PAULINO GO´MEZ-PUERTAS, CARMEN ALBO,ESPERANZA PE´REZ-PASTRANA, AMPARO VIVO, and AGUSTI´N PORTELA
Centro Nacional de Biologı´a Fundamental and Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Majadahonda 28220, Madrid, Spain
Influenza virus that infects humans, birds and other animals falls under the category of Orthomyxoviridae. These are enveloped viruses differing in sizes and shapes. It can be spherical or filamentous in shape. The envelope is made up of lipid bilayer, derived from the host cell in which the virus replicates, assemble the viral proteins. The viral genome consists of 8 single stranded short segmented RNA molecules. It translates into 8 different viral proteins. Nucleocapsid protein covers the RNA and polymerase required for viral replication in host cells. Polymerase has 3 components- PA, PB1, PB2. RNA and the polymerase components together form RNP. There are three transmembrane proteins- Neuraminidase(NA), Hemaglutinin(HA) and M2. Matrix protein (M1) and NS2 are the structural components of the virus particle. M1 has the capability to bind with lipids and interacts strongly with plasma membrane. This paper focuses on the assembly of viral proteins, major driving force behind virus particle formation and tries to find the mechanism behind Influenza A virus morphogenesis.
To solve the mechanism of virus protein assembly and to characterize the virus formation of virus like particles, they infected COS-1 cells with vTF7-3 and transfected with DNA mixtures containing the nine plasmids coding for all structural proteins or mixtures that lacked one of the plasmids or that lacked the plasmids encoding the three polymerase subunits. To determine the presence of VLPs in the transfected cultures, the supernatants of the COS-1 cells clarified by 33% sucrose cushion centrifugation. The pellet and supernatant was analyzed for the presence of the NP, M1, HA, and M2 proteins by immunoblotting, which shows that M1 and NA are essential for biochemical detection of VLPs whereas removal of other proteins does not significantly affect the production of VLPs. Later, EM studies were conducted to check the size and morphology of these VLPs. The particles were identical in shape and size with the true virion. VLPs were observed in cultures without RNA, NA, MS2, HA, NP but were not observed in cultures not expressing M1 protein. This result inferred that all viral structural proteins, except M1, can be removed individually without compromising the formation and budding of VLPs which morphologically resemble wild-type virion. They also proved that RNPs are not required for viral assembly of the VLPs. Lipid floatation assay confirmed the interaction between the plasma membrane and the M1 protein.
Figure 1: Visualization of VLPs in infected COS-1 cells
In this paper it was shown that M1 protein, in the absence of other viral polypeptides, can assemble into virus-like budding particles which are released into the culture medium. Coexpression of the HA glycoprotein modulates the self-association and membrane binding properties of the M1 polypeptide. This paper clearly demonstrated the key role played by the structural protein M1 in the formation, assembly and budding process of influenza virus particles.