| Human Respiratory Syncytial Virus (HRSV) is the leading cause of viral lower respiratory tract infections in infants and young children. The HRSV fusion (F) protein cytoplasmic tail (CT) and matrix protein (M) are key mediators of viral infectivity, but the underlying mechanisms are poorly understood. To systematically examine the role of the F protein CT in infectious virus production, an infectivity assay was developed. Substitutions of F CT amino acid residues 569-572 (CT-R2), as well as deletion of the entire CT, abrogated infectious progeny production and led to accumulation of M and F proteins at inclusion bodies (IBs). The coincidence of altered M and F targeting and strong decrease in virus formation and infectivity, upon CT-R2 mutations shows that F interaction with IBs is an important step in the virion assembly process and suggests that CT residues 569-572 act to facilitate release of M-ribonucleoprotein complexes from IBs. To study the role of M in detail, an experimental system was developed to generate infectious HRSV lacking M expression (M-null). The role of M in virus assembly was then examined by infecting HEp-2 and Vero cells with the M-null virus, and assessing the impact on infectious virus production and viral protein trafficking. In the absence of M, infectious progeny production was strongly impaired and short, stunted, filaments were present at the infected cell surface. This suggests that M is not required for the initial stages of filament formation, but plays an important role in their maturation. Because lipid rafts were shown to be involved in HRSV assembly, we also investigated a potential role for the F protein CT in lipid raft targeting. Using established lipid raft markers and an engineered virus lacking the F protein CT, we found that the CT is not required for F protein association with lipid rafts. Our findings regarding the roles of F and M proteins in HRSV assembly will contribute to the ultimate goal of finding an effective vaccine, and other therapeutic measures against viral infection. |
