Assembly of semliki forest and influenza A viruses

Abstract

Enveloped viruses contain an external lipid membrane with spikes surrounding their nucleocapsid (NC). The envelope is acquired at a late stage of virus assembly by budding of the NC through a virus-modified host membrane. In this thesis, the assembly and budding processes of two enveloped RNA viruses, Semliki Forest virus (SFV) and influenza virus has been studied. It is well established that the budding of SFV at the plasma membrane (PM) requires the binding of its spike proteins to the NC.

In this work I have analysed; i) the spike assembly process, ii) the possible existence of a pool of cell surface spikes that is used for budding and iii) the NC- interacting site in the cytoplasmic domain of the spike protein. In these studies I have used various heterologous gene expression systems including that of SFV and biochemical and cell biological techniques. The SFV spike is composed of three copies of a p62-El heterodimer which is processed intracellularly into an E2-El form by cleavage of an E3-portion from p62. Analyses of a spike deletion variant lacking the E3 region suggested that E3 is very important for the synthesis of a form of p62 that is competent to bind to El. If the E3 region was lacking, heterodimers could not form. Results of studies with a capsid protein deletion variant suggested that the spike heterodimers were syntesized and transported normally but, after arrival at the PM, they were rapidly degraded. This suggested that there is no pool of free spikes at the cell surface of virus infected cells, but that all spikes that arrive at the PM are rapidly captured by the NCs into virus particles.

Finally, we studied the structural requirements of the cytoplasmic domain of the p62/E2 protein for NC-binding during budding. This was done using several mutants that were engineered in vitro and in which changes were introduced into different conserved amino acid residues. The results showed that three conserved cysteine residues and one conserved tyrosine residue represented structural elements of the cytoplasmic domain that were crucial for NC-binding. In contrast to SFV much less is known about the assembly of influenza virus. In this work I have studied the properties of the two most abundant structural proteins, that is Ml and nucleoprotein (NP) of influenza virus. NP constitutes the major protein in the viral ribonucleoprotein (RNP) structures inside the particle whereas Ml forms a protein layer beneath the viral membrane. It is not known how these proteins participate in RNP assembly and virus particle assembly/budding. The two proteins were expressed separately or together in BHK-2 I cells using the SFV expression system. Results of immunofluorescence and cell fractionation analyses showed that NP accumulated in the nucleus and Ml in a perinuclear region. Both proteins underwent extensive self-oligomerization. A minor fraction of Ml was found to be membrane-associated. Ml expressed in cells did not support particle budding at the cell surface. Coexpression of Ml and NP in cells did not result in any detectable molecular interaction between these two proteins.

We conclude that both proteins have a strong tendency to self-polymerize. These features of NP and Ml are probably important during influenza virus infection for the formation of viral RNP particles and virus particles at the PM, respectively. However, additional components, e.g. RNA genomes and viral spikes, are apparently necessary to drive NP-M1 interactions and budding at the cell surface.