scholarly journals Mutations in the nuclear localization signal of nsP2 influencing RNA synthesis, protein expression and cytotoxicity of Semliki Forest virus

2008 ◽  
Vol 89 (3) ◽  
pp. 676-686 ◽  
Author(s):  
Kristi Tamm ◽  
Andres Merits ◽  
Inga Sarand
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Rna Synthesis ◽  
Semliki Forest Virus ◽  
Temperature Sensitive ◽  
Structural Protein ◽  
Arginine Residues ◽  
Localization Signal ◽  
Infected Cells ◽  
Replicase Complex

The cytotoxicity of Semliki Forest virus (SFV) infection is caused partly by the non-structural protein nsP2, an essential component of the SFV replicase complex. Due to the presence of a nuclear localization signal (NLS), nsP2 also localizes in the nucleus of infected cells. The present study analysed recombinant SFV replicons and genomes with various deletions or substitutions in the NLS, or with a proline-to-glycine mutation at position 718 of nsP2 (P718G). Deletion of one or two arginine residues from the NLS or substitution of two of the arginines with aspartic acid resulted in a virus with a temperature-sensitive phenotype, and substitution of all three arginines was lethal. Thus, most of the introduced mutations severely affected nsP2 functioning in viral replication; in addition, they inhibited the ability of SFV to induce translational shut-off and kill infected cells. SFV replicons with a P718G mutation or replacement of the NLS residues 648RRR650 with RDD were found to be the least cytotoxic. Corresponding replicons expressed non-structural proteins at normal levels, but had severely reduced genomic RNA synthesis and were virtually unable to replicate and transcribe co-electroporated helper RNA. The non-cytotoxic phenotype was maintained in SFV full-length genomes harbouring the corresponding mutations; however, during a single cycle of cell culture, these were converted to a cytotoxic phenotype, probably due to the accumulation of compensatory mutations.

scholarly journals Characterization of the Nuclear Localization Signal of the Borna Disease Virus Polymerase

2002 ◽  
Vol 76 (16) ◽  
pp. 8460-8467 ◽  
Author(s):  
Michelle Portlance Walker ◽  
W. Ian Lipkin
Keyword(s):  
Disease Virus ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Rna Virus ◽  
Flag Epitope ◽  
Borna Disease Virus ◽  
Localization Signal ◽  
Infected Cells ◽  
Pass Through ◽  
Borna Disease

ABSTRACT Borna disease virus (BDV) is a nonsegmented negative-strand RNA virus that replicates and transcribes its genome in the nucleus of infected cells. BDV proteins involved in replication and transcription must pass through the nuclear envelope to associate with the genomic viral RNA. The RNA-dependent RNA polymerase (L) of BDV is postulated to be the catalytic enzyme of replication and transcription. We demonstrated previously that BDV L localizes to the nucleus of BDV-infected cells and L-transfected cells. Nuclear localization of the protein presupposes the presence of a nuclear localization signal (NLS) within its primary amino acid sequence or cotransport to the nucleus with another karyophilic protein. Because L localized to the nucleus in the absence of other viral proteins, we investigated the possibility that L contains an NLS. The minimal sequence required for nuclear localization of L was identified by analyzing the subcellular distribution of deletion mutants of L fused to a flag epitope tag or β-galactosidase. Although the majority of the L fusion proteins localized to the cytoplasm of transfected BSR-T7 cells, a strong NLS (844RVVKLRIAP852) with basic and proline residues was identified. Mutation of this sequence resulted in cytoplasmic distribution of L, confirming that this sequence was necessary and sufficient to drive the nuclear localization of L.

scholarly journals Properties of non-structural protein 1 of Semliki Forest virus and its interference with virus replication

2008 ◽  
Vol 89 (6) ◽  
pp. 1457-1466 ◽  
Author(s):  
Kaja Kiiver ◽  
Ingrid Tagen ◽  
Eva Žusinaite ◽  
Nele Tamberg ◽  
John K. Fazakerley ◽  
...  
Keyword(s):  
Cell Lines ◽  
Rna Synthesis ◽  
Semliki Forest Virus ◽  
Detectable Effect ◽  
Structural Protein ◽  
Genomic Rna ◽  
Rna Translation ◽  
Initiation Of Replication ◽  
Fold Reduction ◽  
Infected Cells

