A tyrosine-based motif in the cytoplasmic domain of the alphavirus envelope protein is essential for budding.

Abstract

The budding of enveloped viruses from cellular membranes is believed to be dependent on the specific interaction between transmembrane spike proteins and cytoplasmic core components of the virus. We found that the cytoplasmic domain of the E2 transmembrane spike glycoprotein of Semliki Forest virus contains two essential determinants which are absolutely needed for budding. The first constitutes a single tyrosine residue in the context of a direct pentapeptide repeat. The tyrosine could only partially be substituted for other residues with aromatic or bulky hydrophobic side chains, although these immediately reverted to the original genotype. The second determinant involves palmitylated cysteine residues flanking the tyrosine repeat motif. The function of these is probably to anchor the tail against the inner surface of the membrane so that the tyrosine‐containing motif is properly presented to the nucleocapsid. This is the first example where a membrane virus employs a tyrosine signal for the selective incorporation of spike proteins into budding structures.

Structural Analysis of Sindbis Virus Capsid Mutants Involving Assembly and Catalysis

“…Only very minor adjustments are necessary to place a tyrosine residue into the pocket in place of the observed leucine residue. The involvement of Y400 in E2 binding was demonstrated by mutagenesis, which showed that only hydrophobic residues could replace the tyrosine residue for successful virus propagation (Zhao et al, 1994). Likewise, substitution of L402 of E2 with hydrophobic residues permitted propagation with only a modest alteration of plaque phenotype, whereas hydrophilic substitutions produced crippled or no virus (K. Einterz-Owen & R.J.K., unpublished results).…”

“…Small rod-shaped crystals, 0.1 to 0.2 mm in length, were used as seeds for each new crystallization setup. Crystals grew to about Mutation of Y400 to Trp, Phe, Leu or Met produces a budding-competent virus, whereas substitution to Lys, Ser, Asp, Arg or Thr prevents budding (Zhao et al, 1994). The shaded area shows residues important in binding to the hydrophobic pocket.…”

“…Besides its catalytic activity, SCP plays a role in the assembly of the nucleocapsid by recognizing and packaging the genomic RNA (Weiss et al, 1989) and in viral budding by interacting with the spike proteins (Hahn et al, 1988;Vaux et al, 1988;Metsikkö & Garoff, 1990;Zhao et al, 1994). SCP isolated from virus formed a dimer in at least two different crystal forms (Table 1; and see Choi et al, 1991), as does the SFV core protein (SFCP: unpublished results).…”

Requirements for Budding of Paramyxovirus Simian Virus 5 Virus-Like Particles

“…Much progress has been made in identifying both viral and cellular components that drive this assembly process. For example, alphavirus budding has been found to require both soluble nucleocapsid cores and envelope glycoproteins (9,25,44,45), and a specific interaction between the viral capsid protein and the cytoplasmic tail of the viral E2 glycoprotein is critical for assembly (54). In contrast, the assembly and budding of retroviruses does not require participation of envelope components, and expression of soluble Gag polyprotein in the absence of any other viral component results in efficient budding of virus-like particles (VLPs) that resemble immature virions (46).…”

GP64 of group I nucleopolyhedroviruses cannot readily rescue infectivity of group II f-null nucleopolyhedroviruses

“…The involvement of CTDs of viral envelope proteins in the budding process has indeed been supported for a number of other viruses, including Sendai virus (Ali & Nayak, 2000), influenza A virus (Bilsel et al, 1993), VSV (Robison & Whitt, 2000), Mason-Pfizer monkey virus (Song et al, 2003) and Semliki Forest virus (Zhao et al, 1994). It is plausible that there is an interaction between the CTD of F and one or more viral NC proteins that are specific for group II NPVs.…”