Mapping the domains on the phosphoprotein of bovine respiratory syncytial virus required for N–P and P–L interactions using a minigenome system

Sunil K. Khattar1, Abdul S. Yunus1 and Siba K. Samal1

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


   Abstract
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Abstract
Introduction
References
 
The interaction of bovine respiratory syncytial virus (BRSV) phosphoprotein (P) with nucleocapsid (N) and large polymerase (L) proteins was investigated using an intracellular BRSV–CAT minigenome replication system. Coimmunoprecipitation assays using P-specific antiserum revealed that the P protein can form complexes with N and L proteins. Deletion mutant analysis of the P protein was performed to identify the regions of P protein that interact with N and L proteins. The results indicate that two independent N-binding sites exist on the P protein: an internal region of 161–180 amino acids and a C-terminal region of 221–241 amino acids. The L-binding site was mapped to a region of P protein encompassing amino acids 121–160. The data suggest that N and L protein binding domains on the P protein do not overlap.


   Introduction
Top
Abstract
Introduction
References
 
Bovine respiratory syncytial virus (BRSV) is a major cause of respiratory disease in calves, resulting in substantial economic losses to the cattle industry (Van der Poel et al., 1994 ; Collins et al., 1988 ; Stott & Taylor, 1985 ). BRSV is an enveloped RNA virus which, along with human respiratory syncytial virus (HRSV) and pneumonia virus of mice, belongs to the genus Pneumovirus of the family Paramyxoviridae. BRSV and HRSV are similar in gene and protein compositions. The genome of BRSV is a single-stranded negative-sense RNA of 15140 nt (Buchholz et al., 1999 ). The BRSV genome encodes 11 proteins (Lerch et al., 1989 ; Mallipeddi et al., 1990 ). Four proteins are associated with the genomic RNA, namely nucleocapsid (N) protein, phosphoprotein (P), the large polymerase (L) protein and the transcription anti-termination factor M2-1. Three proteins are transmembrane components of the envelope; namely, fusion (F), attachment glycoprotein (G) and the small hydrophobic (SH) proteins. The matrix (M) protein is an inner virion protein. NS1 and NS2 are nonstructural proteins.

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 (P–L) 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 protein–protein 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 P{Delta}1-20, P{Delta}21-40, P{Delta}41-60, P{Delta}61-80, P{Delta}81-100, P{Delta}101-120, P{Delta}121-140, P{Delta}141-160, P{Delta}161-180, P{Delta}181-200, P{Delta}201-220 and P{Delta}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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Fig. 1. Schematic representation of all the mutant BRSV P proteins that were used for N–P and P–L interaction studies. The names of the mutant P proteins indicate the deleted amino acids and the remaining sequences are shown as solid bars.

 


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Fig. 2. Expression of wild-type or mutant BRSV P proteins in transfected HEp-2 cells. HEp-2 cells were infected with vaccinia virus (MVA-T7) and transfected with plasmids encoding the BRSV–CAT minigenome (0·4 µg), wild-type (WT) or mutant BRSV P protein as indicated (0·2 µg), N protein (0·4 µg) and L protein (0·1 µg). At 48 h post-transfection, cells were harvested and lysates were electrophoresed on a 4–20% gradient gel under reducing conditions. The separated proteins were probed in Western blot with P-specific antiserum. BRSV-specific P polypeptide is indicated to the right.

