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Bacterial Lipopolysaccharide Inhibits Dengue Virus Infection of Primary Human Monocytes/Macrophages by Blockade of Virus Entry via a CD14-Dependent Mechanism

Chen, Yun-Chi; Wang, Sheng-Yuan; King, Chwan-Chuen
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/1999 Português
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Monocytes/macrophages (MO/Mφ) are the major target cells for both dengue virus (DV) and bacterial lipopolysaccharide (LPS), and the aim of this study was to define their interactions. We had found that LPS markedly suppressed DV infection of primary human MO/Mφ when it was added to cultures prior to or together with, but not after, viral adsorption. The inhibitory effect of LPS was direct and specific and was not mediated by LPS-induced secretion of cytokines and chemokines such as tumor necrosis factor alpha, interleukin-1β (IL-1β), IL-6, IL-8, IL-12, alpha interferon, MIP-1α, and RANTES. In fact, productive DV infection was not blocked but was just postponed by LPS, with a time lag equal to one viral replication cycle. Time course studies demonstrated that LPS was only effective in suppressing DV infection of MO/Mφ that had not been previously exposed to the virus. At various time points after viral adsorption, the level of unbound viruses that remained free in the culture supernatants of LPS-pretreated cultures was much higher than that of untreated controls. These observations suggest that the LPS-induced suppression of DV replication was at the level of virus attachment and/or entry. Blockade of the major LPS receptor, CD14...

Measles Virus Infection and Replication in Undifferentiated and Differentiated Human Neuronal Cells in Culture

McQuaid, S.; Campbell, S.; Wallace, I. J. C.; Kirk, J.; Cosby, S. L.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /06/1998 Português
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Measles virus (MV) infection of the human central nervous system (CNS) typically involves widespread infection of neurons. However, little is known about how they become infected, how defective virus arises and accumulates, or how virus spreads among the cells of the CNS. In vitro studies of viral interactions with human neuronal cells may contribute to the resolution of such issues. In mixed cultures containing differentiated human neuronal (hNT2) cells and neuroepithelial cells, immunofluorescence studies show that the neurons, unlike both their NT2 progenitors and the neuroepithelial cells, are not initially susceptible to MV infection. This is possibly due to their lack of expression of CD46, a known cell surface receptor for MV. Later in the course of infection, however, both MV proteins and genomic RNA become detectable in their processes, where they contact infected, fully permissive neuroepithelial cells. Such a mechanism of virus transfer may be involved in the initiation and spread of persistent MV infection in diseases such as subacute sclerosing panencephalitis. Furthermore, mutated defective virus may readily accumulate and spread without the need, at any stage, for viral maturation and budding.

Adenoviruses Activate Human Dendritic Cells without Polarization toward a T-Helper Type 1-Inducing Subset

Rea, Delphine; Schagen, Frederik H. E.; Hoeben, Rob C.; Mehtali, Majid; Havenga, Menzo J. E.; Toes, Rene E. M.; Melief, Cornelis J. M.; Offringa, Rienk
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/1999 Português
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Human monocyte-derived dendritic cells (DC) infected with recombinant adenoviruses (rAd) are promising candidate vaccines for inducing protective immunity against pathogens and tumors. However, since some viruses are known to negatively affect DC function, it is important to investigate the interactions between rAd and DC. We now show that infection by rAd enhances the immunostimulatory capacity of immature human monocyte-derived DC through the upregulation of the costimulatory molecules CD80, CD86, and CD40 and the major histocompatibility complex class I and II molecules. Although rAd infection fails to induce the secretion of interleukin-12 (IL-12) and only marginally induces the expression of the DC maturation marker CD83, it acts in synergy with CD40 triggering in rendering DC fully mature. rAd-infected DC triggered through CD40 produce more IL-12 and are more efficient in eliciting T-helper type 1 responses than DC activated by CD40 triggering only. rAd lacking one or more of the early regions, E1, E2A, E3, and E4, which play an important role in virus-host cell interactions are equally capable of DC activation. Efficient DC infection requires a high multiplicity of infection (>1,000), a fact which can be attributed to the absence of the coxsackievirus and adenovirus receptor on this cell type. Despite the poor ability of DC to be infected by rAd...

