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, 75 (3), 1117-23

Nucleocapsid Incorporation Into Parainfluenza Virus Is Regulated by Specific Interaction With Matrix Protein

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Nucleocapsid Incorporation Into Parainfluenza Virus Is Regulated by Specific Interaction With Matrix Protein

E C Coronel et al. J Virol.

Abstract

The paramyxovirus nucleoproteins (NPs) encapsidate the genomic RNA into nucleocapsids, which are then incorporated into virus particles. We determined the protein-protein interaction between NP molecules and the molecular mechanism required for incorporating nucleocapsids into virions in two closely related viruses, human parainfluenza virus type 1 (hPIV1) and Sendai virus (SV). Expression of NP from cDNA resulted in in vivo nucleocapsid formation. Electron micrographs showed no significant difference in the morphological appearance of viral nucleocapsids obtained from lysates of transfected cells expressing SV or hPIVI NP cDNA. Coexpression of NP cDNAs from both viruses resulted in the formation of nucleocapsid composed of a mixture of NP molecules; thus, the NPs of both viruses contained regions that allowed the formation of mixed nucleocapsid. Mixed nucleocapsids were also detected in cells infected with SV and transfected with hPIV1 NP cDNA. However, when NP of SV was donated by infected virus and hPIV1 NP was from transfected cDNA, nucleocapsids composed of NPs solely from SV or solely from hPIVI were also detected. Although almost equal amounts of NP of the two viruses were found in the cytoplasm of cells infected with SV and transfected with hPIV1 NP cDNA, 90% of the NPs in the nucleocapsids of the progeny SV virions were from SV. Thus, nucleocapsids containing heterologous hPIV1 NPs were excluded during the assembly of progeny SV virions. Coexpression of hPIV1 NP and hPIV1 matrix protein (M) in SV-infected cells increased the uptake of nucleocapsids containing hPIV1 NP; thus, M appears to be responsible for the specific incorporation of the nucleocapsid into virions. Using SV-hPIV1 chimera NP cDNAs, we found that the C-terminal domain of the NP protein (amino acids 420 to 466) is responsible for the interaction with M.

Figures

FIG. 1
FIG. 1
Electron micrographs of nucleocapsid-like structures assembled in cells. Lysates of cells infected with SV or hPIV1 or transfected with NP cDNAs were stained with 1% aqueous uranyl acetate and examined by electron microscopy. (A) Cells infected with SV; (B) cells transfected with pCAGGS-SVNP; (C) cells infected with hPIV1; (D) cells transfected with pCAGGS-hNP; (E) cells transfected with pCAGGS-SVNP and pCAGGS-hNP; (F) cells transfected with pCAGGS-Ch1; (G) cells transfected with pCAGGS-SVNP and pCAGGS-Ch1; (H) mock-transfected cells.
FIG. 2
FIG. 2
Immunoprecipitation of nucleocapsid-like structure with specific MAbs. (A) Cells transfected with pCAGGS-SVNP or pCAGGS-hNP were labeled with Tran35S-label. The nucleocapsid-like structures were purified from cell lysates and immunoprecipitated. Samples purified from cells transfected with pCAGGS-SVNP (lanes 2 and 4) or pCAGGS-hNP (lanes 3 and 5) were immunoprecipitated by MAbs specific for SV NP (lanes 2 and 3) or hPIV1 NP (lanes 4 and 5). Lane 1, purified SV; lane 6, purified hPIV1. (B and C) Serial immunoprecipitation of nucleocapsid-like structures. (B) Cells were cotransfected with pCAGGS-SVNP and pCAGGS-hNP, and 35S-labeled nucleocapsid-like structures were purified and immunoprecipitated. Lanes 2 and 3, two serial radioimmunoprecipitations (RIP) with cross-reactive MAb cocktail (P27/M52); lanes 4 through 7, four serial immunoprecipitations, three with MAb WS16 (specific for SV NP) and the fourth with P27/M52; lanes 8 through 11, four serial immunoprecipitations, three with MAb P35 (specific for hPIV1 NP) and the fourth with P27/M52. Lanes 1 and 12, purified SV and hPIV1, respectively. (C) 35S-labeled nucleocapsid-like structures were prepared from cells infected with SV and transfected with pCAGGS-hNP. Immunoprecipitation was done as for panel B.
FIG. 3
FIG. 3
Incorporation of hPIV1 NP proteins into progeny SV. Cells were infected with SV and transfected with pCAGGS-hNP alone (lane 2) or together with pCAGGS-hM (lane 3), with pCAGGS-hHN (lane 4), with pCAGGS-hF (lane 5), with pCAGGS-hM and pCAGGS-hHN (lane 6), or with pCAGGS-hM and pCAGGS-hF (lane 7). Labeled progeny SV in the culture supernatants was purified and analyzed by SDS-PAGE. Lanes 1 and 8 represent purified SV and hPIV1, respectively. The amounts of SV and hPIV1 NP proteins in purified SV were determined by a PhosphorImager, and the ratios are shown.
FIG. 4
FIG. 4
Incorporation of chimera NP into progeny SV. (A) Immunoprecipitation of the expressed chimeric NP proteins. Cells were labeled after transfection with pCAGGS-SVNP, pCAGGS-Ch1, pCAGGS-Ch2, or pCAGGS-hNP, and the lysates were subjected to immunoprecipitation. A cocktail of cross-reactive MAbs was used for immunoprecipitation. (B) Chimeric NP incorporation into SV virions. Cells were infected with SV and transfected with mock cDNA (lane 1), pCAGGS-hNP alone (lane 2), or pCAGGS-Ch1 or pCAGGS-Ch2, alone (lanes 3 and 4) or together with pCAGGS-hM (lanes 6 to 7). Labeled progeny SV in the culture supernatants, was purified and analyzed by SDS-PAGE. The amounts of SV and hPIV1 NP were quantitated by a PhosphorImager. The arrow indicates the proteolytically digested NP fragment expressed from cDNA, which was also incorporated into SV. w/o hM and w hM, without and with hPIV1 M, respectively.

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