Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, MD 20742, USA1
Author for correspondence: Siba Samal. Fax +1 301 935 6079. e-mail ss5{at}umail.umd.edu
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Abstract |
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Introduction |
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The P protein is a component of the ribonucleoprotein (RNP) complex, which contains the N-RNA nucleocapsid associated with N, P, M2-1 and L proteins. The N protein encapsidates the genomic RNA, and is, therefore, present in large quantities. The P protein is a major phosphorylated protein and is present in smaller quantities. The L protein, which is thought to form the enzymatic component of virus RNA-dependent RNA polymerase, is also present in smaller quantities. The RNP complex is required for transcription and replication of viral genome (Collins et al., 1996 ). During transcription, gene-start and gene-end signals on the viral genome are recognized, and 10 capped and polyadenylated mRNAs are produced. Replication, on the other hand, results in an encapsidated, full-length, antigenomic RNA. It is not completely understood how the switch between transcription and replication occurs. Studies with Sendai and vesicular stomatitis viruses suggest that the polymerase complex (PL) is required for both transcription and replication; whereas, replication requires an additional complex composed of unassembled N and P proteins (Curran et al., 1991
; Horikami et al., 1992
). However, a complete understanding of the transcription and replication will require knowledge about how different proteins of the RNP complex interact with each other.
We have previously used an in vitro protein-blotting protein overlay technique (Samal et al., 1993 ) and a yeast two-hybrid system (Mallipeddi et al., 1996
) to study the interaction of P and N proteins. However, these systems might not reflect the true proteinprotein interactions occurring in virus-infected cells. Recently, we developed a BRSV minigenome system to map the domains of N protein interacting with P protein (Khattar et al., 2000
). In this report, we used this system to map the domains of P protein necessary for interacting with N and L proteins.
In order to identify the regions of P protein required for interaction with N and L proteins, a total of 12 deletions of 20 amino acids each were made throughout the 241 amino acid P protein (mutants P1-20, P
21-40, P
41-60, P
61-80, P
81-100, P
101-120, P
121-140, P
141-160, P
161-180, P
181-200, P
201-220 and P
221-241, shown schematically in Fig. 1
). Plasmid pTM1-P, containing the BRSV P gene (A51908 strain) cloned downstream of the T7 RNA polymerase promoter of plasmid pTM1, has been used to generate these mutants. All the internal deletions of the P gene of BRSV were generated by a single round of PCR using the 5' phosphorylated internal primer pair (Byrappa et al., 1995
). All the P gene mutants were sequenced in their entirety and confirmed for correct P protein expression by in vitro transcription and translation, using the rabbit reticulocyte lysate system (Promega). To determine the amount of expression of these mutant proteins, HEp-2 cells were transfected with either wild-type or mutant pTM1-P plasmids, along with other minigenome system components as described later, and infected with modified vaccinia virus Ankara (MVA) expressing T7 RNA polymerase (a gift from B. Moss, NIH, USA). Expression of the wild-type or mutant P proteins was determined by Western blot assays using P-specific antiserum. As shown in Fig. 2
, all the deletion mutant P proteins accumulated intracellularly to a level comparable to that of wild-type P protein; thus, the defect in the mutant P proteins to interact with N or L protein, described later, was not due to differences in expression or stability of various mutant P proteins.
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To identify domains on the BRSV P protein required for binding to L protein, all the P mutants were tested for their ability to bind L protein in a coimmunoprecipitation assay with P-specific antiserum. We found that the PL interaction occurred in the presence or absence of N support plasmid; however, the PL interaction was stronger in the absence of N support plasmid. Therefore, we omitted N support plasmid from the transfection mixture for PL interaction studies. Deletion of 20 amino acids from the N-terminal end (mutant P1-20) and 21 amino acids from the C-terminal end (mutant P
221-241) had no effect on complex formation between P and L proteins (Fig. 3B
, lanes 4 and 16). Each of the internal deletion mutants strongly interacted with L protein (Fig. 3B
, lanes 59 and 1315) except for P
121-140 (lane 10) and P
141-160 (lane 11). The interaction of mutants P
121-140 and P
141-160 with L protein was weak. This indicated that these two regions are important for interaction with L protein. To further confirm our findings, we constructed deletion mutant P
121-160, in which amino acids 121140 and 141160 were deleted. As shown in Fig. 3(B
, lane 12), interaction of mutant P
121-160 with L protein was completely abolished, which confirms and further delineates the importance of this domain for interaction with L protein. The additional bands immunoprecipitated with anti-P antibody, and detected between P and L protein bands in Fig. 3(B
, lanes 311 and 1316) may be either due to degradation of L protein or due to coimmunoprecipitation of some cellular proteins associated with PL complexes. Our results are in agreement with the previous findings reported for rabies virus (Chenik et al., 1998
) and Sendai virus (Smallwood et al., 1994
) that N and L protein binding sites on P protein do not overlap. This correlates well with the concept that P protein can interact simultaneously with L and N proteins to act as transcription factor when complexed with L protein, and as a replication factor when complexed with N protein.
In conclusion, we have identified amino acids at position 121160 and amino acids at positions 161180 and 221241 of the P protein which are important for binding to L and N proteins, respectively. Although our results probably represent identification of authentic sites of interaction between P and L proteins, it must be considered that N protein was not included in our study due to the reasons mentioned above. Sequence comparison of the P protein of BRSV with other pneumovirus P proteins indicated a high degree of amino acid similarity from residues 128 to 184 (Ling et al., 1995 ). Computer-assisted analysis (Lupas et al., 1991
) of amino acids 100241 of the P protein indicated the formation of coiled coils at the immediate C terminus of the protein. The introduction of two proline residues at positions 220 and 236, which would be expected to prevent the formation of
-helices, has been shown to result in reduced ability of HRSV P protein to interact with N protein (Slack & Easton, 1998
). It appears, thus, that the
-helices and coiled coils present in this region may play an important role in NP and PL interactions. Further, the coiled coil structure has been found to be important for oligomerization in several paramyxoviruses. Various reports in paramyxoviruses indicated the role of oligomerization in P protein functionality. In HRSV, domains for oligomerization have been mapped in the region between 99 and 165 amino acids (Asenjo & Villanueva, 2000
). Whether the same region is important for oligomerization in BRSV is unknown. If it is so, then it is possible to speculate that disruption of oligomerization in this region of BRSV may prevent interaction of P protein with L protein. Further studies are needed to map the region of BRSV P protein important for oligomerization. Finally, it is important to note that the alterations in the P protein could cause conformational changes. Thus, the actual interactive site(s) may not be present within the deleted domains. The change of conformation of the mutant proteins may directly affect the interacting site(s) present elsewhere in the protein. Detailed studies by the use of peptides would certainly help us to understand the interacting domains of the P protein.
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Acknowledgments |
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References |
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Received 30 October 2000;
accepted 3 January 2001.