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 surfac... More
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.
