scholarly journals Effects of inducing or inhibiting apoptosis on Sindbis virus replication in mosquito cells

2008 ◽  
Vol 89 (11) ◽  
pp. 2651-2661 ◽  
Author(s):  
Hua Wang ◽  
Carol D. Blair ◽  
Ken E. Olson ◽  
Rollie J. Clem
Keyword(s):  
Virus Replication ◽  
Persistent Infection ◽  
Sindbis Virus ◽  
Actinomycin D ◽  
Virus Production ◽  
Cell Types ◽  
Lytic Infection ◽  
Mosquito Cells ◽  
Infected Cells ◽  
Apoptotic Genes

Sindbis virus (SINV) is a mosquito-borne virus in the genus Alphavirus, family Togaviridae. Like most alphaviruses, SINVs exhibit lytic infection (apoptosis) in many mammalian cell types, but are generally thought to cause persistent infection with only moderate cytopathic effects in mosquito cells. However, there have been several reports of apoptotic-like cell death in mosquitoes infected with alphaviruses or flaviviruses. Given that apoptosis has been shown to be an antiviral response in other systems, we have constructed recombinant SINVs that express either pro-apoptotic or anti-apoptotic genes in order to test the effects of inducing or inhibiting apoptosis on SINV replication in mosquito cells. Recombinant SINVs expressing the pro-apoptotic genes reaper (rpr) from Drosophila or michelob_x (mx) from Aedes aegypti caused extensive apoptosis in cells from the mosquito cell line C6/36, thus changing the normal persistent infection observed with SINV to a lytic infection. Although the infected cells underwent apoptosis, high levels of virus replication were still observed during the initial infection. However, virus production subsequently decreased compared with persistently infected cells, which continued to produce high levels of virus over the next several days. Infection of C6/36 cells with SINV expressing the baculovirus caspase inhibitor P35 inhibited actinomycin D-induced caspase activity and protected infected cells from actinomycin D-induced apoptosis, but had no observable effect on virus replication. This study is the first to test directly whether inducing or inhibiting apoptosis affects arbovirus replication in mosquito cells.

Sindbis virus maturation in cultured mosquito cells is sensitive to actinomycin D

Virology ◽  
1981 ◽  
Vol 110 (2) ◽  
pp. 292-301 ◽  
Author(s):  
Ursula Scheefers-Borchel ◽  
Hans Scheefers ◽  
Judy Edwards ◽  
Dennis T. Brown
Keyword(s):  
Sindbis Virus ◽  
Actinomycin D ◽  
Virus Maturation ◽  
Mosquito Cells

scholarly journals Effects of Palmitoylation of Replicase Protein nsP1 on Alphavirus Infection

2000 ◽  
Vol 74 (15) ◽  
pp. 6725-6733 ◽  
Author(s):  
Tero Ahola ◽  
Pekka Kujala ◽  
Minna Tuittila ◽  
Titta Blom ◽  
Pirjo Laakkonen ◽  
...  
Keyword(s):  
Sindbis Virus ◽  
Semliki Forest Virus ◽  
Rna Replication ◽  
Cell Types ◽  
Alkaline Solutions ◽  
Nonstructural Proteins ◽  
Replication Complex ◽  
Replication Sites ◽  
Infected Cells ◽  
Alphavirus Infection

ABSTRACT The membrane-associated alphavirus RNA replication complex contains four virus-encoded subunits, the nonstructural proteins nsP1 to nsP4. Semliki Forest virus (SFV) nsP1 is hydrophobically modified by palmitoylation of cysteines 418 to 420. Here we show that Sindbis virus nsP1 is also palmitoylated on the same site (cysteine 420). When mutations preventing nsP1 palmitoylation were introduced into the genomes of these two alphaviruses, the mutant viruses remained viable and replicated to high titers, although their growth was slightly delayed. The subcellular distribution of palmitoylation-defective nsP1 was altered in the mutant: it no longer localized to filopodial extensions, and a fraction of it was soluble. The ultrastructure of the alphavirus replication sites appeared normal, and the localization of the other nonstructural proteins was unaltered in the mutants. In both wild-type- and mutant-virus-infected cells, SFV nsP3 and nsP4 could be extracted from membranes only by alkaline solutions whereas the nsP2-membrane association was looser. Thus, the membrane binding properties of the alphavirus RNA replication complex were not determined by the palmitoylation of nsP1. The nsP1 palmitoylation-defective alphaviruses produced normal plaques in several cell types, but failed to give rise to plaques in HeLa cells, although they induced normal apoptosis of these cells. The SFV mutant was apathogenic in mice: it caused blood viremia, but no infectious virus was detected in the brain.