 
We have utilized a BRSV minigenome system to identify the regions of BRSV P protein that interact with N and L proteins. The advantage of this system is that protein–protein interactions occur under conditions of reconstituted BRSV transcription and replication, and thus, it is probably a close facsimile of the authentic situation. In this system, a plasmid-synthesized negative-sense BRSV minigenome containing the CAT gene is transcribed and replicated by BRSV N, P and L proteins supplied in trans from plasmids, using the vaccinia virus/T7 RNA polymerase expression system (Yunus et al., 1998 ). Construction of cDNA encoding the BRSV–CAT minigenome and construction of support plasmids pTM1-P, pTM1-N and pTM1-L containing P, N and L genes of the BRSV A51908 strain, respectively, cloned downstream of the T7 RNA polymerase promoter of plasmid pTM1 have been described previously (Yunus et al., 1998 ). Specifically, HEp-2 cells were infected with the MVA strain of vaccinia virus and transfected with a mixture of plasmids encoding the BRSV minigenome (0·4 µg), wild-type or mutant BRSV P protein (0·2 µg), N protein (0·4 µg) and L protein (0·1 µg) by using LipoFECTAMINE (Life Technologies). At 24 h post-transfection, cells were incubated in the presence of [35S]methionine for 5 h, and lysates were prepared in a buffer containing 50 mM Tris–HCl (pH 7·5), 150 mM NaCl and 0·7% Nonidet P-40. The cell lysates were subjected to immunoprecipitation with polyclonal P-specific antiserum (Fig. 3). Preparation of P-specific antiserum was described earlier (Khattar et al., 2000 ). Using P-specific antiserum, N protein (Fig. 3A, lane 3) and L protein (Fig. 3B, lane 3), coprecipitated with the P protein, indicating N–P and P–L complex formation, respectively, in transfected cells. These coimmunoprecipitations were not due to nonspecific precipitation of either N or L protein, since P-specific antiserum did not precipitate N protein (Fig. 3A, lane 1) or L protein (Fig. 3B, lane 1) in the absence of P protein.



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Fig. 3. Identification of BRSV P protein regions involved in interaction with N and L proteins. HEp-2 cells were transfected with the minigenome system components, as described in the legend to Fig. 2, and with plasmids encoding wild-type or mutant P proteins as indicated. The cells were pulse-labelled at 24 h post-infection or -transfection for 5 h with [35S]methionine and lysates were processed and subjected to immunoprecipitation with P-specific serum. Under these conditions, N protein (A) or L protein (B) coprecipitated due to its interaction with P protein. Immunoprecipitated proteins were separated by 10% SDS–PAGE under reducing conditions. As negative controls, the cells in lanes 1 and 2 in (A) did not receive plasmids encoding WT P or N proteins, respectively, and the cells in lanes 1 and 2 in (B) did not receive plasmids encoding WT P or L proteins, respectively. The molecular mass markers are indicated to the left (in kDa). BRSV-specific P, N or L polypeptides are indicated to the right.

 
To identify domains on the BRSV P protein required for binding to N protein, all the P deletion mutants generated above were tested for their ability to bind N protein in a coimmunoprecipitation assay with P-specific antiserum (Fig. 3A). Whereas the amount of labelled coimmunoprecipitated P protein varied in these mutants due to deletion of a variable number of methionine residues, the amount of N protein brought down by P-specific antiserum was essentially the same for all the P mutants. Deletion of 21 amino acids at the C terminus of the P protein (mutant P{Delta}221-241) completely abolished the interaction with the N protein (Fig. 3A, lane 15). On the other hand, a deletion of 20 amino acids at the N terminus (mutant P{Delta}1-20) did not abolish the interaction with the N protein (Fig. 3A, lane 4). Each of the internal deletion mutants formed a complex with the N protein (Fig. 3A, lanes 5–11, 13 and 14), except for P{Delta}161-180 (Fig. 3A, lane 12). Thus, an internal region of 161–180 amino acids is also important for interaction with P protein. Taken together, these results indicated that two independent N-binding regions exist on P protein: one is located between amino acids 161 and 180, the other is located in the C-terminal part between amino acids 221 and 241. A similar study of N–P interactions in various other non-segmented RNA viruses has suggested that the C terminus of P protein is particularly important in interaction with the nucleocapsid protein N or NP (Ryan & Portner, 1990 ; Takacs et al., 1993 ; Chenik et al., 1994 ; Zhao & Banerjee, 1995 ; Barr & Easton, 1995 ; Garcia-Barreno et al., 1996 ; Mallipeddi et al., 1996 ). Studies in rabies virus (Chenik et al., 1994 ) and Sendai virus (Ryan & Portner, 1990 ) indicated the existence of an internal region, in addition to a C-terminal region, important for binding to P protein. Using a yeast two-hybrid system for BRSV (Mallipeddi et al., 1996 ), and protein overlay blot system for pneumonia virus of mice (Barr & Easton, 1995 ), the N terminus of P protein has been shown to be important for N–P interactions. Our data did not indicate the involvement of the N terminus in interactions with P protein. This may be due to the fact that these interaction studies were performed in different systems. Moreover, in the yeast two-hybrid system, the N terminus of the protein may not be available to interact efficiently with the target protein.

