scholarly journals Improved growth of enteric adenovirus type 40 in a modified cell line that can no longer respond to interferon stimulation

2007 ◽  
Vol 88 (1) ◽  
pp. 71-76 ◽  
Author(s):  
Victoria Sherwood ◽  
Hans-Gerhard Burgert ◽  
Yun-Hsiang Chen ◽  
Sandeep Sanghera ◽  
Socrates Katafigiotis ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Adenovirus Type ◽  
Simian Virus ◽  
Response Analysis ◽  
V Protein ◽  
Simian Virus 5 ◽  
293 Cells ◽  
Enteric Adenovirus ◽  
Enteric Adenoviruses

Human enteric adenoviruses propagate poorly in conventional human cell lines used to grow other adenovirus serotypes. As human enteric adenoviruses have a defect in counteracting the cellular interferon (IFN) response in cell culture, to aid in growth of the virus, a 293-based cell line defective in its ability to respond to IFN was constructed. This cell line (293-SV5/V) constitutively expresses V-protein of the paramyxovirus Simian virus 5, which degrades the signal transducer and activator of transcription 1 (STAT1) and thereby prevents the STAT1-mediated IFN response. Analysis of human enteric adenovirus type 40 (HAdV-40)-infected 293-SV5/V cells compared with parental 293 cells shows that the recombinant line allows more rapid production of virus and results in higher titres. These results suggest that the defect in HAdV-40 in counteracting the IFN response can be overcome at least partially through the use of 293-SV5/V cell lines.

scholarly journals Degradation of STAT1 and STAT2 by the V Proteins of Simian Virus 5 and Human Parainfluenza Virus Type 2, Respectively: Consequences for Virus Replication in the Presence of Alpha/Beta and Gamma Interferons

2002 ◽  
Vol 76 (5) ◽  
pp. 2159-2167 ◽  
Author(s):  
J. Andrejeva ◽  
D. F. Young ◽  
S. Goodbourn ◽  
R. E. Randall
Keyword(s):  
Protein Synthesis ◽  
Cell Lines ◽  
Simian Virus ◽  
Parainfluenza Virus ◽  
V Protein ◽  
Simian Virus 5 ◽  
Ifn Γ ◽  
Alpha Beta ◽  
Human Parainfluenza Virus

ABSTRACT Human cell lines were isolated that express the V protein of either simian virus 5 (SV5) or human parainfluenza virus type 2 (hPIV2); the cell lines were termed 2f/SV5-V and 2f/PIV2-V, respectively. STAT1 was not detectable in 2f/SV5-V cells, and the cells failed to signal in response to either alpha/beta interferons (IFN-α and IFN-β, or IFN-α/β) or gamma interferon (IFN-γ). In contrast, STAT2 was absent from 2f/PIV2-V cells, and IFN-α/β but not IFN-γ signaling was blocked in these cells. Treatment of both 2f/SV5-V and 2f/PIV2-V cells with a proteasome inhibitor allowed the respective STAT levels to accumulate at rates similar to those seen in 2fTGH cells, indicating that the V proteins target the STATs for proteasomal degradation. Infection with SV5 can lead to a complete loss of both phosphorylated and nonphosphorylated forms of STAT1 by 6 h postinfection. Since the turnover of STAT1 in uninfected cells is longer than 24 h, we conclude that degradation of STAT1 is the main mechanism by which SV5 blocks interferon (IFN) signaling. Pretreatment of 2fTGH cells with IFN-α severely inhibited both SV5 and hPIV2 protein synthesis. However, and in marked contrast, pretreatment of 2fTGH cells with IFN-γ had little obvious effect on SV5 protein synthesis but did significantly reduce the replication of hPIV2. Pretreament with IFN-α or IFN-γ did not induce an antiviral state in 2f/SV5-V cells, indicating either that the induction of an antiviral state is completely dependent on STAT signaling or that the V protein interferes with other, STAT-independent cell signaling pathways that may be induced by IFNs. Even though SV5 blocked IFN signaling, the addition of exogenous IFN-α to the culture medium of 2fTGH cells 12 h after a low-multiplicity infection with SV5 significantly reduced the subsequent cell-to-cell spread of virus. The significance of the results in terms of the strategy that these viruses have evolved to circumvent the IFN response is discussed.

scholarly journals Naturally Occurring Substitutions in the P/V Gene Convert the Noncytopathic Paramyxovirus Simian Virus 5 into a Virus That Induces Alpha/Beta Interferon Synthesis and Cell Death

2002 ◽  
Vol 76 (20) ◽  
pp. 10109-10121 ◽  
Author(s):  
Elizabeth K. Wansley ◽  
Griffith D. Parks
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Simian Virus ◽  
Human Cells ◽  
V Protein ◽  
Beta Interferon ◽  
Simian Virus 5 ◽  
Naturally Occurring ◽  
V Gene ◽  
Alpha Beta