scholarly journals Development of Sindbis Viruses Encoding nsP2/GFP Chimeric Proteins and Their Application for Studying nsP2 Functioning

2007 ◽  
Vol 81 (10) ◽  
pp. 5046-5057 ◽  
Author(s):  
Svetlana Atasheva ◽  
Rodion Gorchakov ◽  
Robert English ◽  
Ilya Frolov ◽  
Elena Frolova
Keyword(s):  
Virus Replication ◽  
Sindbis Virus ◽  
Fluorescent Protein ◽  
Protein Complexes ◽  
Chimeric Protein ◽  
Specific Protein ◽  
Heterologous Proteins ◽  
Peptide Tags ◽  
Infected Cells ◽  
Green Fluorescent

ABSTRACT Sindbis virus (SINV) is one of almost 30 currently known alphaviruses. In infected cells, it produces only a few proteins that function in virus replication and interfere with the development of the antiviral response. One of the viral nonstructural proteins, nsP2, not only exhibits protease and RNA helicase activities that are directly involved in viral RNA replication but also plays critical roles in the development of transcriptional and translational shutoffs in the SINV-infected cells. These multiple activities of nsP2 complicate investigations of this protein's functions and further understanding of its structure. Using a transposon-based approach, we generated a cDNA library of SINV genomes with a green fluorescent protein (GFP) gene randomly inserted into nsP2 and identified a number of sites that can be used for GFP cloning without a strong effect on virus replication. Recombinant SIN viruses encoding nsP2/GFP chimeric protein were capable of growth in tissue culture and interfering with cellular functions. SINV, expressing GFP in the nsP2, was used to isolate nsP2-specific protein complexes formed in the cytoplasm of the infected cells. These complexes contained viral nsPs, all of the cellular proteins that we previously coisolated with SINV nsP3, and some additional protein factors that were not found before in detectable concentrations. The random insertion library-based approach, followed by the selection of the viable variants expressing heterologous proteins, can be applied for mapping the domain structure of the viral nonstructural and structural proteins, cloning of peptide tags for isolation of the protein-specific complexes, and studying their formation by using live-cell imaging. This approach may also be applicable to presentation of additional antigens and retargeting of viruses to new receptors.

scholarly journals IRF-3 Activation by Sendai Virus Infection Is Required for Cellular Apoptosis and Avoidance of Persistence

2008 ◽  
Vol 82 (7) ◽  
pp. 3500-3508 ◽  
Author(s):  
Kristi Peters ◽  
Saurabh Chattopadhyay ◽  
Ganes C. Sen
Keyword(s):  
Virus Infection ◽  
Persistent Infection ◽  
Cellular Response ◽  
Sendai Virus ◽  
Virus Production ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Infected Cells ◽  
Jnk Activation ◽  
Human Parainfluenza Virus

