Functional requirements of the yellow fever virus capsid protein

Abstract

Although it is known that the flavivirus capsid protein is essential for genome packaging and formation of infectious particles, the minimal requirements of the dimeric capsid protein for virus assembly/disassembly have not been characterized. By use of a trans-packaging system that involved packaging a yellow fever virus (YFV) replicon into pseudo-infectious particles by supplying the YFV structural proteins using a Sindbis virus helper construct, the functional elements within the YFV capsid protein (YFC) were characterized. Various N- and C-terminal truncations, internal deletions, and point mutations of YFC were analyzed for their ability to package the YFV replicon. Consistent with previous reports on the tick-borne encephalitis virus capsid protein, YFC demonstrates remarkable functional flexibility. Nearly 40 residues of YFC could be removed from the N terminus while the ability to package replicon RNA was retained. Additionally, YFC containing a deletion of approximately 27 residues of the C terminus, including a complete deletion of C-terminal helix 4, was functional. Internal deletions encompassing the internal hydrophobic sequence in YFC were, in general, tolerated to a lesser extent. Site-directed mutagenesis of helix 4 residues predicted to be involved in intermonomeric interactions were also analyzed, and although single mutations did not affect packaging, a YFC with the double mutation of leucine 81 and valine 88 was nonfunctional. The effects of mutations in YFC on the viability of YFV infection were also analyzed, and these results were similar to those obtained using the replicon packaging system, thus underscoring the flexibility of YFC with respect to the requirements for its functioning.

FIG. 1.

(A) Multiple sequence alignment of flavivirus C proteins. Residues with high similarity (>50%) are shown in red, and the conserved residues are indicated by the solid red bar. The IHS is shaded in gray. The secondary structure for DENC showng helices 1 through 4 is indicated at the top. JEV, Japanese encephalitis virus. (B) Ribbon diagram of the DENC dimer, residues 21 to 100 (26). Helix α1 is blue, α2 is green, α3 is yellow, and α4 is red (image generated using Visual Molecular Dynamics [VMD]; University of Illinois, Urbana-Champaign). (C) Schematic representation of the YFV genome, with open boxes denoting regions coding for the structural (white) and nonstructural (light gray) proteins that are translated as a polyprotein. The 5′- and 3′-untranslated regions (UTR) are depicted as solid lines. Also shown is a schematic representation of the YFV replicon construct, YF-R.Luc2A-RP (14). A light gray box denotes the YFV nonstructural protein coding sequence. The star indicates the location of the RNA cyclization sequence within the coding sequence for the first 20 residues of the C protein, which is retained in the replicon. The black box indicates the NS1 signal sequence. R.luc, Renilla luciferase; 2A, a 17-amino-acid residue autoproteolytic peptide from foot-and-mouth disease virus. Also shown is a schematic representation of the SIN replicon constructs that were used to express YFV structural proteins, SIN-CprME and SIN-prME. The coding sequence for the SIN nonstructural proteins, nsP1-nsP4, is denoted by a dark gray box. The YFV structural proteins (white box) are expressed from the SIN subgenomic promoter (black arrow). Lines indicate 5′- and 3′-UTRs, and the poly A tail is shown.

FIG. 2.

Packaging of YF-R.luc2A-RP using truncated YFC. A schematic diagram of mutated YFC is shown, with white boxes indicating deleted residues. The helices are colored as described in the legend to Fig. 1. Graphs indicate log values of Renilla luciferase activity (Log RLU) in BHK-15 cells infected with PIPs at 24 h postinfection. PIPs were generated by electroporation of BHK cells with the YF-R.luc2A-RP replicon followed by transfection of full-length (Wt) or mutated SIN-CprME or SIN-prME (prME) replicon RNAs (see Materials and Methods). (A) Activity of PIPs produced using YFC containing truncations within the N-terminal unstructured region (first 20 residues). (B) Activity of PIPs produced using YFC containing truncations of the N-terminal regions (including α1 and α2). (C) Activity of PIPs produced using YFC containing truncations within the C-terminal domain (including α4). The activity of PIPs produced using the C(Δ77-96) construct containing the Q45L revertant, labeled as Δ77-96/Q45L, is also shown. (D) Activity of PIPs produced using YFC containing both N- and C-terminal truncations.

FIG. 3.

Packaging of YF-R.luc2A-RP using YFC harboring internal deletions. A schematic diagram of mutated YFC is shown, with white boxes indicating deleted residues. The helices are colored as described in the legend to Fig. 1. The graph indicates log values of Renilla luciferase activity (Log RLU) in BHK-15 cells infected with PIPs at 24 h postinfection. PIPs were generated by electroporation of BHK cells with YF-R.Luc2A-RP replicon followed by transfection of full-length (Wt) or mutated SIN-CprME or SIN-prME (prME) replicon RNAs (see Materials and Methods). The activity of PIPs produced using YFC containing either a deletion of α1 [C(Δ26-36)], a deletion of α1 and the loop between α1 and α2 [C(Δ26-42)], smaller deletions within α2 [C(Δ43-48) and C(Δ49-56)], a deletion of the entire α2 [C(Δ43-56)], or a deletion of entire α1 and α2 [C(Δ26-56)] is shown.

FIG. 4.

(A) Helical wheel representations of the C-terminal helix, α4, of DENC and YFC. α4 shows characteristics of a coiled coil with hydrophobic residues at positions a and d of the helical wheel; however, R85 in DENC (K85 in YFC) in the a position is an unlikely amino acid for coiled-coil structures. The side chains of L78, L81, V88, M92, and L95 in α4 of one monomer are involved in intermonomeric interactions with their counterparts in α4 of the other monomer. (B) Packaging of YF-R.luc2A-RP using YFC containing point mutations in α4. The activity of PIPs produced using YFC containing point mutations of residues L78, L81, V88, M92, or L95 that are involved in intermonomeric hydrophobic interactions is shown. Wt, wild type.

FIG. 5.

(A) Velocity gradient sedimentation analysis of PIPs. PIPs were centrifuged on a continuous OptiPrep gradient. The gradient was fractionated, and BHK-15 cells were infected using 100 μl of the fractions. Renilla luciferase activity in infected cells at 24 postinfection (on the y axis) was plotted against the corresponding fraction number (on the x axis). Renilla luciferase activity is expressed in RLU. PIPs generated using C(Δ4-40) or C(Δ26-42) sedimented similarly to wild-type (Wt) PIPs. (B) The thermal stability of the PIPs was assayed by incubating PIPs at 48°C. Aliquots (100 μl) were taken at 10, 20, 30, 40, and 50 min and used to infect BHK-15 cells. Renilla luciferase activity in infected cells at 24 postinfection (on the y axis) was plotted against the time of incubation (on the x axis). Renilla luciferase activity is expressed in RLU. The dashed line indicates background luciferase levels.

FIG. 6.

Cumulative growth curves of wild-type (WT) and mutant YFV viruses. BHK cells were transfected with transcripts of cDNAs containing either wild-type or mutated C protein. Culture supernatants were harvested at 12, 24, 36, and 48 h posttransfection (p.t.), and titers of infectious virus released were calculated.