactorial. Direct viral infection likely accounts for the earliest loss of CD4+T cells. Subsequently, ongoing infection of susceptible CD4+T cells, along with activation-induced cellular death and host cytotoxic cellular response, are responsible for the persistence of the lesion.
Evidence for Persistent, Occult Infection in Neonatal Macaques following Perinatal Transmission of Simian-Human Immunodeficiency Virus SF162P3
Authors: Jayaraman Pushpa , Zhu Tuofu , Misher Lynda , Mohan Deepika , Kuller LaRene , Polacino Patricia , Richardson Barbra A. , Bielefeldt-Ohmann Helle , Anderson David , Hu Shiu-Lok , Haigwood Nancy L.
Published: December 2006

To model human immunodeficiency virus (HIV) perinatal transmission, we studied infection of simian-human immunodeficiency virus (SHIV) SF162P3 in 10 pregnant
Macaca nemestrina
females and their offspring. Four of nine infants born to and suckled by these dams had evidence of infection, a transmission rate of 44.4% (95% confidence interval, 13.7% to 78.8%). We quantified transplacentally acquired and de novo Env-specific immunoglobulin G (IgG), IgM, and neutralizing antibodies in newborns. Transmission of escape variants was confirmed. In utero infection (
n
= 1) resulted in high viremia, depletion of peripheral CD4
+
T cells, and rapid evolution of
env
in blood and tissues. Peripartum or postpartum SHIV infection (
n
= 3) resulted in postacute viral control that was undetectable by very sensitive multiplex PCR, despite increasing antibodies. Seropositive infants with highly controlled viremia had homogeneous peripheral blood
env
sequences, and their tissues had <3 copies per million cells. A high incidence of seropositive virus-low or -negative SHIV infection in infant macaques has implications for HIV type 1 perinatal transmission and detection.
The Switch from Latent to Productive Infection in Epstein-Barr Virus-Infected B Cells Is Associated with Sensitization to NK Cell Killing
Authors: Pappworth Isabel Y. , Wang Eddie C. , Rowe Martin
Published: December 2006

Following activation of Epstein-Barr virus (EBV)-infected B cells from latent to productive (lytic) infection, there is a concomitant reduction in the level of cell surface major histocompatibility complex (MHC) class I molecules and an impaired antigen-presenting function that may facilitate evasion from EBV-specific CD8+cytotoxic T cells. In some other herpesviruses studied, most notably human cytomegalovirus (HCMV), evasion of virus-specific CD8+effector responses via downregulation of surface MHC class I molecules is supplemented with specific mechanisms for evading NK cells. We now report that EBV differs from HCMV in this respect. While latently infected EBV-positive B cells were resistant to lysis by two NK lines and by primary polyclonal NK cells from peripheral blood, these effectors efficiently killed cells activated into the lytic cycle. Susceptibility to NK lysis coincided not only with downregulation of HLA-A, -B, and -C molecules that bind to the KIR family of inhibitory receptors on NK cells but also with downregulation of HLA-E molecules binding the CD94/NKG2A inhibitory receptors. Conversely, ULBP-1 and CD112, ligands for the NK cell-activating receptors NKG2D and DNAM-1, respectively, were elevated. Susceptibility of the virus-producing target cells to NK cell lysis was partially reversed by blocking ULBP-1 or CD112 with specific antibodies. These results highlight a fundamental difference between EBV and HCMV with regards to evasion of innate immunity.
C5 Modulates Airway Hyperreactivity and Pulmonary Eosinophilia during Enhanced Respiratory Syncytial Virus Disease by Decreasing C3a Receptor Expression
Authors: Melendi Guillermina A. , Hoffman Scott J. , Karron Ruth A. , Irusta Pablo M. , Laham Federico R. , Humbles Alison , Schofield Brian , Pan Chien-Hsiung , Rabold Richard , Thumar Bhagvanji , Thumar Adeep , Gerard Norma P. , Mitzner Wayne , Barnum Scott R. , Gerard Craig , Kleeberger Steven R. , Polack Fernando P.
Published: December 2006

Enhanced respiratory syncytial virus disease, a serious pulmonary disorder that affected recipients of an inactivated vaccine against respiratory syncytial virus in the 1960s, has delayed the development of vaccines against the virus. The enhanced disease was characterized by immune complex-mediated airway hyperreactivity and a severe pneumonia associated with pulmonary eosinophilia. In this paper, we show that complement factors contribute to enhanced-disease phenotypes. Mice with a targeted disruption of complement component C5 affected by the enhanced disease displayed enhanced airway reactivity, lung eosinophilia, and mucus production compared to wild-type mice and C5-deficient mice reconstituted with C5. C3aR expression in bronchial epithelial and smooth muscle cells in the lungs of C5-deficient mice was enhanced compared to that in wild-type and reconstituted rodents. Treatment of C5-deficient mice with a C3aR antagonist significantly attenuated airway reactivity, eosinophilia, and mucus production. These results indicate that C5 plays a crucial role in modulating the enhanced-disease phenotype, by affecting expression of C3aR in the lungs. These findings reveal a novel autoregulatory mechanism for the complement cascade that affects the innate and adaptive immune responses.
Tubulovesicular Structures within Vesicular Stomatitis Virus G Protein-Pseudotyped Lentiviral Vector Preparations Carry DNA and Stimulate Antiviral Responses via Toll-Like Receptor 9
Authors: Pichlmair Andreas , Diebold Sandra S. , Gschmeissner Stephen , Takeuchi Yasuhiro , Ikeda Yasuhiro , Collins Mary K. , Reis e Sousa Caetano
Published: December 2006