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 P–L interaction occurred in the presence or absence of N support plasmid; however, the P–L interaction was stronger in the absence of N support plasmid. Therefore, we omitted N support plasmid from the transfection mixture for P–L interaction studies. Deletion of 20 amino acids from the N-terminal end (mutant P{Delta}1-20) and 21 amino acids from the C-terminal end (mutant P{Delta}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 5–9 and 13–15) except for P{Delta}121-140 (lane 10) and P{Delta}141-160 (lane 11). The interaction of mutants P{Delta}121-140 and P{Delta}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{Delta}121-160, in which amino acids 121–140 and 141–160 were deleted. As shown in Fig. 3(B, lane 12), interaction of mutant P{Delta}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 3–11 and 13–16) may be either due to degradation of L protein or due to coimmunoprecipitation of some cellular proteins associated with P–L 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 121–160 and amino acids at positions 161–180 and 221–241 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 100–241 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 {alpha}-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 {alpha}-helices and coiled coils present in this region may play an important role in N–P and P–L 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.


   Acknowledgments
 
We thank Daniel Rockemann for excellent technical assistance. This work was supported by USDA grant 9702414.


   References
Top
Abstract
Introduction
References
 
Asenjo, A. & Villanueva, N. (2000). Regulated but not constitutive human respiratory syncytial virus (HRSV) P protein phosphorylation is essential for oligomerization. FEBS Letters 467, 279-284.[Medline]

Barr, J. & Easton, A. J. (1995). Characterization of the interaction between the nucleoprotein and phosphoprotein of pneumonia virus of mice. Virus Research 39, 221-235.[Medline]

Buchholz, U. J., Finke, S. & Conzelmann, K.-K. (1999). Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. Journal of Virology 73, 251-259.[Abstract/Free Full Text]

Byrappa, S., Gavin, D. K. & Gupta, K. C. (1995). A highly efficient procedure for site-specific mutagenesis of full-length plasmids using Vent DNA polymerase. Genome Research 5, 404-407.[Abstract]

Chenik, M., Chebli, K., Gaudin, Y. & Blondel, D. (1994). In vivo interaction of rabies virus phosphoprotein (P) and nucleoprotein (N): existence of two N-binding sites on P protein. Journal of General Virology 75, 2889-2896.[Abstract]

Chenik, M., Schnell, M., Conzelmann, K. K. & Blondel, D. (1998). Mapping the interacting domains between the rabies virus polymerase and phosphoprotein. Journal of Virology 72, 1925-1930.[Abstract/Free Full Text]

Collins, J. K., Teegarden, R. M., Macvean, D. W., Smith, G. H., Frank, G. & Salman, S. (1988). Prevalence and specificity of antibodies to bovine respiratory syncytial virus in sera from feedlot and range cattle. American Journal of Veterinary Research 49, 1316-1319.[Medline]

Collins, P. L., Hill, M. G., Cristina, J. & Grosfeld, H. (1996). Transcription elongation factor of respiratory syncytial virus, a nonsegmented negative strand RNA virus. Proceedings of the National Academy of Sciences, USA 93, 81-85.[Abstract/Free Full Text]

Curran, J., Boeck, R. & Kolakofsky, D. (1991). The Sendai virus P gene expresses both an essential protein and an inhibitor of RNA synthesis by shuffling modules via mRNA editing. EMBO Journal 10, 3079-3085.[Abstract]

Garcia-Barreno, B., Delgado, T. & Malero, J. (1996). Identification of protein regions involved in the interaction of human respiratory syncytial virus phosphoprotein and nucleoprotein: significance for nucleocapsid assembly and formation of cytoplasmic inclusion. Journal of Virology 70, 801-808.[Abstract]

