Aromatic interactions define the binding of the alphavirus spike to its nucleocapsid.

BACKGROUND: Most enveloped viruses bud from infected cells by a process in which viral intracellular core components interact with cytoplasmic domains of transmembrane spike glycoproteins. We have demonstrated previously that a tyrosine motif in the cytoplasmic domain of the Semliki Forest virus (SFV) spike glycoprotein E2 is absolutely essential for budding. In contrast, hardly anything is known regarding which region of the capsid protein is involved in spike binding. Therefore, the mechanism by which spikes are selectively sorted into the viral bud or by which energy is provided for envelopment, remains unclear. RESULTS: Molecular models of the SFV capsid protein (SFCP) and the cytoplasmic domain of the spike protein were fitted as a basis for a reverse genetics approach to characterizing the interaction between these two proteins. Biochemical analysis of mutants defined a hydrophobic pocket of the capsid protein that is involved both in spike binding and nucleocapsid assembly. CONCLUSIONS: We suggest that aromatic residues in the capsid protein serve to bind the side chain of the essential E2 tyrosine providing both specificity for spike incorporation and energy for budding. The same hydrophobic pocket also appears to play a role in capsid assembly. Furthermore, the results suggest that budding may occur in the absence of preformed nucleocapsids. This is the first demonstration of the molecular mechanisms of spike-nucleocapsid interactions during virus budding.

Role of the C-terminal tryptophan residue for the structure-function of the alphavirus capsid protein.

The Semliki Forest virus capsid protein is a multifunctional protein which packages genomic RNA into nucleocapsid structures and binds to viral spike protein during budding. In addition, the capsid protein has an autoproteolytic activity whereby the C-terminal tryptophan is used as the substrate for cotranslational cleavage of the viral structure polyprotein. The autoproteolytic domain of the capsid protein has a chymotrypsin-like fold but has two additional short beta-strands which place the tryptophan into the active site. Here, we have substituted the C-terminal tryptophan of Semliki Forest virus capsid protein for alanine, arginine and phenylalanine and analysed the effects on different functions of the C protein such as nucleocapsid formation, spike binding and autoproteolytic activity. We found that (i) tryptophan is a better substrate for the autoproteolytic activity, (ii) the wild-type tryptophan is the only residue that allows efficient viral growth and (iii) an aromatic residue is important for correct initial folding and stability of the protein.

Budding of enveloped viruses from the plasma membrane.

Many enveloped viruses are released from infected cells by maturing and budding at the plasma membrane. During this process, viral core components are incorporated into membrane vesicles that contain viral transmembrane proteins, termed 'spike' proteins. For many years these spike proteins, which are required for infectivity, were believed to be incorporated into virions via a direct interaction between their cytoplasmic domains and viral core components. More recent evidence shows that, while such direct interactions drive budding of alphaviruses, this may not be the case for negative strand RNA viruses and retroviruses. These viruses can bud particles in the absence of spike proteins, using only viral core components to drive the process. In some cases the spike proteins, without the viral core, can be released as virus-like particles. Optimal budding and release may, therefore, depend on a 'push-and-pull' concerted action of core and spike, where oligomerization of both components plays a crucial role.