ABSTRACT Here, we report that specific manipulations of the cellular response to virus infection can cause prevention of apoptosis and consequent establishment of persistent infection. Infection of several human cell lines with Sendai virus (SeV) or human parainfluenza virus 3, two prototypic paramyxoviruses, caused slow apoptosis, which was markedly accelerated upon blocking the action of phosphatidylinositol 3-kinases (PI3 kinases) in the infected cells. The observed apoptosis required viral gene expression and the action of the caspase 8 pathway. Although virus infection activated PI3 kinase, as indicated by AKT activation, its blockage did not inhibit JNK activation or IRF-3 activation. The action of neither the Jak-STAT pathway nor the NF-κB pathway was required for apoptosis. In contrast, IRF-3 activation was essential, although induction of the proapototic protein TRAIL by IRF-3 was not required. When IRF-3 was absent or its activation by the RIG-I pathway was blocked, SeV established persistent infection, as documented by viral protein production and infectious virus production. Introduction of IRF-3 in the persistently infected cells restored the cells' ability to undergo apoptosis. These results demonstrated that in our model system, IRF-3 controlled the fate of the SeV-infected cells by promoting apoptosis and preventing persistence.

scholarly journals Alpha/Beta Interferon Protects Adult Mice from Fatal Sindbis Virus Infection and Is an Important Determinant of Cell and Tissue Tropism

2000 ◽  
Vol 74 (7) ◽  
pp. 3366-3378 ◽  
Author(s):  
Kate D. Ryman ◽  
William B. Klimstra ◽  
Khuong B. Nguyen ◽  
Christine A. Biron ◽  
Robert E. Johnston
Keyword(s):  
Virus Replication ◽  
Sindbis Virus ◽  
High Titer ◽  
Cell Types ◽  
Interleukin 12 ◽  
Tissue Tropism ◽  
Necrosis Factor Alpha ◽  
Beta Interferon ◽  
Alpha Beta

ABSTRACT Infection of adult 129 Sv/Ev mice with consensus Sindbis virus strain TR339 is subclinical due to an inherent restriction in early virus replication and viremic dissemination. By comparing the pathogenesis of TR339 in 129 Sv/Ev mice and alpha/beta interferon receptor null (IFN-α/βR−/−) mice, we have assessed the contribution of IFN-α/β in restricting virus replication and spread and in determining cell and tissue tropism. In adult 129 Sv/Ev mice, subcutaneous inoculation with 100 PFU of TR339 led to extremely low-level virus replication and viremia, with clearance under way by 96 h postinoculation (p.i.). In striking contrast, adult IFN-α/βR−/− mice inoculated subcutaneously with 100 PFU of TR339 succumbed to the infection within 84 h. By 24 h p.i. a high-titer serum viremia had seeded infectious virus systemically, coincident with the systemic induction of the proinflammatory cytokines interleukin-12 (IL-12) p40, IFN-γ, tumor necrosis factor alpha, and IL-6. Replicating virus was located in macrophage-dendritic cell (DC)-like cells at 24 h p.i. in the draining lymph node and in the splenic marginal zone. By 72 h p.i. virus replication was widespread in macrophage-DC-like cells in the spleen, liver, lung, thymus, and kidney and in fibroblast-connective tissue and periosteum, with sporadic neuroinvasion. IFN-α/β-mediated restriction of TR339 infection was mimicked in vitro in peritoneal exudate cells from 129 Sv/Ev versus IFN-α/βR−/− mice. Thus, IFN-α/β protects the normal adult host from viral infection by rapidly conferring an antiviral state on otherwise permissive cell types, both locally and systemically. Ablation of the IFN-α/β system alters the apparent cell and tissue tropism of the virus and renders macrophage-DC-lineage cells permissive to infection.

scholarly journals Serum extracellular vesicle depletion processes affect release and infectivity of HIV-1 in culture

2016 ◽  
Author(s):  
Zhaohao Liao ◽  
Dillon C. Muth ◽  
Erez Eitan ◽  
Meghan Travers ◽  
Elin Lehrmann ◽  
...  
Keyword(s):  
Culture Media ◽  
Virus Production ◽  
Cell Types ◽  
Extracellular Vesicle ◽  
Substantial Effect ◽  
Primary Cells ◽  
Antiviral Responses ◽  
Health And Disease ◽  
Infected Cells ◽  
Hiv 1