Horikami, S. M., Curran, J., Kolakofsky, D. & Moyer, S. A. (1992). Complexes of Sendai virus NP–P and P–L proteins are required for defective interfering particle genome replication in vitro. Journal of Virology 66, 4901-4908.[Abstract]

Khattar, S. K., Yunus, A. S., Collins, P. L. & Samal, S. K. (2000). Mutational analysis of the bovine respiratory syncytial virus nucleocapsid protein using a minigenome system: mutations that affect encapsidation, RNA synthesis, and interaction with the phosphoprotein. Virology 270, 215-228.[Medline]

Lerch, R. A., Stott, E. J. & Wertz, G. W. (1989). Characterization of bovine respiratory syncytial virus proteins and mRNAs and generation of cDNA clones to the viral mRNAs. Journal of Virology 63, 833-840.[Medline]

Ling, R., Davis, P. J., Qingzhong, Y., Wood, C. M., Pringle, C. R., Cavanagh, D. & Easton, A. J. (1995). Sequence and in vitro expression of the phosphoprotein gene of avian pneumovirus. Virus Research 36, 247-257.[Medline]

Lupas, A., Van Dyke, M. & Stock, J. (1991). Predicting coiled coils from protein sequences. Science 252, 1162-1164.[Medline]

Mallipeddi, S. K., Samal, S. K. & Mohanty, S. B. (1990). Analysis of polypeptides synthesized in bovine respiratory syncytial virus-infected cells. Archives of Virology 115, 23-36.[Medline]

Mallipeddi, S. K., Lupiani, B. & Samal, S. K. (1996). Mapping the domains on the phosphoprotein of bovine respiratory syncytial virus required for N–P interaction using a two-hybrid system. Journal of General Virology 77, 1019-1023.[Abstract]

Ryan, K. W. & Portner, A. (1990). Separate domains of Sendai virus P protein are required for binding to viral nucleocapsid. Virology 174, 515-521.[Medline]

Samal, S. K., Pastey, M. K., McPhillips, T. H. & Mohanty, S. B. (1993). Bovine respiratory syncytial virus nucleocapsid protein expressed in insect cells specifically interacts with the phosphoprotein and the M2 protein. Virology 193, 470-473.[Medline]

Slack, M. S. & Easton, A. J. (1998). Characterization of the interaction of the human respiratory syncytial virus phosphoprotein and nucleocapsid protein using the two-hybrid system. Virus Research 55, 167-176.[Medline]

Smallwood, S. K., Ryan, K. W. & Moyer, S. A. (1994). Deletion analysis defines a carboxyl-proximal region of Sendai virus P protein that binds to the polymerase L protein. Virology 202, 154-163.[Medline]

Stott, E. J. & Taylor, G. (1985). Respiratory syncytial virus: brief review. Archives of Virology 84, 1-52.[Medline]

Takacs, A. M., Das, T. & Banerjee, A. K. (1993). Mapping of interacting domains between nucleocapsid protein and the phosphoprotein of vesicular stomatitis virus by using a two-hybrid system. Proceedings of the National Academy of Sciences, USA 90, 10375-10379.[Abstract]

Van der Poel, W. H., Brand, A., Kramps, J. A. & Van Oirshot, J. T. (1994). Respiratory syncytial virus infections in human beings and cattle. An epidemiological review. Journal of Infection 29, 215-228.[Medline]

Yunus, A. S., Collins, P. L. & Samal, S. K. (1998). Sequence analysis of functional polymerase (L) gene of bovine respiratory syncytial virus: determination of minimal trans-acting requirements for RNA replication. Journal of General Virology 79, 2231-2238.[Abstract]

Zhao, H. & Banerjee, A. K. (1995). Interaction between the nucleocapsid protein and the phosphoprotein of human parainfluenza virus 3. Journal of Biological Chemistry 21, 12485-12490.

Received 30 October 2000; accepted 3 January 2001.