ABSTRACT The V protein of the paramyxovirus simian virus 5 (SV5) is responsible for targeted degradation of STAT1 and the block in alpha/beta interferon (IFN-α/β) signaling that occurs after SV5 infection of human cells. We have analyzed the growth properties of a recombinant SV5 that was engineered to be defective in targeting STAT1 degradation. A recombinant SV5 (rSV5-P/V-CPI−) was engineered to contain six naturally occurring P/V protein mutations, three of which have been shown in previous transfection experiments to disrupt the V-mediated block in IFN-α/β signaling. In contrast to wild-type (WT) SV5, human cells infected with rSV5-P/V-CPI− had STAT1 levels similar to those in mock-infected cells. Cells infected with rSV5-P/V-CPI− were found to express higher-than-WT levels of viral proteins and mRNA, suggesting that the P/V mutations had disrupted the regulation of viral RNA synthesis. Despite the inability to target STAT1 for degradation, single-step growth assays showed that the rSV5-P/V-CPI− mutant virus grew better than WT SV5 in all cell lines tested. Unexpectedly, cells infected with rSV5-P/V-CPI− but not WT SV5 showed an activation of a reporter gene that was under control of the IFN-β promoter. The secretion of IFN from cells infected with rSV5-P/V-CPI− but not WT SV5 was confirmed by a bioassay for IFN. The rSV5-P/V-CPI− mutant grew to higher titers than did WT rSV5 at early times in multistep growth assays. However, rSV5-P/V-CPI− growth quickly reached a final plateau while WT rSV5 continued to grow and produced a final titer higher than that of rSV5-P/V-CPI− by late times postinfection. In contrast to WT rSV5, infection of a variety of cell lines with rSV5-P/V-CPI− induced cell death pathways with characteristics of apoptosis. Our results confirm a role for the SV5 V protein in blocking IFN signaling but also suggest new roles for the P/V gene products in controlling viral gene expression, the induction of IFN-α/β synthesis, and virus-induced apoptosis.

scholarly journals Hepatitis B Virus X Protein and Simian Virus 5 V Protein Exhibit Similar UV-DDB1 Binding Properties To Mediate Distinct Activities

2003 ◽  
Vol 77 (11) ◽  
pp. 6274-6283 ◽  
Author(s):  
Olivier Leupin ◽  
Séverine Bontron ◽  
Michel Strubin
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Simian Virus ◽  
Binding Activity ◽  
X Protein ◽  
Binding Properties ◽  
V Protein ◽  
Dna Binding Activity ◽  
Simian Virus 5 ◽  
B Virus

ABSTRACT The UV-damaged DNA-binding activity protein (UV-DDB) consists of two subunits, DDB1 and DDB2, and functions in DNA repair and cell cycle regulation. The DDB1 subunit is a target for the hepatitis B virus X protein (HBx). Binding of HBx to DDB1 interferes with cell growth and viability in culture and has been implicated in the establishment of viral infection. DDB1 also interacts with the V proteins encoded by several paramyxoviruses including simian virus 5 (SV5), which prevent interferon signaling by targeting either STAT1 or STAT2 proteins for proteolysis. The role of V binding to DDB1, however, remains unclear. Here we show that the V protein of SV5 (SV5-V) and HBx exhibit strikingly similar DDB1 binding properties. Thus, SV5-V and HBx bind to DDB1 in a mutually exclusive manner, and SV5-V shares with HBx the ability to enhance the steady-state levels of DDB1 and to inhibit its association with DDB2. Yet only HBx induces cell death, and SV5-V can prevent HBx from doing so by blocking its interaction with DDB1. Binding of SV5-V to DDB1 may serve another function, since SV5-V shows a decreased ability to induce STAT1 degradation in cells expressing reduced amounts of DDB1. These findings demonstrate that HBx performs a unique function through its association with DDB1 for which SV5-V cannot substitute and suggest that SV5-V and HBx have evolved to bind DDB1 to achieve distinct functions, both by a mechanism that does not involve DDB2.

scholarly journals Growth sensitivity of a recombinant simian virus 5 P/V mutant to type I interferon differs between tumor cell lines and normal primary cells

Virology ◽  
2005 ◽  
Vol 335 (1) ◽  
pp. 131-144 ◽  
Author(s):  
Elizabeth K. Wansley ◽  
Patrick J. Dillon ◽  
Maria D. Gainey ◽  
James Tam ◽  
Scott D. Cramer ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Lines ◽  
Type I Interferon ◽  
Simian Virus ◽  
Type I ◽  
Tumor Cell Lines ◽  
Primary Cells ◽  
Simian Virus 5

scholarly journals The Paramyxovirus Simian Virus 5 V Protein Slows Progression of the Cell Cycle

