title: Interaction of Human Parainfluenza Virus Type 3 Nucleoprotein with Matrix Protein Mediates Internal Viral Protein Assembly.
abstract: Human parainfluenza virus type 3 (HPIV3) belongs to the Paramyxoviridae family. Its three internal viral proteins, the nucleoprotein (N), the phosphoprotein (P), and the polymerase (L), form the ribonucleoprotein (RNP) complex, which encapsidates the viral genome and associates with the matrix protein (M) for virion assembly. We previously showed that the M protein expressed alone is sufficient to assemble and release virus-like particles (VLPs) and a mutant with the L305A point mutation in the M protein (ML305A) has a VLP formation ability similar to that of wild-type M protein. In addition, recombinant HPIV3 (rHPIV3) containing the ML305A mutation (rHPIV3-ML305A) could be successfully recovered. In the present study, we found that the titer of rHPIV3-ML305A was at least 10-fold lower than the titer of rHPIV3. Using VLP incorporation and coimmunoprecipitation assays, we found that VLPs expressing the M protein (M-VLPs) can efficiently incorporate N and P via an N-M or P-M interaction and ML305A-VLPs had an ability to incorporate P via a P-M interaction similar to that of M-VLPs but were unable to incorporate N and no longer interacted with N. Furthermore, we found that the incorporation of P into ML305A-VLPs but not M-VLPs was inhibited in the presence of N. In addition, we provide evidence that the C-terminal region of P is involved in its interaction with both N and M and N binding to the C-terminal region of P inhibits the incorporation of P into ML305A-VLPs. Our findings provide new molecular details to support the idea that the N-M interaction and not the P-M interaction is critical for packaging N and P into infectious viral particles.
  
  
  OBJECTIVE
  Human parainfluenza virus type 3 (HPIV3) is a nonsegmented, negative-sense, single-stranded RNA virus that belongs to the Paramyxoviridae family and can cause lower respiratory tract infections in infants and young children as well as elderly or immunocompromised individuals. However, no effective vaccine has been developed or licensed. We used virus-like particle (VLP) incorporation and coimmunoprecipitation assays to determine how the M protein assembles internal viral proteins. We demonstrate that both nucleoprotein (N) and phosphoprotein (P) can incorporate into M-VLPs and N inhibits the M-P interaction via the binding of N to the C terminus of P. We also provide additional evidence that the N-M interaction but not the P-M interaction is critical for the regulation of HPIV3 assembly. Our studies provide a more complete characterization of HPIV3 virion assembly and substantiation that N interaction with M regulates internal viral organization.
authors.affiliation: State Key Laboratory of Virology and Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Guangyuan
authors.initials: G
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Virology and Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhong
authors.affiliation: State Key Laboratory of Virology and Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Yali
authors.initials: Y
authors.lastname: Qin
authors.affiliation: State Key Laboratory of Virology and Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China chenmz@whu.edu.cn.
authors.forename: Mingzhou
authors.initials: M
authors.lastname: Chen
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D006801:Humans; D047468:Immunoprecipitation; D009698:Nucleoproteins / Q000378:metabolism*; D010224:Parainfluenza Virus 3, Human / Q000502:physiology*; D011485:Protein Binding; D055503:Protein Multimerization; D014763:Viral Matrix Proteins / Q000378:metabolism*; D022701:Virosomes / Q000737:chemistry / Q000378:metabolism; D019065:Virus Assembly
chemical_list: D009698:Nucleoproteins; D014763:Viral Matrix Proteins; D022701:Virosomes