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 The V Protein of Simian Virus 5 Inhibits Interferon Signalling by Targeting STAT1 for Proteasome-Mediated Degradation

1999 ◽  
Vol 73 (12) ◽  
pp. 9928-9933 ◽  
Author(s):  
L. Didcock ◽  
D. F. Young ◽  
S. Goodbourn ◽  
R. E. Randall
Keyword(s):  
Protein Synthesis ◽  
Defense Mechanisms ◽  
Simian Virus ◽  
Virus Protein ◽  
Structural Protein ◽  
V Protein ◽  
Antiviral State ◽  
Simian Virus 5 ◽  
Transcription Complexes

ABSTRACT To replicate in vivo, viruses must circumvent cellular antiviral defense mechanisms, including those induced by the interferons (IFNs). Here we demonstrate that simian virus 5 (SV5) blocks IFN signalling in human cells by inhibiting the formation of the IFN-stimulated gene factor 3 and gamma-activated factor transcription complexes that are involved in activating IFN-α/β- and IFN-γ-responsive genes, respectively. SV5 inhibits the formation of these complexes by specifically targeting STAT1, a component common to both transcription complexes, for proteasome-mediated degradation. Expression of the SV5 structural protein V, in the absence of other virus proteins, also inhibited IFN signalling and induced the degradation of STAT1. Following infection with SV5, STAT1 was degraded in the absence of virus protein synthesis and remained undetectable for up to 4 days postinfection. Furthermore, STAT1 was also degraded in IFN-pretreated cells, even though the cells were in an antiviral state. Since pretreatment of cells with IFN delayed but did not prevent virus replication and protein synthesis, these observations suggest that following infection of IFN-pretreated cells, SV5 remains viable within the cells until they eventually go out of the antiviral state.

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 Sendai Virus and Simian Virus 5 Block Activation of Interferon-Responsive Genes: Importance for Virus Pathogenesis

1999 ◽  
Vol 73 (4) ◽  
pp. 3125-3133 ◽  
Author(s):  
L. Didcock ◽  
D. F. Young ◽  
S. Goodbourn ◽  
R. E. Randall
Keyword(s):  
Protein Synthesis ◽  
Culture Medium ◽  
Sendai Virus ◽  
Simian Virus ◽  
Human Cells ◽  
Virus Protein ◽  
Marked Contrast ◽  
Rnase Protection ◽  
Highly Pathogenic ◽  
Simian Virus 5

ABSTRACT Sendai virus (SeV) is highly pathogenic for mice. In contrast, mice (including SCID mice) infected with simian virus 5 (SV5) showed no overt signs of disease. Evidence is presented that a major factor which prevented SV5 from productively infecting mice was its inability to circumvent the interferon (IFN) response in mice. Thus, in murine cells that produce and respond to IFN, SV5 protein synthesis was rapidly switched off. In marked contrast, once SeV protein synthesis began, it continued, even if the culture medium was supplemented with alpha/beta IFN (IFN-α/β). However, in human cells, IFN-α/β did not inhibit the replication of either SV5 or SeV once virus protein synthesis was established. To begin to address the molecular basis for these observations, the effects of SeV and SV5 infections on the activation of an IFN-α/β-responsive promoter and on that of the IFN-β promoter were examined in transient transfection experiments. The results demonstrated that (i) SeV, but not SV5, inhibited an IFN-α/β-responsive promoter in murine cells; (ii) both SV5 and SeV inhibited the activation of an IFN-α/β-responsive promoter in human cells; and (iii) in both human and murine cells, SeV was a strong inducer of the IFN-β promoter, whereas SV5 was a poor inducer. The ability of SeV and SV5 to inhibit the activation of IFN-responsive genes in human cells was confirmed by RNase protection experiments. The importance of these results in terms of paramyxovirus pathogenesis is discussed.