Plasma Membrane Topology of Syncytial Domains of Herpes Simplex Virus Type 1 Glycoprotein K (gK): the UL20 Protein Enables Cell Surface Localization of gK but Not gK-Mediated Cell-to-Cell Fusion

Foster, Timothy P.; Alvarez, Xavier; Kousoulas, Konstantin G.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/2003 Português
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Most spontaneously occurring mutations that cause extensive herpes simplex virus type 1 (HSV-1)-induced cell fusion are single amino acid changes within glycoprotein K (gK). Despite the strong genetic association of gK with virus-induced cell fusion, its direct involvement in cellular membrane fusion has been controversial, largely due to previously unsuccessful efforts to detect gK expression on virion and cellular surfaces. Recently, we showed that gK is expressed on HSV-1 virions and functioned in virus entry (T. P. Foster, G. V. Rybachuk, and K. G. Kousoulas, J. Virol. 75:12431-12438, 2001). To determine whether gK is expressed on cellular surfaces, as well as its membrane topology, we generated the recombinant viruses gKV5DI, gKV5DII, gKV5DIII, and gKV5DIVcontaining insertions of the V5 antigenic epitope within each of four domains of gK predicted to localize either in the cytoplasmic side or in the extracytoplasmic side of cellular membranes. Immunohistochemical and confocal microscopy analyses of infected cells showed that both wild-type and syncytial forms of gK were expressed on cell surfaces. Analysis of the topology of the V5-tagged gK revealed that gK domains I and IV were located extracellularly, whereas domains II and III were localized intracellularly. Transiently expressed gK failed to localize in cellular plasma membranes. In contrast...

Binding of Human Immunodeficiency Virus Type 1 to Immature Dendritic Cells Can Occur Independently of DC-SIGN and Mannose Binding C-Type Lectin Receptors via a Cholesterol-Dependent Pathway

Gummuluru, Suryaram; Rogel, Mark; Stamatatos, Leonidas; Emerman, Michael
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2003 Português
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Interactions of human immunodeficiency virus type 1 (HIV-1) with immature dendritic cells (DC) are believed to be multifactorial and involve binding to the CD4 antigen, DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), mannose binding C-type lectin receptors (MCLR), and heparan sulfate proteoglycans (HSPG). In this study we assessed the relative contributions of these previously defined virus attachment factors to HIV binding and accumulation in DC and the subsequent transfer of the bound virus particle to CD4+ T cells. Using competitive inhibitors of HIV-1 attachment to DC, we have identified the existence of DC-SIGN-, MCLR-, and HSPG-independent mechanism(s) of HIV attachment and internalization. Furthermore, virus particles bound by DC independently of CD4, DC-SIGN, MCLR, and HSPG are efficiently transmitted to T cells. Treatment of virus particles with the protease subtilisin or treatment of immature DC with trypsin significantly reduced virus binding, thus demonstrating the role of HIV envelope glycoprotein interactions with unidentified DC-surface factor(s). Finally, this DC-mediated virus binding and internalization are dependent on lipid rafts. We propose that pathways to HIV-1 attachment and uptake in DC exhibit functional redundancy; that is...

Postinternalization Inhibition of Adenovirus Gene Expression and Infectious Virus Production in Human T-Cell Lines

McNees, Adrienne L.; Mahr, Jeff A.; Ornelles, David; Gooding, Linda R.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/2004 Português
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Detection of adenovirus DNA in human tonsillar T cells in the absence of active virus replication suggests that T cells may be a site of latency or of attenuated virus replication in persistently infected individuals. The lytic replication cycle of Ad5 in permissive epithelial cells (A549) was compared to the behavior of Ad5 in four human T-cell lines, Jurkat, HuT78, CEM, and KE37. All four T-cell lines expressed the integrin coreceptors for Ad2 and Ad5, but only Jurkat and HuT78 express detectable surface levels of the coxsackie adenovirus receptor (CAR). Jurkat and HuT78 cells supported full lytic replication of Ad5, albeit at a level ∼10% of that of A549, while CAR-transduced CEM and KE37 cells (CEM-CARhi and KE37-CARhi, respectively) produced no detectable virus following infection. All four T-cell lines bind and internalize fluorescently labeled virus. In A549, Jurkat, and HuT78 cells, viral proteins were detected in 95% of cells. In contrast, only a small subpopulation of CEM-CARhi and KE37-CARhi cells contained detectable viral proteins. Interestingly, Jurkat and HuT78 cells synthesize four to six times more copies of viral DNA per cell than did A549 cells, indicating that these cells produce infectious virions with much lower efficiency than A549. Similarly...