Semliki Forest virus (SFV) non-structural protein 1 (nsP1) is a major component of the virus replicase complex. It has previously been studied in cells infected with virus or using transient or stable expression systems. To extend these studies, tetracycline-inducible stable cell lines expressing SFV nsP1 or its palmitoylation-negative mutant (nsP16D) were constructed. The levels of protein expression and the subcellular localization of nsP1 in induced cells were similar to those in virus-infected cells. The nsP1 expressed by stable, inducible cell lines or by SFV-infected HEK293 T-REx cells was a stable protein with a half-life of approximately 5 h. In contrast to SFV infection, induction of nsP1 expression had no detectable effect on cellular transcription, translation or viability. Induction of expression of nsP1 or nsP16D interfered with multiplication of SFV, typically resulting in a 5–10-fold reduction in virus yields. This reduction was not due to a decrease in the number of infected cells, indicating that nsP1 expression does not block virus entry or initiation of replication. Expression of nsP1 interfered with virus genomic RNA synthesis and delayed accumulation of viral subgenomic RNA translation products. Expression of nsP1 with a mutation in the palmitoylation site reduced synthesis of genomic and subgenomic RNAs and their products of translation, and this effect did not resolve with time. These results are in agreement with data published previously, suggesting a role for nsP1 in genomic RNA synthesis.

scholarly journals Overproduction of v-Myc in the nucleus and its excess over Max are not required for avian fibroblast transformation

1993 ◽  
Vol 13 (6) ◽  
pp. 3623-3631
Author(s):  
A T Tikhonenko ◽  
A R Hartman ◽  
M L Linial
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Point Mutations ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Localization Signal ◽  
Infected Cells ◽  
Fibroblast Transformation

The cellular proto-oncogene c-myc can acquire transforming potential by a number of different means, including retroviral transduction. The transduced allele generally contains point mutations relative to c-myc and is overexpressed in infected cells, usually as a v-Gag-Myc fusion protein. Upon synthesis, v-Gag-Myc enters the nucleus, forms complexes with its heterodimeric partner Max, and in this complex binds to DNA in a sequence-specific manner. To delineate the role for each of these events in fibroblast transformation, we introduced several mutations into the myc gene of the avian retrovirus MC29. We observed that Gag-Myc with a mutated nuclear localization signal is confined predominantly in the cytoplasm and only about 5% of the protein could be detected in the nucleus (less than the amount of endogenous c-Myc). Consequently, only a small fraction of Max is associated with Myc. However, cells infected with this mutant exhibit a completely transformed phenotype in vitro, suggesting that production of enough v-Gag-Myc to tie up all cellular Max is not needed for transformation. While the nuclear localization signal is dispensable for transformation, minimal changes in the v-Gag-Myc DNA-binding domain completely abolish its transforming potential, consistent with a role of Myc as a transcriptional regulator. One of its potential targets might be the endogenous c-myc, which is repressed in wild-type MC29-infected cells. Our experiments with MC29 mutants demonstrate that c-myc down-regulation depends on the integrity of the v-Myc DNA-binding domain and occurs at the RNA level. Hence, it is conceivable that v-Gag-Myc, either directly or circuitously, regulates c-myc transcription.

scholarly journals Intracellular localization and determination of a nuclear localization signal of the core protein of dengue virus

2002 ◽  
Vol 83 (12) ◽  
pp. 3093-3102 ◽  
Author(s):  
Shao-Hung Wang ◽  
Wan-Jr Syu ◽  
Kao-Jean Huang ◽  
Huan-Yao Lei ◽  
Chen-Wen Yao ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Core Protein ◽  
Intracellular Localization ◽  
Intracellular Location ◽  
The Core ◽  
Core Proteins ◽  
Localization Signal ◽  
Infected Cells

