Analysis of a large-T-antigen variant expressed in simian virus 40-transformed mouse cell line mKS-A

Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA.

Section: Downloaded Fromsupporting

confidence: 80%

Section: Downloaded Fromsupporting

confidence: 80%

cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication

Hartl¹,

Willnow²,

Fanning³

1990

“…Transformation of MEFs. MEFs were transfected with the plasmid SV2, which encodes the large T antigen of simian virus 40 (SV40) polyomavirus (65). Cells were passaged ϳ10 times and used for subsequent experiments.…”

Section: Ethics Statementmentioning

confidence: 99%

The Interferon-Stimulated GeneIfitm3Restricts West Nile Virus Infection and Pathogenesis

Gorman

Poddar

Farzan

et al. 2016

T he interferon (IFN)-induced transmembrane protein (IFITM) genes consist of a family of genes encoding related proteins: Ifitm1,2,3,5,6,7, and 10 in mice and IFITM1, 2, 3, 5, and 10 in humans (1, 2). The expression of several IFITM genes (e.g., those encoding IFITM1, 2, and 3) can be induced by type I, II, or III IFNs (3, 4). Although initial studies described possible roles of IFITM1, IFITM2, and IFITM3 in development, apoptosis, cell proliferation, and cell signaling (5-13), a subsequent report suggested that ectopic expression of IFITM1 in mouse L cells could restrict infection of vesicular stomatitis virus (VSV) (14). A decade later, gene silencing and ectopic expression studies established that IFITM proteins have antiviral activity in cell culture against members of the Flaviviridae, Orthomyxoviridae, Filoviridae, Rhabdoviridae, Retroviridae, Bunyaviridae, Reoviridae, Togaviridae,. Nonetheless, some enveloped and nonenveloped viruses appear to be resistant to the actions of IFITM proteins, including arenaviruses, papillomaviruses, cytomegaloviruses, and adenoviruses (16,17,29).IFITM proteins are transmembrane proteins. Although their precise membrane topology remains uncertain (5,6,15,(30)(31)(32)(33)(34)(35)(36), recent studies suggest that they are type II membrane proteins (35)(36)(37). Moreover, the cellular sublocalization of the IFITM proteins varies among family members, with IFITM1 expressed primarily at the plasma membrane, and IFITM2 and IFITM3 colocalizing predominantly with late endosomes (20,32). Based on the cellular localization and effects on specific steps in viral life cycles, IFITM1, IFITM2, and IFITM3 appear to restrict fusion and uncoating of viruses into the cytoplasm (33,38,39), with different IFITM proteins inhibiting specific viruses in distinct membrane compartments. Despite the intensive study of the IFITM proteins in cell culture, the precise mechanism of restriction of viral fusion has remained elusive. It has been suggested that IFITM proteins

“…Primary WT, Ifitm-del, Ifitm2 Ϫ/Ϫ , and Ifitm3 Ϫ/Ϫ mouse-derived mouse embryonic fibroblasts (MEFs) and bone marrowderived macrophages were generated according to published methods (57). Transformed MEFs were generated by transfection of the SV2 plasmid, which encodes the large T antigen of SV2 polyomavirus (58), and passaged ϳ10 times. All MEFs were cultured in complete Dulbecco's modified Eagle's medium (DMEM), which was supplemented with 10% fetal bovine serum and 10 mM (each) GlutaMAX, sodium pyruvate, nonessential amino acids, and HEPES, pH 7.3.…”

mentioning

confidence: 99%

The Interferon-Stimulated Gene IFITM3 Restricts Infection and Pathogenesis of Arthritogenic and Encephalitic Alphaviruses

Poddar

Hyde

Gorman

et al. 2016

Host cells respond to viral infections by producing type I interferon (IFN), which induces the expression of hundreds of interferon-stimulated genes (ISGs). Although ISGs mediate a protective state against many pathogens, the antiviral functions of the majority of these genes have not been identified. IFITM3 is a small transmembrane ISG that restricts a broad range of viruses, including orthomyxoviruses, flaviviruses, filoviruses, and coronaviruses. Here, we show that alphavirus infection is increased in Ifitm3 ؊/؊ and Ifitm locus deletion (Ifitm-del) fibroblasts and, reciprocally, reduced in fibroblasts transcomplemented with Ifitm3. Mechanistic studies showed that Ifitm3 did not affect viral binding or entry but inhibited pH-dependent fusion. In a murine model of chikungunya virus arthritis, Ifitm3 ؊/؊ mice sustained greater joint swelling in the ipsilateral ankle at days 3 and 7 postinfection, and this correlated with higher levels of proinflammatory cytokines and viral burden. Flow cytometric analysis suggested that Ifitm3 ؊/؊ macrophages from the spleen were infected at greater levels than observed in wild-type (WT) mice, results that were supported by experiments with Ifitm3 ؊/؊ bone marrow-derived macrophages. Ifitm3 ؊/؊ mice also were more susceptible than WT mice to lethal alphavirus infection with Venezuelan equine encephalitis virus, and this was associated with greater viral burden in multiple organs. Collectively, our data define an antiviral role for Ifitm3 in restricting infection of multiple alphaviruses. IMPORTANCE The interferon-induced transmembrane protein 3 (IFITM3) inhibits infection of multiple families of viruses in cell culture.Compared to other viruses, much less is known about the antiviral effect of IFITM3 on alphaviruses. In this study, we characterized the antiviral activity of mouse Ifitm3 against arthritogenic and encephalitic alphaviruses using cells and animals with a targeted gene deletion of Ifitm3 as well as deficient cells transcomplemented with Ifitm3. Based on extensive virological analysis, we demonstrate greater levels of alphavirus infection and disease pathogenesis when Ifitm3 expression is absent. Our data establish an inhibitory role for Ifitm3 in controlling infection of alphaviruses. The type I interferon (IFN) response is a critical factor that orchestrates innate protection against viral pathogens. Upon detection of pathogen-associated molecular patterns (PAMPs), host cells produce type I IFN, which in turn induces expression of hundreds of IFN-stimulated genes (ISGs). ISGs can inhibit multiple steps of the viral life cycle (e.g., entry, protein translation, assembly, or egress) or modulate the immune response, such as by enhancing the recruitment of leukocytes or promoting B and T cell maturation (1).IFN-induced transmembrane (IFITM) proteins 1, 2, and 3 were among the first IFN-stimulated genes (ISGs) to be identified (2) and initially were studied for their roles in germ cell homing and maturation. IFITM proteins are approximately 130 amino acids in l...