Establishment of a Subgenomic Replicon for Bovine Viral Diarrhea Virus in Huh-7 Cells and Modulation of Interferon-Regulated Factor 3-Mediated Antiviral Response

Horscroft, Nigel; Bellows, Dan; Ansari, Israrul; Lai, Vicky C. H.; Dempsey, Shannon; Liang, Delin; Donis, Ruben; Zhong, Weidong; Hong, Zhi
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /03/2005 Português
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We describe the development of a selectable, bi-cistronic subgenomic replicon for bovine viral diarrhea virus (BVDV) in Huh-7 cells, similar to that established for hepatitis C virus (HCV). The selection marker and reporter (Luc-Ubi-Neo) in the BVDV replicon was fused with the amino-terminal protease Npro, and expression of the nonstructural proteins (NS3 to NS5B) was driven by an encephalomyocarditis virus internal ribosome entry site. This BVDV replicon allows us to compare RNA replication of these two related viruses in a similar cellular background and to identify antiviral molecules specific for HCV RNA replication. The BVDV replicon showed similar sensitivity as the HCV replicon to interferons (alpha, beta, and gamma) and 2′-β-C-methyl ribonucleoside inhibitors. Known nonnucleoside inhibitor molecules specific for either HCV or BVDV can be easily distinguished by using the parallel replicon systems. The HCV replicon has been shown to block, via the NS3/4A serine protease, Sendai virus-induced activation of interferon regulatory factor 3 (IRF-3), a key antiviral signaling molecule. Similar suppression of IRF-3-mediated responses was also observed with the Huh-7-BVDV replicon but was independent of NS3/4A protease activity. Instead...

Herpes Simplex Virus Entry Mediator Associates in Infected Cells in a Complex with Viral Proteins gD and at Least gH

Perez-Romero, Pilar; Perez, Aleida; Capul, Althea; Montgomery, Rebecca; Fuller, A. Oveta
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/2005 Português
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We examined herpes simplex virus (HSV)-infected human HEp-2 cells or porcine cells that express herpes virus entry mediator (HVEM) for virus and receptor protein interactions. Antibody to HVEM, or its viral ligand gD, coimmunoprecipitated several similar proteins. A prominent 110-kDa protein that coprecipitated was identified as gH. The HVEM/gD/gH complex was detected with mild or stringent cell lysis conditions. It did not form in cells infected with HSV-1(KOS)Rid1 virus or with null virus lacking gD, gH, or gL. Thus, in cells a complex forms through physical associations of HVEM, gD, and at least gH.

Identification of an Essential Domain in the Herpes Simplex Virus 1 UL34 Protein That Is Necessary and Sufficient To Interact with UL31 Protein

Liang, Li; Baines, Joel D.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /03/2005 Português
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Previous results have indicated that the herpes simplex virus 1 UL31 and UL34 proteins interact and form a complex at the inner nuclear membranes of infected cells, where both play important roles in the envelopment of nucleocapsids at the inner nuclear membrane. In the work described here, mapping studies using glutathione S-transferase pull-down assays indicated that amino acids 137 to 181 of the UL34 protein are sufficient to mediate an interaction with the UL31 protein. A recombinant virus (v3480) lacking UL34 codons 138 to 181 was constructed. Similar to a UL34 null virus, v3480 failed to replicate on Vero cells and grew to a limited extent on rabbit skin cells. A UL34-expressing cell line restored v3480 growth and plaque formation. Similar to the localization of UL31 protein in cells infected with a UL34 null virus, the UL31 protein was present in the nuclei of Hep2 cells infected with v3480. Hep2 cells infected with v3480 contained the UL34 protein in the cytoplasm, the nucleus, and the nuclear membrane, and this was noted to be similar to the appearance of cells infected with a UL31 null virus. In transient expression assays, the interaction between UL34 amino acids 137 to 181 and the UL31 protein was sufficiently robust to target green fluorescent protein and emerin to intranuclear sites that contained the UL31 protein. These data indicate that amino acids 137 to 181 of the UL34 protein are (i) sufficient to mediate interactions with the UL31 protein in vitro and in vivo...