Recombinant lentiviral vectors (LVs) are commonly used as research tools and are being tested in the clinic as delivery agents for gene therapy. Here, we show that Vesicular stomatitis virus G protein (VSV-G)-pseudotyped LV preparations produced by transient transfection are heavily contaminated with tubulovesicular structures (TVS) of cellular origin, which carry nucleic acids, including the DNA plasmids originally used for LV generation. The DNA carried by TVS can act as a stimulus for innate antiviral responses, triggering Toll-like receptor 9 and inducing alpha/beta interferon production by plasmacytoid dendritic cells (pDC). Removal of TVS markedly reduces the ability of VSV-G-pseudotyped LV preparations to activate pDC. Conversely, virus-free TVS are sufficient to stimulate pDC and act as potent adjuvants in vivo, eliciting T- and B-cell responses to coadministered proteins. These results highlight the role of by-products of virus production in determining the immunostimulatory properties of recombinant virus preparations and suggest possible strategies for diminishing responses to LVs in gene therapy and in research use.
Hijacking Components of the Cellular Secretory Pathway for Replication of Poliovirus RNA
Authors: Belov George A. , Altan-Bonnet Nihal , Kovtunovych Gennadiy , Jackson Catherine L. , Lippincott-Schwartz Jennifer , Ehrenfeld Ellie
Published: December 2006

Infection of cells with poliovirus induces a massive intracellular membrane reorganization to form vesicle-like structures where viral RNA replication occurs. The mechanism of membrane remodeling remains unknown, although some observations have implicated components of the cellular secretory and/or autophagy pathways. Recently, we showed that some members of the Arf family of small GTPases, which control secretory trafficking, became membrane-bound after the synthesis of poliovirus proteins in vitro and associated with newly formed membranous RNA replication complexes in infected cells. The recruitment of Arfs to specific target membranes is mediated by a group of guanine nucleotide exchange factors (GEFs) that recycle Arf from its inactive, GDP-bound state to an active GTP-bound form. Here we show that two different viral proteins independently recruit different Arf GEFs (GBF1 and BIG1/2) to the new structures that support virus replication. Intracellular Arf-GTP levels increase ∼4-fold during poliovirus infection. The requirement for these GEFs explains the sensitivity of virus growth to brefeldin A, which can be rescued by the overexpression of GBF1. The recruitment of Arf to membranes via specific GEFs by poliovirus proteins provides an important clue toward identifying cellular pathways utilized by the virus to form its membranous replication complex.
Compensatory Mutations in E1, p7, NS2, and NS3 Enhance Yields of Cell Culture-Infectious Intergenotypic Chimeric Hepatitis C Virus
Authors: Yi MinKyung , Ma Yinghong , Yates Jeremy , Lemon Stanley M.
Published: December 2006

There is little understanding of mechanisms underlying the assembly and release of infectious hepatitis C virus (HCV) from cultured cells. Cells transfected with synthetic genomic RNA from a unique genotype 2a virus (JFH1) produce high titers of virus, while virus yields are much lower with a prototype genotype 1a RNA containing multiple cell culture-adaptive mutations (H77S). To characterize the basis for this difference in infectious particle production, we constructed chimeric genomes encoding the structural proteins of H77S within the background of JFH1. RNAs encoding polyproteins fused at the NS2/NS3 junction (“H-NS2/NS3-J”) and at a site of natural, intergenotypic recombination within NS2 [“H-(NS2)-J”] produced infectious virus. In contrast, no virus was produced by a chimera fused at the p7-NS2 junction. Chimera H-NS2/NS3-J virus (vH-NS2/NS3-J) recovered from transfected cultures contained compensatory mutations in E1 and NS3 that were essential for the production of infectious virus, while yields of infectious vH-(NS2)-J were enhanced by mutations within p7 and NS2. These compensatory mutations were chimera specific and did not enhance viral RNA replication or polyprotein processing; thus, they likely compensate for incompatibilities between proteins of different genotypes at sites of interactions essential for virus assembly and/or release. Mutations in p7 and NS2 acted additively and increased the specific infectivity of vH-(NS2)-J particles, while having less impact on the numbers of particles released. We conclude that interactions between NS2 and E1 and p7 as well as between NS2 and NS3 are essential for virus assembly and/or release and that each of these viral proteins plays an important role in this process.
Replicase Genes of Murine Coronavirus Strains A59 and JHM Are Interchangeable: Differences in Pathogenesis Map to the 3′ One-Third of the Genome
Authors: Navas-Martin Sonia , Brom Maarten , Chua Ming-Ming , Watson Richard , Qiu Zhaozhu , Weiss Susan R.
Published: December 2006

The important roles of the spike protein and other structural proteins in murine coronavirus (MHV) pathogenesis have been demonstrated; however, the role of the replicase gene remains unexplored. We assessed the influence of the replicase genes of the highly neurovirulent MHV-JHM strain and the hepatotropic and mildly neurovirulent A59 strain in acute infection of the mouse. Analysis of chimeric A59/JHM recombinant viruses indicates that the replicase genes are interchangeable and that it is the 3′ end of the genome, encoding the structural proteins, rather than the replicase gene, that determines the pathogenic properties of these chimeras.