Requirement for cyclophilin A for the replication of vesicular stomatitis virus New Jersey serotype

Immunoprecipitation and subsequent mass spectrometry analysis of the cellular proteins from cells expressing the vesicular stomatitis virus (VSV) P protein identified the poly(C) binding protein 2 (PCBP2) as one of the P protein-interacting proteins. To investigate the role of PCBP2 in the viral life cycle, we examined the effects of depletion or overexpression of this protein on VSV growth. Small interfering RNA-mediated silencing of PCBP2 promoted VSV replication. Conversely, overexpression of PCBP2 in transfected cells suppressed VSV growth. Further studies revealed that PCBP2 negatively regulates overall viral mRNA accumulation and subsequent genome replication. Coimmunoprecipitation and immunofluorescence microscopic studies showed that PCBP2 interacts and colocalizes with VSV P protein in virus-infected cells. The P-PCBP2 interaction did not result in reduced levels of protein complex formation with the viral N and L proteins, nor did it induce degradation of the P protein. In addition, PCBP1, another member of the poly(C) binding protein family with homology to PCBP2, was also found to interact with the P protein and inhibit the viral mRNA synthesis at the level of primary transcription without affecting secondary transcription or genome replication. The inhibitory effects of PCBP1 on VSV replication were less pronounced than those of PCBP2. Overall, the results presented here suggest that cellular PCBP2 and PCBP1 antagonize VSV growth by affecting viral gene expression and highlight the importance of these two cellular proteins in restricting virus infections. Vesicular stomatitis virus (VSV)is an enveloped RNA virus in the family Rhabdoviridae. The negative-sense RNA genome of VSV is 11,161 nucleotides in length and encodes 5 proteins: the nucleocapsid protein (N), the phosphoprotein (P), the matrix (M), the glycoprotein (G), and the large protein (L). Inside the virion, the viral genome is tightly encapsidated with the N protein to form the ribonucleocapsid complex (RNP or NC) with which the viral RNA-dependent RNA polymerase (RdRp) complex of P-L proteins (41) is associated. The G protein forms spikes on the surface of the VSV virion and mediates virus attachment as well as entry by clathrin-mediated endocytosis. Based on the low-pH environment of the endosome, the VSV G protein mediates fusion of the viral envelope with the endosomal membrane, releasing the viral RNP into the cytoplasm of the infected cell. Once inside the cytosol, the RNP undergoes primary transcription by the associated RdRp activity of the L protein along with its cofactor P protein. Translation of the transcripts by the cellular translation machinery generates viral proteins, which are used for further steps in the infection process, including genome replication, secondary transcription, assembly, and budding (53).The viral P is a multifunctional protein that modulates the association of viral replicative proteins and is indispensable for viral growth. It is required for viral genome transcription and replication (18) and also pl...

Section: Discussionmentioning

confidence: 99%

Antagonistic Effects of Cellular Poly(C) Binding Proteins on Vesicular Stomatitis Virus Gene Expression

Dinh

Beura

Panda³

et al. 2011

“…VSV N mRNA accumulation owing to primary transcription was measured as described previously (8). KO2 and KI cells were pretreated with 1,000 U of recombinant human IFN-␣ A/D (Hoffmann LaRoche, Inc.) per ml for 16 h and washed with PBS once.…”

Section: Methodsmentioning

confidence: 99%

“…RNA (10 g) was separated on 1.2% agarose-formaldehyde gels, transferred to a Hybond-N ϩ membrane (Amersham Biosciences), and cross-linked with UV. The blots were incubated with ULTRAhyb (Ambion) hybridization buffer at 42°C for 4 h, prior to the addition of 32 P-cDNA, encoding the VSV N protein (8,20,33), which was labeled by using the Prime-a-Gene system (Promega) at 42°C for 16 h. The autoradiograms of the blots were prepared after washing four times at 50°C in 2ϫ SSC (1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate) with 0.1% SDS. The blots were stripped and reprobed with 32 P-labeled ␤-actin cDNA.…”

Section: Methodsmentioning

confidence: 99%

Phospholipid Scramblase 1 Potentiates the Antiviral Activity of Interferon

Dong

Zhou

Zhao

et al. 2004

Self Cite

112

116

“…Cyclophilin A (CypA), a chaperon protein possessing peptidyl prolyl cis-trans isomerase activity, binds to the N protein of the vesicular stomatitis virus (VSV) and is required for efficient viral replication (1). Hsp72 (20,29), an inducible heat shock protein, binds to N TAIL of MeV-Ed and competes with the P protein in binding to it (35).…”

mentioning

confidence: 99%

Peroxiredoxin 1 Is Required for Efficient Transcription and Replication of Measles Virus

Watanabe

Yoneda

Ikeda

et al. 2011

Measles is a highly contagious human disease caused by the measles virus (MeV). In this study, by proteomic analysis, we identified peroxiredoxin 1 (Prdx1) as a host factor that binds to the C-terminal region of the nucleoprotein (N; N TAIL ) of MeV. Glutathione S-transferase (GST) pulldown experiments showed that the Prdx1-binding site overlapped with the MeV phosphoprotein (P)-binding site on N TAIL and that Prdx1 competed for the binding to N TAIL with the P protein, which is a component of RNA-dependent RNA polymerase (RdRp). Furthermore, RNA interference for Prdx1 resulted in a significant reduction in MeV growth in HEK293-SLAM cells. A minigenome assay indicated that Prdx1 suppression affected the viral RNA transcription and/or replication step. Relative quantification of viral RNA by real-time PCR (RT-PCR) showed that Prdx1 suppression not only reduced viral RNA transcription and replication but also enhanced polar attenuation in viral mRNA transcription. Surface plasmon resonance analysis showed that the binding affinity of Prdx1 to MeV-N was 40-fold lower than that of MeV-P to MeV-N, which suggested that Prdx1 might be involved in the early stage of MeV infection, when the expression level of Prdx1 was much higher than that of MeV-P. Since Prdx1 was expressed abundantly and constitutively in various cells, the results in this study indicate that Prdx1 is one of the inherent host factors implicated in MeV RNA synthesis.Measles is a highly contagious and very serious human disease caused by the measles virus (MeV). MeV infection causes immunosuppression, which induces secondary infection and several complications, such as diarrhea, pneumonia, and encephalitis. Presently, measles can be prevented by administering live vaccines that are derived mainly from the MeV Edmonston strain (MeV-Ed); however, measles has still been the leading cause of death in children, particularly in developing countries, for the past 40 years (6).MeV is an enveloped virus with a negative single-stranded RNA genome and belongs to the genus Morbillivirus in the family Paramyxoviridae. MeV is composed of six structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin protein (H), and large protein (L). Among these structural proteins, the N, P, and L proteins are essential for viral RNA transcription and replication. The L protein exhibits RNA dependent-RNA polymerase (RdRp) activity and forms a complex with the P protein, which acts as a cofactor of RdRp. The N protein consists of two portions: the N-terminal portion, which is termed the core region, is a highly conserved region and is involved in the oligomerization and encapsidation of genomic RNA, and the C-terminal portion, which is termed the tail region, is a relatively variable and disordered region that binds to the P protein. In paramyxoviruses, the N-genomic RNA complex serves as a template for viral RNA transcription and replication by RdRp composed of P and L proteins. During viral RNA synthesis, the P protein binds ...