The Extracellular Domain of Herpes Simplex Virus gE Is Indispensable for Efficient Cell-to-Cell Spread: Evidence for gE/gI Receptors

Polcicova, Katarina; Goldsmith, Kim; Rainish, Barb L.; Wisner, Todd W.; Johnson, David C.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /09/2005 Português
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Herpes simplex virus (HSV) spreads rapidly and efficiently within epithelial and neuronal tissues. The HSV glycoprotein heterodimer gE/gI plays a critical role in promoting cell-to-cell spread but does not obviously function during entry of extracellular virus into cells. Thus, gE/gI is an important molecular handle on the poorly understood process of cell-to-cell spread. There was previous evidence that the large extracellular (ET) domains of gE/gI might be important in cell-to-cell spread. First, gE/gI extensively accumulates at cell junctions, consistent with being tethered there. Second, expression of gE/gI in trans interfered with HSV spread between epithelial cells. To directly test whether the gE ET domain was necessary for gE/gI to promote virus spread, a panel of gE mutants with small insertions in the ET domain was constructed. Cell-to-cell spread was reduced when insertions were made within either of two regions, residues 256 to 291 or 348 to 380. There was a strong correlation between loss of cell-to-cell spread function and binding of immunoglobulin. gE ET domain mutants 277, 291, and 348 bound gI, produced mature forms of gE that reached the cell surface, and were incorporated into virions yet produced plaques similar to gE null mutants. Moreover...

Potassium Ion Channels of Chlorella Viruses Cause Rapid Depolarization of Host Cells during Infection

Frohns, Florian; Käsmann, Anja; Kramer, Detlef; Schäfer, Britta; Mehmel, Mario; Kang, Ming; Van Etten, James L.; Gazzarrini, Sabrina; Moroni, Anna; Thiel, Gerhard
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /03/2006 Português
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Previous studies have established that chlorella viruses encode K+ channels with different structural and functional properties. In the current study, we exploit the different sensitivities of these channels to Cs+ to determine if the membrane depolarization observed during virus infection is caused by the activities of these channels. Infection of Chlorella NC64A with four viruses caused rapid membrane depolarization of similar amplitudes, but with different kinetics. Depolarization was fastest after infection with virus SC-1A (half time [t1/2], about 9 min) and slowest with virus NY-2A (t1/2, about 12 min). Cs+ inhibited membrane depolarization only in viruses that encode a Cs+-sensitive K+ channel. Collectively, the results indicate that membrane depolarization is an early event in chlorella virus-host interactions and that it is correlated with viral-channel activity. This suggestion was supported by investigations of thin sections of Chlorella cells, which show that channel blockers inhibit virus DNA release into the host cell. Together, the data indicate that the channel is probably packaged in the virion, presumably in its internal membrane. We hypothesize that fusion of the virus internal membrane with the host plasma membrane results in an increase in K+ conductance and membrane depolarization; this depolarization lowers the energy barrier for DNA release into the host.

Postentry Events Are Responsible for Restriction of Productive Varicella-Zoster Virus Infection in Chinese Hamster Ovary Cells

Finnen, Renée L.; Mizokami, Kara R.; Banfield, Bruce W.; Cai, Guang-Yun; Simpson, Scott A.; Pizer, Lewis I.; Levin, Myron J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /11/2006 Português
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Productive infection of varicella-zoster virus (VZV) in vitro is restricted almost exclusively to cells derived from humans and other primates. We demonstrate that the restriction of productive VZV infection in CHO-K1 cells occurs downstream of virus entry. Entry of VZV into CHO-K1 cells was characterized by utilizing an ICP4/β-galactosidase reporter gene that has been used previously to study herpes simplex virus type 1 entry. Entry of VZV into CHO-K1 cells involved cell surface interactions with heparan sulfate glycosaminoglycans and a cation-independent mannose-6-phosphate receptor. Lysosomotropic agents inhibited the entry of VZV into CHO-K1 cells, consistent with a low-pH-dependent endocytic mechanism of entry. Infection of CHO-K1 cells by VZV resulted in the production of both immediate early and late gene products, indicating that a block to progeny virus production occurs after the initiation of virus gene expression.