In dengue virus (DEN) particles, the core protein is a structural protein of the nucleocapsid. The core protein is known to be present in the nucleus of DEN-infected cells but there have been conflicting reports as to whether it is also present in the nucleolus. To clarify this, the intracellular location of the core protein was examined using a monoclonal antibody, 15B11, which was produced in this study. Immunofluorescence staining with this antibody demonstrated that the core protein first appeared in the cytoplasm and then in the nuclei and nucleoli of infected cells. Nuclear localization of the core protein was determined to be independent of other DEN proteins, since recombinant core proteins still entered the nuclei and nucleoli of cells transfected with only the core protein gene. Three putative nuclear localization signal motifs have been predicted to be present on the core protein. Deletion of the first one (KKAR), located at aa 6–9, and mutation of the second one (KKSK), located at aa 73–76, did not eliminate the nuclear localization property of the core protein. The third motif with a bipartite structure, RKeigrmlnilnRRRR, located at aa 85–100, was determined to be responsible for the nuclear localization of the core protein, since the core protein without this motif was located exclusively in the cytoplasm of DEN-infected cells and that this motif mediated nuclear localization of a normally cytoplasmic protein.

scholarly journals Human Cytomegalovirus UL84 Localizes to the Cell Nucleus via a Nuclear Localization Signal and Is a Component of Viral Replication Compartments

2002 ◽  
Vol 76 (17) ◽  
pp. 8931-8938 ◽  
Author(s):  
Yiyang Xu ◽  
Kelly S. Colletti ◽  
Gregory S. Pari
Keyword(s):  
Amino Acids ◽  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Human Fibroblasts ◽  
Reading Frame ◽  
Early Protein ◽  
Localization Signal ◽  
Infected Cells

ABSTRACT The UL84 open reading frame encodes a protein that is required for origin-dependent DNA replication and interacts with the immediate-early protein IE2 in lytically infected cells. Transfection of UL84 expression constructs showed that UL84 localized to the nucleus of transfected cells in the absence of any other viral proteins and displayed a punctate speckled fluorescent staining pattern. Cotransfection of all the human cytomegalovirus replication proteins and oriLyt, along with pUL84-EGFP, showed that UL84 colocalized with UL44 (polymerase accessory protein) in replication compartments. Experiments using infected human fibroblasts demonstrated that UL84 also colocalized with UL44 and IE2 in viral replication compartments in infected cells. A nuclear localization signal was identified using plasmid constructs expressing truncation mutants of the UL84 protein in transient transfection assays. Transfection assays showed that UL84 failed to localize to the nucleus when 200 amino acids of the N terminus were deleted. Inspection of the UL84 amino acid sequence revealed a consensus putative nuclear localization signal between amino acids 160 and 171 (PEKKKEKQEKK) of the UL84 protein.

scholarly journals Identification of a Highly Conserved, Functional Nuclear Localization Signal within the N-Terminal Region of Herpes Simplex Virus Type 1 VP1-2 Tegument Protein

2008 ◽  
Vol 82 (11) ◽  
pp. 5234-5244 ◽  
Author(s):  
F. Abaitua ◽  
P. O'Hare
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Pseudorabies Virus ◽  
Single Amino Acid ◽  
Functional Assay ◽  
Structural Protein ◽  
Localization Signal ◽  
Simplex Virus

