Mapping of interacting domains between the nucleocapsid protein and the phosphoprotein of vesicular stomatitis virus by using a two-hybrid system.

Abstract

Specific interaction between the nucleocapsid protein (N) and the phosphoprotein (P) of vesicular stomatitis virus (VSV), an important step in the life-cycle ofthe virus, was studied by using a two-hybrid system. Plasmids encoding P fused with the yeast GAL4 DNA-binding domain (pGALP) and N fused with the herpes simplex virus VP16 transactivating region (pVPN) were transfected into CHO cells along with a reporter plasmid encoding chloramphenicol acetyltransferase (CAT). The ability of N and P to associate in vivo was measured by activation of the CAT gene by the VP16 transactivating region. Transfection of plasmids pGALP and pVPN resulted in a high level of CAT activity, indicating that the N and P portions of the fusion proteins associated very strongly with each other. Progressive C-terminal deletions of the P protein revealed two regions that are important for association with the N protein: the N-terminal acidic domain and the C-terminal basic domain. Phosphorylation of P protein was not required for N-P association. Various deletions and mutations of the N protein revealed the C-terminal 5 amino acids (Val-Glu-PheAsp-Lys), in particular the amino acids Val-Glu-Phe, to be critical for N association with P. This two-hybrid system can be used in other viral systems to study the interaction between proteins involved in transcription and replication.The nucleocapsid protein N of vesicular stomatitis virus (VSV) tightly wraps the RNA genome and maintains the structural integrity and the template function of the negativestrand genome RNA (1). Within the virion this N-RNA template is associated with the RNA polymerase L and the transcription factor, phosphoprotein P, to form the transcribing ribonucleoprotein (RNP) complex. During transcription, the RNA polymerase complex (L and P) interacts with the N protein of the N-RNA template to transiently displace N and gain access to the genome RNA. During replication of the genome RNA, a soluble form of the N protein is required (2). This form of the N protein has been proposed to interact with nascently transcribed RNA chains from the RNP complex and to switch the mode of the RNA polymerase from transcription to replication (2). This results in the formation of an N-RNA complex containing full-length sense or antisense genome RNA. The precise mechanism by which the N protein recognizes the cognate sequence within the nascent RNA chain and continues to encapsidate during transcription remains unclear.Development of in vitro replication systems and studies on the structure and function of the N protein have provided some insight into the replication step of the virus' life-cycle (1). Although the N protein alone can initiate replication in vitro, albeit inefficiently, the presence of the P protein was found to greatly stimulate the reaction (3-5). Moreover, itThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact...

N-Terminal Phosphorylation of Phosphoprotein of Vesicular Stomatitis Virus Is Required for Preventing Nucleoprotein from Binding to Cellular RNAs and for Functional Template Formation

“…This finding confirms those of several studies demonstrating that phosphorylation of the P's of rabies virus and Sendai virus is not required for their oligomerization (31-33), indicating that P of VSV behaves like the P's of rabies virus and Sendai virus. Furthermore, our in vivo coimmunoprecipitation assay also confirmed that N-terminal phosphorylation of P is dispensable for the interaction of P with N and L, although Gao and Lenard (14,15) proposed that N-terminal phosphorylation may be involved in interaction of P with L. Moreover, our results also support a previous study revealing that phosphorylation of VSV P is not required for N-P complex formation (34). Thus, the transcriptional inactivity of P3A in vivo may not be caused by its selfassociation, as it interacted with L and N.…”

Altered Activation of Protein Kinase PKR and Enhanced Apoptosis in Dystonia Cells Carrying a Mutation in PKR Activator Protein PACT

“…Mammalian Two-hybrid Interaction Assay-The WT PACT and P222L ORFs were subcloned into pSG424 (Addgene) such that it produced an in-frame fusion to GAL4 DBD and in VP16 AD vector pVP16AASV19N (8,34) such that it produced an in-frame fusion to VP16 AD. Fusion proteins were tested for interaction in various combinations.…”

Measles virus phosphoprotein (P) requires the NH2- and COOH-terminal domains for interactions with the nucleoprotein (N) but only the COOH terminus for interactions with itself

“…Similar systems based upon Gal4 and VP16 fusion proteins have been described recently whereby interactive domains of other viral proteins have been identified and mapped (Takacs et al, 1993). Our data support and extend those of Huber et al (1991), who utilized a biochemical approach to show that the Cterminal half of the P protein was important for binding to N. Although the involvement of the C-terminal region of VP16/P in binding to Gal4/N may have been anticipated, the importance of the NH2-terminal region of P in binding to N was unforeseen, since experiments to ascertain the importance of the NHz terminus of P in binding to N or to other measles virus proteins had yet to be performed.…”

“…A more thorough understanding of the structural and functional domains of these key regulatory proteins of measles virus, and their homologues encoded by other negativestranded RNA viruses, will undoubtedly enhance our knowledge of paramyxovirus replication and pathogenesis. Toward this end, interactions among the N, P and L proteins of Sendai virus (Buchholz et al, 1994;Curran et al, 1994;Homann et al, 1991 ;Ryan & Portner, 1990;Ryan et al, 1991;Smallwood et al, 1994), vesicular stomatitis virus (Howard & Wertz, 1989;Takacs et al, 1993), rabies virus (Fu et al, 1994) and measles virus (Horikami et al, 1994;Huber et al, 1991) have been the focus of recent investigations. The COOH-* Author for correspondence.…”