Altered Interaction of the Matrix Protein with the Cytoplasmic Tail of Hemagglutinin Modulates Measles Virus Growth by Affecting Virus Assembly and Cell-Cell Fusion▿

Tahara, Maino; Takeda, Makoto; Yanagi, Yusuke
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Português
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Clinical isolates of measles virus (MV) use signaling lymphocyte activation molecule (SLAM) as a cellular receptor, whereas vaccine and laboratory strains may utilize the ubiquitously expressed CD46 as an additional receptor. MVs also infect, albeit inefficiently, SLAM− cells, via a SLAM- and CD46-independent pathway. Our previous study with recombinant chimeric viruses revealed that not only the receptor-binding hemagglutinin (H) but also the matrix (M) protein of the Edmonston vaccine strain can confer on an MV clinical isolate the ability to grow well in SLAM− Vero cells. Two substitutions (P64S and E89K) in the M protein which are present in many vaccine strains were found to be responsible for the efficient growth of recombinant virus in Vero cells. Here we show that the P64S and E89K substitutions allow a strong interaction of the M protein with the cytoplasmic tail of the H protein, thereby enhancing the assembly of infectious particles in Vero cells. These substitutions, however, are not necessarily advantageous for MVs, as they inhibit SLAM-dependent cell-cell fusion, thus reducing virus growth in SLAM+ B-lymphoblastoid B95a cells. When the cytoplasmic tail of the H protein is deleted, a virus with an M protein possessing the P64S and E89K substitutions no longer grows well in Vero cells yet causes cell-cell fusion and replicates efficiently in B95a cells. These results reveal a novel mechanism of adaptation and attenuation of MV in which the altered interaction of the M protein with the cytoplasmic tail of the H protein modulates MV growth in different cell types.

Linker Histones Are Mobilized during Infection with Herpes Simplex Virus Type 1▿

Conn, Kristen L.; Hendzel, Michael J.; Schang, Luis M.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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Histones interact with herpes simplex virus type 1 (HSV-1) genomes and localize to replication compartments early during infections. However, HSV-1 genomes do not interact with histones in virions and are deposited in nuclear domains devoid of histones. Moreover, late viral replication compartments are also devoid of histones. The processes whereby histones come to interact with HSV-1 genomes, to be later displaced, remain unknown. However, they would involve the early movement of histones to the domains containing HSV-1 genomes and the later movement away from them. Histones unbind from chromatin, diffuse through the nucleoplasm, and rebind at different sites. Such mobility is upregulated by, for example, phosphorylation or acetylation. We evaluated whether HSV-1 infection modulates histone mobility, using fluorescence recovery after photobleaching. All somatic H1 variants were mobilized to different degrees. H1.2, the most mobilized, was mobilized at 4 h and further so at 7 h after infection, resulting in increases in its “free” pools. H1.2 was mobilized to a “basal” degree under conditions of little to no HSV-1 protein expression. This basal mobilization required nuclear native HSV-1 genomes but was independent of HSV-1 proteins and most likely due to cellular responses. Mobilization above this basal degree...

E1 Mutants Identify a Critical Region in the Trimer Interface of the Semliki Forest Virus Fusion Protein▿

Liu, Catherine Y.; Kielian, Margaret
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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The alphavirus Semliki Forest virus (SFV) uses a membrane fusion reaction to infect host cells. Fusion of the virus and cell membranes is triggered by low pH in the endosome and is mediated by the viral membrane protein E1. During fusion, E1 inserts into the target membrane, trimerizes, and refolds into a hairpin conformation. Formation of the E1 homotrimer is critical to membrane fusion, but the mechanism of trimerization is not understood. The crystal structure of the postfusion E1 trimer shows that an aspartate residue, D188, is positioned in the central core trimer interface. D188 is conserved in all reported alphavirus E1 sequences. We tested the contribution of this amino acid to trimerization and fusion by replacing D188 with alanine (D188A) or lysine (D188K) in an SFV infectious clone. These mutations were predicted to disrupt specific interactions at this position and/or change their pH dependence. Our results indicated that the D188K mutation blocked SFV fusion and infection. At low pH, D188K E1 inserted into target membranes but was trapped as a target membrane-inserted monomer that did not efficiently form the stable core trimer. In contrast, the D188A mutant was infectious, although trimerization and fusion required a lower pH. While there are extensive contacts between E1 subunits in the homotrimer...