ABSTRACT VP1-2 is a large structural protein assembled into the tegument compartment of the virion, conserved across the herpesviridae, and essential for virus replication. In herpes simplex virus (HSV) and pseudorabies virus, VP1-2 is tightly associated with the capsid. Studies of its assembly and function remain incomplete, although recent data indicate that in HSV, VP1-2 is recruited onto capsids in the nucleus, with this being required for subsequent recruitment of additional structural proteins. Here we have developed an antibody to characterize VP1-2 localization, observing the protein in both cytoplasmic and nuclear compartments, frequently in clusters in both locations. Within the nucleus, a subpopulation of VP1-2 colocalized with VP26 and VP5, though VP1-2-positive foci devoid of these components were observed. We note a highly conserved basic motif adjacent to the previously identified N-terminal ubiquitin hydrolase domain (DUB). The DUB domain in isolation exhibited no specific localization, but when extended to include the adjacent motif, it efficiently accumulated in the nucleus. Transfer of the isolated motif to a test protein, β-galactosidase, conferred specific nuclear localization. Substitution of a single amino acid within the motif abolished the nuclear localization function. Deletion of the motif from intact VP1-2 abrogated its nuclear localization. Moreover, in a functional assay examining the ability of VP1-2 to complement growth of a VP1-2-ve mutant, deletion of the nuclear localization signal abolished complementation. The nuclear localization signal may be involved in transport of VP1-2 early in infection or to late assembly sites within the nucleus or, considering the potential existence of VP1-2 cleavage products, in selective localization of subdomains to different compartments.

scholarly journals Overproduction of v-Myc in the nucleus and its excess over Max are not required for avian fibroblast transformation.

1993 ◽  
Vol 13 (6) ◽  
pp. 3623-3631 ◽  
Author(s):  
A T Tikhonenko ◽  
A R Hartman ◽  
M L Linial
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Point Mutations ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Localization Signal ◽  
Infected Cells ◽  
Fibroblast Transformation

The cellular proto-oncogene c-myc can acquire transforming potential by a number of different means, including retroviral transduction. The transduced allele generally contains point mutations relative to c-myc and is overexpressed in infected cells, usually as a v-Gag-Myc fusion protein. Upon synthesis, v-Gag-Myc enters the nucleus, forms complexes with its heterodimeric partner Max, and in this complex binds to DNA in a sequence-specific manner. To delineate the role for each of these events in fibroblast transformation, we introduced several mutations into the myc gene of the avian retrovirus MC29. We observed that Gag-Myc with a mutated nuclear localization signal is confined predominantly in the cytoplasm and only about 5% of the protein could be detected in the nucleus (less than the amount of endogenous c-Myc). Consequently, only a small fraction of Max is associated with Myc. However, cells infected with this mutant exhibit a completely transformed phenotype in vitro, suggesting that production of enough v-Gag-Myc to tie up all cellular Max is not needed for transformation. While the nuclear localization signal is dispensable for transformation, minimal changes in the v-Gag-Myc DNA-binding domain completely abolish its transforming potential, consistent with a role of Myc as a transcriptional regulator. One of its potential targets might be the endogenous c-myc, which is repressed in wild-type MC29-infected cells. Our experiments with MC29 mutants demonstrate that c-myc down-regulation depends on the integrity of the v-Myc DNA-binding domain and occurs at the RNA level. Hence, it is conceivable that v-Gag-Myc, either directly or circuitously, regulates c-myc transcription.

scholarly journals Identification and characterization of complex dual nuclear localization signals in human bocavirus NP1

2013 ◽  
Vol 94 (6) ◽  
pp. 1335-1342 ◽  
Author(s):  
Qian Li ◽  
Zhenfeng Zhang ◽  
Zhenhua Zheng ◽  
Xianliang Ke ◽  
Huanle Luo ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Human Bocavirus ◽  
Structural Protein ◽  
Nuclear Localization Signals ◽  
Importin Α ◽  
Localization Signal ◽  
Bovine Parvovirus

Human bocavirus (HBoV), closely related to canine minute virus (MVC) and bovine parvovirus (BPV), is a new member of the Bocavirus genus within the Parvoviridae family. The non-structural protein NP1 of HBoV is a nuclear localized protein and plays an important role in DNA replication as well as in the evasion of host innate immunity. In the current study, we provide the first evidence that NP1 possesses a non-classical nuclear localization signal (ncNLS) (amino acids 7–50). Embedded within this ncNLS is a classical bipartite nuclear localization signal (cNLS) (amino acids 14–28), capable of transporting a heterologous cytoplasmic protein β-galactosidase fusion protein (β-gal-EGFP) to the nucleus via the classical importin α/β1-mediated pathway. Amino acids 7–50 containing the cNLS and the ncNLS of NP1 or full-length NP1 interact with importin α1, importin β1 and importin β1Δ, which lacks the importin α binding domain, indicating that the nuclear import of NP1 is through both conventional importin α/β1 heterodimer- and non-classical importinß1-mediated pathways. Given that the arrangement of a cNLS embedded within an ncNLS is unusual in viral proteins, our data together reveal a novel molecular mechanism underlying the nuclear import of HBoV NP1, providing a basis for further understanding its biological function.

