A heterologous coiled coil can substitute for helix I of the Sindbis virus capsid protein

Abstract

Alphavirus core assembly proceeds along an assembly pathway involving a dimeric assembly intermediate. Several regions of the alphavirus capsid protein have been implicated in promoting and stabilizing this dimerization, including a putative heptad repeat sequence named helix I. This sequence, which spans residues 38 to 55 of the Sindbis virus capsid protein, was implicated in stabilizing dimeric contacts initiated through the C-terminal two-thirds of the capsid protein and nucleic acid. The studies presented here demonstrate that helix I can be functionally replaced by the corresponding sequence of a related alphavirus, western equine encephalitis virus, and also by an unrelated sequence from the yeast transcription activator, GCN4, that was previously shown to form a dimeric coiled coil. Replacing helix I with the entire leucine zipper domain of GCN4 (residues 250 to 281) produced a virus with the wild-type phenotype as determined by plaque assay and one-step growth analysis. However, replacement of helix I with a GCN4 sequence that favored trimer formation produced a virus that exhibited approximately 40-fold reduction in virus replication compared to the wild-type Sindbis virus. Changing residues within the Sindbis virus helix I sequence to favor trimer formation also produced a virus with reduced replication. Peptides corresponding to helix I inhibited core-like particle assembly in vitro. On the basis of these studies, it is proposed that helix I favors capsid protein-capsid protein interactions through the formation of dimeric coiled-coil interactions and may stabilize assembly intermediates in the alphavirus nucleocapsid core assembly pathway.

FIG. 1.

Schematic of the Sindbis virus capsid protein and sequences of helix I mutants. (A) The SINV CP is shown schematically. Residues 1 to 76 are predominantly positively charged, except for residues 38 to 55 which are uncharged and form helix I. Residues 76 to 132 (hatched box) are implicated in specific binding of the genomic RNA. This region is also important for CP-CP interactions during core assembly. Residues 114 to 264 form the protease domain responsible for autocatalytic cleavage of the CP from the structural polyprotein. This domain is also involved in CP-CP and CP-glycoprotein interactions. (B) Sequences for chimeric viruses and mutants within helix I and their corresponding plaque phenotypes determined 48 h postinfection on BHK cells. The amino acids in bold type are the amino acids of the foreign sequence inserted instead of amino acids 35 to 57 of the SINV CP (helix I). The numbers in parentheses in the virus designations indicate the amino acids of the foreign sequence that was inserted or substituted. Identical amino acids at a given position are indicated by dashes. SINV/GCN4(255-277)(L268D) is identical in sequence to SINV/GCN4(255-277) except for the change at residue 268. Plaque phenotypes are as follows: VSP, very small plaque (<2.0 mm in diameter); SP, small plaque (2.0 to 2.5 mm); MP, medium plaque (2.5 to 3.5 mm); LP, large plaque (3.5 to 4 mm); NR, not recovered.

FIG. 2.

One-step growth analysis of chimeric viruses in BHK cells. BHK cells were infected with the indicated virus at a MOI of 1. The medium over the cells was replaced every 30 min for the first 2 h and then every hour for 12 h postinfection. Supernatant was collected at the indicated times and assayed for released virus by titration on BHK cell monolayers at 37°C. Values are averages from two independent experiments.

FIG. 3.

Thermal inactivation of chimeric viruses. Thermal stability of chimeric virus particles was determined by incubation of 5,000 PFU (diluted in PBS supplemented with Ca²⁺ and Mg ²⁺) at 56°C for the times indicated. Aliquots were taken at the indicated time points, and virus titers were determined by plaque titration on BHK cell monolayers. The wild-type (wt) virus, Toto71, was included for comparison. Values are averages from four independent experiments.

FIG. 4.

Electron microscopy of in vitro-assembled CLPs. E. coli-expressed capsid protein was mixed with a 48-mer DNA oligonucleotide at a molar ratio of 1:1 or 1:2 in assembly buffer and incubated at room temperature for 15 min. The assembly reaction mixtures without further purification were negatively stained and examined by electron microscopy by the method of Mukhopadhyay et al. (27).

FIG. 5.

Inhibition of wild-type CLP assembly in vitro with peptides corresponding to helix I and the cytoplasmic domain of E2. (A) In vitro assembly with the helix I peptide. A 0.8% agarose gel stained for nucleic acid with ethidium bromide shows in vitro-assembled CLPs and unincorporated oligonucleotides (Oligo). E. coli-expressed wild-type (WT) CP and helix I peptide were incubated together at the indicated molar ratios and allowed to equilibrate at room temperature. The samples were then mixed with a 48-mer oligonucleotide and incubated at room temperature for 15 to 30 min. CLP assembly was assayed by agarose gel analysis as previously described (37). The data show that increasing amounts of helix I peptide (left to right) inhibit wild-type CLP assembly. (B) In vitro assembly with a peptide corresponding to the C-terminal 24 residues of the cytoplasmic domain of E2 (cdE2) of SINV. The data show that increasing amounts of the cdE2 peptide (left to right) do not inhibit wild-type CLP assembly.