The Requirement for Cellular Transportin 3 (TNPO3 or TRN-SR2) during Infection Maps to Human Immunodeficiency Virus Type 1 Capsid and Not Integrase ▿

Krishnan, Lavanya; Matreyek, Kenneth A.; Oztop, Ilker; Lee, Kyeongeun; Tipper, Christopher H.; Li, Xiang; Dar, Mohd J.; KewalRamani, Vineet N.; Engelman, Alan
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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Recent genome-wide screens have highlighted an important role for transportin 3 in human immunodeficiency virus type 1 (HIV-1) infection and preintegration complex (PIC) nuclear import. Moreover, HIV-1 integrase interacted with recombinant transportin 3 protein under conditions whereby Moloney murine leukemia virus (MLV) integrase failed to do so, suggesting that integrase-transportin 3 interactions might underscore active retroviral PIC nuclear import. Here we correlate infectivity defects in transportin 3 knockdown cells with in vitro protein binding affinities for an expanded set of retroviruses that include simian immunodeficiency virus (SIV), bovine immunodeficiency virus (BIV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), and Rous sarcoma virus (RSV) to critically address the role of integrase-transportin 3 interactions in viral infection. Lentiviruses, with the exception of FIV, display a requirement for transportin 3 in comparison to MLV and RSV, yielding an infection-based dependency ranking of SIV > HIV-1 > BIV and EIAV > MLV, RSV, and FIV. In vitro pulldown and surface plasmon resonance assays, in contrast, define a notably different integrase-transportin 3 binding hierarchy: FIV, HIV-1, and BIV > SIV and MLV > EIAV. Our results therefore fail to support a critical role for integrase binding in dictating transportin 3 dependency during retrovirus infection. In addition to integrase...

Generation and Comprehensive Analysis of an Influenza Virus Polymerase Cellular Interaction Network▿†§

Tafforeau, Lionel; Chantier, Thibault; Pradezynski, Fabrine; Pellet, Johann; Mangeot, Philippe E.; Vidalain, Pierre-Olivier; Andre, Patrice; Rabourdin-Combe, Chantal; Lotteau, Vincent
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2011 Português
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The influenza virus transcribes and replicates its genome inside the nucleus of infected cells. Both activities are performed by the viral RNA-dependent RNA polymerase that is composed of the three subunits PA, PB1, and PB2, and recent studies have shown that it requires host cell factors to transcribe and replicate the viral genome. To identify these cellular partners, we generated a comprehensive physical interaction map between each polymerase subunit and the host cellular proteome. A total of 109 human interactors were identified by yeast two-hybrid screens, whereas 90 were retrieved by literature mining. We built the FluPol interactome network composed of the influenza virus polymerase (PA, PB1, and PB2) and the nucleoprotein NP and 234 human proteins that are connected through 279 viral-cellular protein interactions. Analysis of this interactome map revealed enriched cellular functions associated with the influenza virus polymerase, including host factors involved in RNA polymerase II-dependent transcription and mRNA processing. We confirmed that eight influenza virus polymerase-interacting proteins are required for virus replication and transcriptional activity of the viral polymerase. These are involved in cellular transcription (C14orf166...

