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 (M L305A ) has a VLP formation ability similar to that of wild-type M protein. In addition, recombinant HPIV3 (rHPIV3) containing the M L305A mutation (rHPIV3-M L305A ) could be successfully recovered. In the present study, we found that the titer of rHPIV3-M L305A 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 M L305A -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 M L305A -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 M L305A -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. IMPORTANCEHuman 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. Human parainfluenza virus type 3 (HPIV3) is a negativestrand RNA virus (NSV) that belongs to the Paramyxoviridae family and often causes lower respiratory tract infections in infants and young children. The HPIV3 genome consists of 6 open reading frames that encode 6 structural proteins: the nucleoprotein (N), phosphoprotein (P), RNA-dependent RNA polymerase (L), matrix protein (M), and two glycoprotei...

Viral protein requirements for assembly and release of human parainfluenza virus type 3 virus-like particles

“…The P protein did not release from cells when coexpressed with M (Fig. 1d), in contrast to recent HPIV3 results (Zhang et al, 2015). Additionally, none of the HPIV3 proteins increased M release efficiency, though coexpression of M with high concentrations of F resulted in decreased M release.…”

“…As shown in Fig. 1d, co-expression of the HPIV3 M and P proteins did not result in P release, in contrast to observations by Zhang et al (2015). To determine whether the N protein could mediate P protein incorporation into VLPs in our system, N protein was expressed along with the M and P proteins (Fig.…”

“…Recently, it was shown that a tagged HPIV3 M protein, alone and in combination with the HIPV3 N, F and HN proteins, was capable of inducing VLP formation (Zhang et al, 2014(Zhang et al, , 2015. However, the ability of the F and HN proteins to release from cells, and the role of the C protein were not investigated.…”

“…Co-expression of the M protein with N, HN and N+HN resulted in detection of the proteins in the floated fraction, indicating all proteins were lipid associated. For the Mononegavirales, the only investigation of P protein incorporation into VLPs was reported recently with HPIV3 (Zhang et al, 2015). As shown in Fig.…”

Measles Virus N and P Proteins Find Interacting Partners in Hela Cells.

“…The NTAIL is associated with the formation of ribonucleoprotein (RNP) complex 3 . The NTAIL is proposed to interact with Hsp72, IRF3, FygRII and NR [4][5][6][7] .…”