scholarly journals Nuclear Localization of Avian Polyomavirus Structural Protein VP1 Is a Prerequisite for the Formation of Virus-Like Particles

2004 ◽  
Vol 78 (2) ◽  
pp. 930-937 ◽  
Author(s):  
Reimar Johne ◽  
Hermann Müller
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Influenza Viruses ◽  
Structural Proteins ◽  
Structural Protein ◽  
Virus Like Particles ◽  
Localization Signal ◽  
Avian Polyomavirus

ABSTRACT Virions of polyomaviruses consist of the major structural protein VP1, the minor structural proteins VP2 and VP3, and the viral genome associated with histones. An additional structural protein, VP4, is present in avian polyomavirus (APV) particles. As it had been reported that expression of APV VP1 in insect cells did not result in the formation of virus-like particles (VLP), the prerequisites for particle formation were analyzed. To this end, recombinant influenza viruses were created to (co)express the structural proteins of APV in chicken embryo cells, permissive for APV replication. VP1 expressed individually or coexpressed with VP4 did not result in VLP formation; both proteins (co)localized in the cytoplasm. Transport of VP1, or the VP1-VP4 complex, into the nucleus was facilitated by the coexpression of VP3 and resulted in the formation of VLP. Accordingly, a mutant APV VP1 carrying the N-terminal nuclear localization signal of simian virus 40 VP1 was transported to the nucleus and assembled into VLP. These results support a model of APV capsid assembly in which complexes of the structural proteins VP1, VP3 (or VP2), and VP4, formed within the cytoplasm, are transported to the nucleus using the nuclear localization signal of VP3 (or VP2); there, capsid formation is induced by the nuclear environment.

scholarly journals Functional Characterization of the Nuclear Localization Signal for a Suppressor of Posttranscriptional Gene Silencing

2003 ◽  
Vol 77 (12) ◽  
pp. 7026-7033 ◽  
Author(s):  
Xiangli Dong ◽  
Rene van Wezel ◽  
John Stanley ◽  
Yiguo Hong
Keyword(s):  
Amino Acid ◽  
Gene Silencing ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Potato Virus X ◽  
Posttranscriptional Gene Silencing ◽  
Arginine Residues ◽  
Localization Signal

ABSTRACT The nucleus-localized C2 protein of Tomato yellow leaf curl virus-China (TYLCV-C) is an active suppressor of posttranscriptional gene silencing (PTGS). Consistently, infection with TYLCV-C resulted in PTGS arrest in plants. The C2 protein possesses a functional, arginine-rich nuclear localization signal within the basic amino acid-rich region 17KVQHRIAKKTTRRRR31. When expressed from potato virus X, C2-RRRR31DVGG (in which the four consecutive arginine residues 28RRRR31 were replaced with DVGG) that had been tagged with a green fluorescent protein (GFP) failed to transport GFP into nuclei and was dysfunctional in inducing necrosis and suppressing PTGS in plants. Amino acid substitution mutants C2-K17D-GFP, C2-HR21DV-GFP, and C2-KK25DI-GFP localized to nuclei and produced necrosis, but only C2-K17D-GFP suppressed PTGS. The N-terminal portions C21-31 and C217-31 fused in frame to GFP were capable of targeting GFP to nuclei, but neither caused necrosis nor affected PTGS. Our data establish that nuclear localization is likely required for C2 protein to function in C2-mediated induction of necrosis and suppression of PTGS, which may follow diverse pathways in plants. Possible mechanisms of how the C2 protein involves these biological functions are discussed.

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