2000 ◽  
Vol 74 (19) ◽  
pp. 9152-9166 ◽  
Author(s):  
Grace Y. Lin ◽  
Robert A. Lamb
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Simian Virus ◽  
S Phase ◽  
V Protein ◽  
Simian Virus 5 ◽  
C Terminus ◽  
M Phase ◽  
Infected Cells ◽  
Affect Cell Cycle Progression

ABSTRACT Infection of cells by many viruses affects the cell division cycle of the host cell to favor viral replication. We examined the ability of the paramyxovirus simian parainfluenza virus 5 (SV5) to affect cell cycle progression, and we found that SV5 slows the rate of proliferation of HeLa T4 cells. The SV5-infected cells had a delayed transition from G1 to S phase and prolonged progression through S phase, and some of the infected cells were arrested in G2 or M phase. The levels of p53 and p21CIP1were not increased in SV5-infected cells compared to mock-infected cells, suggesting that the changes in the cell cycle occur through a p53-independent mechanism. However, the phosphorylation of the retinoblastoma protein (pRB) was delayed and prolonged in SV5-infected cells. The changes in the cell cycle were also observed in cells expressing the SV5 V protein but not in the cells expressing the SV5 P protein or the V protein lacking its unique C terminus (VΔC). The unique C terminus of the V protein of SV5 was shown previously to interact with DDB1, which is the 127-kDa subunit of the multifunctional damage-specific DNA-binding protein (DDB) heterodimer. The coexpression of DDB1 with V can partially restore the changes in the cell cycle caused by expression of the V protein.

scholarly journals Enhancement of Enteric Adenovirus Cultivation by Viral Transactivator Proteins

2010 ◽  
Vol 76 (8) ◽  
pp. 2509-2516 ◽  
Author(s):  
Misoon Kim ◽  
Mi Young Lim ◽  
GwangPyo Ko
Keyword(s):  
Cell Line ◽  
Cytopathic Effect ◽  
Cell Cultivation ◽  
Promoter Regions ◽  
Viral Dna ◽  
Viral Transactivator ◽  
Food Borne ◽  
B Virus ◽  
Enteric Adenovirus ◽  
Enteric Adenoviruses

ABSTRACT Human enteric adenoviruses (HAdVs; serotypes 40 and 41) are important waterborne and food-borne pathogens. However, HAdVs are fastidious, are difficult to cultivate, and do not produce a clear cytopathic effect during cell culture within a reasonable time. Thus, we examined whether the viral transactivator proteins cytomegalovirus (CMV) IE1 and hepatitis B virus (HBV) X promoted the multiplication of HAdVs. Additionally, we constructed a new 293 cell line expressing CMV IE1 protein for cultivation assays. We analyzed the nucleic acid sequences of the promoter regions of both E1A and hexon genes, which are considered to be the most important regions for HAdV replication. Expression of either HBV X or CMV IE1 protein significantly increased the promoter activities of E1A and hexon genes of HAdVs by as much as 14-fold during cell cultivation. The promotion of HAdV expression was confirmed by increased levels of both adenoviral DNA and mRNA expression. Finally, the newly developed 293 cell line expressing CMV IE1 protein showed an increase in viral DNA ranging from 574% to 619% compared with the conventional 293 cell line. These results suggest that the newly constructed cell line could be useful for efficient cultivation and research of fastidious HAdVs.

scholarly journals Single Amino Acid Substitution in the V Protein of Simian Virus 5 Differentiates Its Ability To Block Interferon Signaling in Human and Murine Cells

2001 ◽  
Vol 75 (7) ◽  
pp. 3363-3370 ◽  
Author(s):  
D. F. Young ◽  
N. Chatziandreou ◽  
B. He ◽  
S. Goodbourn ◽  
R. A. Lamb ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Aspartic Acid ◽  
Amino Acid Substitution ◽  
Simian Virus ◽  
Virus Protein ◽  
V Protein ◽  
Persistently Infected ◽  
P Gene ◽  
Simian Virus 5