Large-Scale Chromatin Immunoprecipitation with Promoter Sequence Microarray Analysis of the Interaction of the NSs Protein of Rift Valley Fever Virus with Regulatory DNA Regions of the Host Genome

Benferhat, Rima; Josse, Thibaut; Albaud, Benoit; Gentien, David; Mansuroglu, Zeyni; Marcato, Vasco; Souès, Sylvie; Le Bonniec, Bernard; Bouloy, Michèle; Bonnefoy, Eliette
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/2012 Português
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Rift Valley fever virus (RVFV) is a highly pathogenic Phlebovirus that infects humans and ruminants. Initially confined to Africa, RVFV has spread outside Africa and presently represents a high risk to other geographic regions. It is responsible for high fatality rates in sheep and cattle. In humans, RVFV can induce hepatitis, encephalitis, retinitis, or fatal hemorrhagic fever. The nonstructural NSs protein that is the major virulence factor is found in the nuclei of infected cells where it associates with cellular transcription factors and cofactors. In previous work, we have shown that NSs interacts with the promoter region of the beta interferon gene abnormally maintaining the promoter in a repressed state. In this work, we performed a genome-wide analysis of the interactions between NSs and the host genome using a genome-wide chromatin immunoprecipitation combined with promoter sequence microarray, the ChIP-on-chip technique. Several cellular promoter regions were identified as significantly interacting with NSs, and the establishment of NSs interactions with these regions was often found linked to deregulation of expression of the corresponding genes. Among annotated NSs-interacting genes were present not only genes regulating innate immunity and inflammation but also genes regulating cellular pathways that have not yet been identified as targeted by RVFV. Several of these pathways...

Implication of Bemisia tabaci Heat Shock Protein 70 in Begomovirus-Whitefly Interactions

Götz, Monika; Popovski, Smadar; Kollenberg, Mario; Gorovits, Rena; Brown, Judith K.; Cicero, Joseph M.; Czosnek, Henryk; Winter, Stephan; Ghanim, Murad
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2012 Português
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The whitefly Bemisia tabaci (Gennadius) is a major cosmopolitan pest capable of feeding on hundreds of plant species and transmits several major plant viruses. The most important and widespread viruses vectored by B. tabaci are in the genus Begomovirus, an unusual group of plant viruses owing to their small, single-stranded DNA genome and geminate particle morphology. B. tabaci transmits begomoviruses in a persistent circulative nonpropagative manner. Evidence suggests that the whitefly vector encounters deleterious effects following Tomato yellow leaf curl virus (TYLCV) ingestion and retention. However, little is known about the molecular and cellular basis underlying these coevolved begomovirus-whitefly interactions. To elucidate these interactions, we undertook a study using B. tabaci microarrays to specifically describe the responses of the transcriptomes of whole insects and dissected midguts following TYLCV acquisition and retention. Microarray, real-time PCR, and Western blot analyses indicated that B. tabaci heat shock protein 70 (HSP70) specifically responded to the presence of the monopartite TYLCV and the bipartite Squash leaf curl virus. Immunocapture PCR, protein coimmunoprecipitation, and virus overlay protein binding assays showed in vitro interaction between TYLCV and HSP70. Fluorescence in situ hybridization and immunolocalization showed colocalization of TYLCV and the bipartite Watermelon chlorotic stunt virus virions and HSP70 within midgut epithelial cells. Finally...

Remote Activation of Host Cell DNA Synthesis in Uninfected Cells Signaled by Infected Cells in Advance of Virus Transmission

Schmidt, Nora; Hennig, Thomas; Serwa, Remigiusz A.; Marchetti, Magda; O'Hare, Peter
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em 26/08/2015 Português
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Viruses modulate cellular processes and metabolism in diverse ways, but these are almost universally studied in the infected cell itself. Here, we study spatial organization of DNA synthesis during multiround transmission of herpes simplex virus (HSV) using pulse-labeling with ethynyl nucleotides and cycloaddition of azide fluorophores. We report a hitherto unknown and unexpected outcome of virus-host interaction. Consistent with the current understanding of the single-step growth cycle, HSV suppresses host DNA synthesis and promotes viral DNA synthesis in spatially segregated compartments within the cell. In striking contrast, during progressive rounds of infection initiated at a single cell, we observe that infection induces a clear and pronounced stimulation of cellular DNA replication in remote uninfected cells. This induced DNA synthesis was observed in hundreds of uninfected cells at the extended border, outside the perimeter of the progressing infection. Moreover, using pulse-chase analysis, we show that this activation is maintained, resulting in a propagating wave of host DNA synthesis continually in advance of infection. As the virus reaches and infects these activated cells, host DNA synthesis is then shut off and replaced with virus DNA synthesis. Using nonpropagating viruses or conditioned medium...