The Respiratory Syncytial Virus Phosphoprotein, Matrix Protein, and Fusion Protein Carboxy-Terminal Domain Drive Efficient Filamentous Virus-Like Particle Formation

ABSTRACT Virus-like particles (VLPs) are attractive as a vaccine concept. For human respiratory syncytial virus (hRSV), VLP assembly is poorly understood and appears inefficient. Hence, hRSV antigens are often incorporated into foreign VLP systems to generate anti-RSV vaccine candidates. To better understand the assembly, and ultimately to enable efficient production, of authentic hRSV VLPs, we examined the associated requirements and mechanisms. In a previous analysis in HEp-2 cells, the nucleoprotein (N), phosphoprotein (P), matrix protein (M), and fusion protein (F) were required for formation of filamentous VLPs, which, similar to those of wild-type virus, were associated with the cell surface. Using fluorescence and electron microscopy combined with immunogold labeling, we examined the surfaces of transfected HEp-2 cells and further dissected the process of filamentous VLP formation. Our results show that N is not required. Coexpression of P plus M plus F, but not P plus M, M plus F, or P plus F, induced both viral protein coalescence and formation of filamentous VLPs that resembled wild-type virions. Despite suboptimal coalescence in the absence of P, the M and F proteins, when coexpressed, formed cell surface-associated filaments with abnormal morphology, appearing longer and thinner than wild-type virions. For F, only the carboxy terminus (Fstem) was required, and addition of foreign protein sequences to Fstem allowed incorporation into VLPs. Together, the data show that P, M, and the F carboxy terminus are sufficient for robust viral protein coalescence and filamentous VLP formation and suggest that M-F interaction drives viral filament formation, with P acting as a type of cofactor facilitating the process and exerting control over particle morphology. IMPORTANCE hRSV is responsible for >100,000 deaths in children worldwide, and a vaccine is not available. Among the potential anti-hRSV approaches are virus-like particle (VLP) vaccines, which, based on resemblance to virus or viral components, can induce protective immunity. For hRSV, few reports are available concerning authentic VLP production or testing, in large part because VLP production is inefficient and the mechanisms underlying particle assembly are poorly understood. Here, we took advantage of the cell-associated nature of RSV particles and used high-resolution microscopy analyses to examine the viral proteins required for formation of wild-type-virus-resembling VLPs, the contributions of these proteins to morphology, and the domains involved in incorporation of the antigenically important viral F protein. The results provide new insights that will facilitate future production of hRSV VLPs with defined shapes and compositions and may translate into improved manufacture of live-attenuated hRSV vaccines.