ABSTRACT Previous work has demonstrated that the V protein of simian virus 5 (SV5) targets STAT1 for proteasome-mediated degradation (thereby blocking interferon [IFN] signaling) in human but not in murine cells. In murine BF cells, SV5 establishes a low-grade persistent infection in which the virus fluxes between active and repressed states in response to local production of IFN. Upon passage of persistently infected BF cells, virus mutants were selected that were better able to replicate in murine cells than the parental W3 strain of SV5 (wild type [wt]). Viruses with mutations in the Pk region of the N-terminal domain of the V protein came to predominate the population of viruses carried in the persistently infected cell cultures. One of these mutant viruses, termed SV5 mci-2, was isolated. Sequence analysis of the V/P gene of SV5 mci-2 revealed two nucleotide differences compared to wt SV5, only one of which resulted in an amino acid substitution (asparagine [N], residue 100, to aspartic acid [D]) in V. Unlike the protein of wt SV5, the V protein of SV5 mci-2 blocked IFN signaling in murine cells. Since the SV5 mci-2 virus had additional mutations in genes other than the V/P gene, a recombinant virus (termed rSV5-V/P N100D) was constructed that contained this substitution alone within the wt SV5 backbone to evaluate what effect the asparagine-to-aspartic-acid substitution in V had on the virus phenotype. In contrast to wt SV5, rSV5-V/P N100D blocked IFN signaling in murine cells. Furthermore, rSV5-V/P N100D virus protein synthesis in BF cells continued for significantly longer periods than that for wt SV5. However, even in cells infected with rSV5-V/P N100D, there was a late, but significant, inhibition in virus protein synthesis. Nevertheless, there was an increase in virus yield from BF cells infected with rSV5-V/P N100D compared to wt SV5, demonstrating a clear selective advantage to SV5 in being able to block IFN signaling in these cells.

scholarly journals Establishment of two rabbit mammary epithelial cell lines with distinct oncogenic potential and differentiated phenotype after microinjection of transforming genes.

1986 ◽  
Vol 6 (6) ◽  
pp. 1974-1982 ◽  
Author(s):  
I Garcia ◽  
B Sordat ◽  
E Rauccio-Farinon ◽  
M Dunand ◽  
J P Kraehenbuhl ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Doubling Time ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Oncogenic Potential ◽  
Transformed Cell Line ◽  
Epithelial Cell Lines ◽  
Transforming Genes

The goal of this work was to establish an assay for transformation of epithelial cells. Two epithelial cell lines were obtained after microinjecting transforming genes into primary rabbit mammary secretory cells. The cell lines were analyzed for their oncogenic potential and for the maintenance of a differentiated phenotype. A fully transformed cell line, which retained epithelial cell organization, was obtained by coinjecting simian virus 40 DNA and the activated human c-Ha-ras gene. The proliferation rate of these cells was high, with a doubling time of 16 h. Their growth was anchorage independent, and they had lost contact inhibition. The cells were tumorigenic in nude mice, but had no metastatic potential. Both microinjected DNAs were efficiently transcribed and translated, in contrast to the casein genes, which were expressed in primary cells but not in the transformed cell line. An immortalized cell line established after injection with simian virus 40 DNA alone was characterized by a moderate rate of proliferation with a doubling time of approximately 30 h. The growth of these cells was contact inhibited and anchorage dependent. The cells were not tumorigenic in nude mice. The viral DNA was expressed during early passages, as shown by the presence of the large T antigen in cell nuclei, but not at later passages. A high number of lactogenic hormone receptors were found associated with the cell surface. Despite the presence of these receptors, no induction of genes coding for milk proteins was observed after addition of prolactin. These data demonstrate that this assay system can be used to assess the immortalizing and transforming potential of candidate oncogenes in epithelial cells.

scholarly journals Neoplastic transformation of rat 3Y1 cells by a transcriptionally activated human c-myc gene and stabilization of p53 cellular tumor antigen in the transformed cells.

1986 ◽  
Vol 6 (12) ◽  
pp. 4379-4386 ◽  
Author(s):  
K Shiroki ◽  
K Segawa ◽  
Y Koita ◽  
M Shibuya
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Tumor Antigen ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
Transformed Cells ◽  
Agar Culture ◽  
Myc Gene ◽  
Tumorigenic Cell Line

Transformed foci were obtained in rat 3Y1 fibroblasts cotransfected with pRmyc 27 (transcriptionally activated c-myc) and pSV2neo DNA. RmycY cell lines (1 to 7) were established from these foci. RmycY cells were small and round and contained enlarged nucleoli in the nucleus. The myc gene was expressed in these cell lines at a much higher level than in 3Y1 cells and at a level similar to that in HL-60 cells. These cell lines formed colonies in soft-agar culture and tumors in syngeneic rats transplanted with RmycY cells. Expression of the gene and colony formation in soft-agar culture were analyzed in subclones from RmycY cell line 1. A correlation between myc gene expression and the ability to form colonies in soft-agar culture was observed in these cells. Antibody against p53 cellular tumor antigen was detected in some sera from tumor-bearing rats. p53 cellular tumor antigen stabilized and accumulated in RmycY cells to the same extent as in simian virus 40-transformed cells. The results suggest that elevated c-myc expression and an increased amount of p53 cause 3Y1 cells to become a more tumorigenic cell line.

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