ABSTRACTExtracellular vesicles (EVs, including exosomes and microvesicles) are involved in intercellular communication in health and disease and affect processes including immune and antiviral responses. Ultracentrifuged serum is depleted of EVs and, when used in culture media, reduces growth and viability of some cell types. In this study, we examined the effects of serum EV depletion processes on HIV-1 replication in primary cells and cell lines, including two HIV-1 latency models. Increased HIV-1 production was observed in certain EV-depleted conditions, along with cell morphology changes and decreased cell viability. Add-back of ultracentrifuge pellets rescued baseline HIV-1 production. Primary cells appeared to be less sensitive to EV depletion. ACH-2 and U1 latency models produced more HIV-1 under EV-depleted conditions, while virus produced under processed serum conditions was more infectious. Finally, changes in cellular metabolism and gene expression were associated with EV-depleted culture. In conclusion, the EV environment of HIV-1 infected cells has a substantial effect on virus production and infectivity. EV-dependence of cell cultures should be examined carefully along with other experimental variables. However, EVs may not be the only particles depleted by ultracentrifugation or other processes. Effects of EVs may be accompanied by or confused with those of closely associated or physically similar particles.

scholarly journals Cell-to-Cell Spread of the RNA Interference Response Suppresses Semliki Forest Virus (SFV) Infection of Mosquito Cell Cultures and Cannot Be Antagonized by SFV

2009 ◽  
Vol 83 (11) ◽  
pp. 5735-5748 ◽  
Author(s):  
Ghassem Attarzadeh-Yazdi ◽  
Rennos Fragkoudis ◽  
Yi Chi ◽  
Ricky W. C. Siu ◽  
Liane Ülper ◽  
...  
Keyword(s):  
Rna Interference ◽  
Semliki Forest Virus ◽  
Cultured Cells ◽  
Virus Production ◽  
Mosquito Cells ◽  
Infected Cells ◽  
Interference Response ◽  
Interfering Rna ◽  
Rnai Response ◽  
Trigger Cell Death

ABSTRACT In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.

scholarly journals Roles of Nonstructural Protein nsP2 and Alpha/Beta Interferons in Determining the Outcome of Sindbis Virus Infection

2002 ◽  
Vol 76 (22) ◽  
pp. 11254-11264 ◽  
Author(s):  
Elena I. Frolova ◽  
Rafik Z. Fayzulin ◽  
Susan H. Cook ◽  
Diane E. Griffin ◽  
Charles M. Rice ◽  
...  
Keyword(s):  
Virus Infection ◽  
Host Cell ◽  
Virus Replication ◽  
Mammalian Cells ◽  
Sindbis Virus ◽  
Nonstructural Protein ◽  
Virus Propagation ◽  
Virus Growth ◽  
Infected Cells ◽  
Alpha Beta

ABSTRACT Alphaviruses productively infect a variety of vertebrate and insect cell lines. In vertebrate cells, Sindbis virus redirects cellular processes to meet the needs of virus propagation. At the same time, cells respond to virus replication by downregulating virus growth and preventing dissemination of the infection. The balance between these two mechanisms determines the outcome of infection at the cellular and organismal levels. In this report, we demonstrate that a viral nonstructural protein, nsP2, is a significant regulator of Sindbis virus-host cell interactions. This protein not only is a component of the replicative enzyme complex required for replication and transcription of viral RNAs but also plays a role in suppressing the antiviral response in Sindbis virus-infected cells. nsP2 most likely acts by decreasing interferon (IFN) production and minimizing virus visibility. Infection of murine cells with Sindbis virus expressing a mutant nsP2 leads to higher levels of IFN secretion and the activation of 170 cellular genes that are induced by IFN and/or virus replication. Secreted IFN protects naive cells against Sindbis virus infection and also stops viral replication in productively infected cells. Mutations in nsP2 can also attenuate Sindbis virus cytopathogenicity. Such mutants can persist in mammalian cells with defects in the alpha/beta IFN (IFN-α/β) system or when IFN activity is neutralized by anti-IFN-α/β antibodies. These findings provide new insight into the alphavirus-host cell interaction and have implications for the development of improved alphavirus expression systems with better antigen-presenting potential.

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