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 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 A single amino acid substitution in the V protein of Nipah virus alters its ability to block interferon signalling in cells from different species

2006 ◽  
Vol 87 (12) ◽  
pp. 3649-3653 ◽  
Author(s):  
Kathrin Hagmaier ◽  
Nicola Stock ◽  
Steve Goodbourn ◽  
Lin-Fa Wang ◽  
Richard Randall
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Molecular Basis ◽  
Nipah Virus ◽  
Human Cells ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
V Protein ◽  
Major Constraint ◽  
Interferon Signalling

The V protein of the paramyxovirus Nipah virus (NiV) has been shown to antagonize the interferon (IFN) response in human cells via sequestration of STAT1 and STAT2. This study describes a mutant of the NiV V protein, referred to as V(AAHL), that is unable to antagonize IFN signalling and demonstrates that a single amino acid substitution is responsible for its inactivity. The molecular basis for this was identified as a failure to interact with STAT1 and STAT2. It was also shown that NiV V, but not V(AAHL), was functional as an IFN antagonist in human, monkey, rabbit, dog, horse, pig and bat cells, which suggests that the ability of NiV to block IFN signalling is not a major constraint that prevents this virus from crossing species barriers.

Synthesis of aspartic acid derivatives useful for the preparation of misacylated transfer RNAs

2000 ◽  
Vol 78 (6) ◽  
pp. 884-891 ◽  
Author(s):  
Michiel Lodder ◽  
Curtis F Crasto ◽  
Andrei L Laikhter ◽  
Haoyun An ◽  
Tuncer Arslan ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Aspartic Acid ◽  
Side Chain ◽  
Transfer Rnas ◽  
Rna Ligase ◽  
T4 Rna Ligase ◽  
In Vitro Transcripts ◽  
Derivatives Of

Several derivatives of aspartic acid were protected on Nα as their NVOC derivatives, and on the side chain carboxylates as nitroveratryl esters. Following activation as the cyanomethyl esters, these fully protected aspartate derivatives were converted to the respective pdCpA esters. The protected aspartyl-pdCpA esters were then utilized as substrates for T4 RNA ligase in the presence of in vitro transcripts of tRNA lacking the pCpA dinucleotide normally found at the 3'-end. In this fashion, several misacylated tRNAs were prepared; following photolytic deprotection, these were employed successfully for incorporation into proteins at predetermined positions.Key words: aminoacylated nucleotides, amino acid protection, protein synthesis, tRNA activation.

scholarly journals Mutations in Multiple Domains Activate Paramyxovirus F Protein-Induced Fusion

2004 ◽  
Vol 78 (16) ◽  
pp. 8513-8523 ◽  
Author(s):  
Shaguna Seth ◽  
Andrew L. Goodman ◽  
Richard W. Compans
Keyword(s):  
Amino Acid ◽  
Transmembrane Domain ◽  
Critical Role ◽  
Syncytium Formation ◽  
Simian Virus ◽  
Wild Type ◽  
F Protein ◽  
Simian Virus 5 ◽  
Partial Suppression ◽  
Multiple Domains

ABSTRACT SER virus, a paramyxovirus that is closely related to simian virus 5 (SV5), is unusual in that it fails to induce syncytium formation. The SER virus F protein has an unusually long cytoplasmic tail (CT), and it was previously observed that truncations or specific mutations of this domain result in enhanced syncytium formation. In addition to the long CT, the SER F protein has nine amino acid differences from the F protein of SV5. We previously observed only a partial suppression of fusion in a chimeric SV5 F protein with a CT derived from SER virus, indicating that these other amino acid differences between the SER and SV5 F proteins also play a role in regulating the fusion phenotype. To examine the effects of individual amino acid differences, we mutated the nine SER residues individually to the respective residues of the SV5 F protein. We found that most of the mutants were expressed well and were transported to the cell surface at levels comparable to that of the wild-type SER F protein. Many of the mutants showed enhanced lipid mixing, calcein transfer, and syncytium formation even in the presence of the long SER F protein CT. Some mutants, such as the I310 M, T438S, M489I, T516V, and N529K mutants, also showed fusion at lower temperatures of 32, 25, and 18°C. The residue Asn529 plays a critical role in the suppression of fusion activity, as the mutation of this residue to lysine caused a marked enhancement of fusion. The effect of the N529K mutation on the enhancement of fusion by a previously described mutant, L539,548A, as well as by chimeric SV5/SER F proteins was also dramatic. These results indicate that activation to a fusogenic conformation is dependent on the interplay of residues in the ectodomain, the transmembrane domain, and the CT domain of paramyxovirus F proteins.

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