Direct interaction between two viral proteins, the nonstructural protein 2C and the capsid protein VP3, is required for enterovirus morphogenesis - PubMed (original) (raw)
Direct interaction between two viral proteins, the nonstructural protein 2C and the capsid protein VP3, is required for enterovirus morphogenesis
Ying Liu et al. PLoS Pathog. 2010.
Abstract
In spite of decades-long studies, the mechanism of morphogenesis of plus-stranded RNA viruses belonging to the genus Enterovirus of Picornaviridae, including poliovirus (PV), is not understood. Numerous attempts to identify an RNA encapsidation signal have failed. Genetic studies, however, have implicated a role of the non-structural protein 2C(ATPase) in the formation of poliovirus particles. Here we report a novel mechanism in which protein-protein interaction is sufficient to explain the specificity in PV encapsidation. Making use of a novel "reporter virus", we show that a quasi-infectious chimera consisting of the capsid precursor of C-cluster coxsackie virus 20 (C-CAV20) and the nonstructural proteins of the closely related PV translated and replicated its genome with wild type kinetics, whereas encapsidation was blocked. On blind passages, encapsidation of the chimera was rescued by a single mutation either in capsid protein VP3 of CAV20 or in 2C(ATPase) of PV. Whereas each of the single-mutation variants expressed severe proliferation phenotypes, engineering both mutations into the chimera yielded a virus encapsidating with wild type kinetics. Biochemical analyses provided strong evidence for a direct interaction between 2C(ATPase) and VP3 of PV and CAV20. Chimeras of other C-CAVs (CAV20/CAV21 or CAV18/CAV20) were blocked in encapsidation (no virus after blind passages) but could be rescued if the capsid and 2C(ATPase) coding regions originated from the same virus. Our novel mechanism explains the specificity of encapsidation without apparent involvement of an RNA signal by considering that (i) genome replication is known to be stringently linked to translation, (ii) morphogenesis is known to be stringently linked to genome replication, (iii) newly synthesized 2C(ATPase) is an essential component of the replication complex, and (iv) 2C(ATPase) has specific affinity to capsid protein(s). These conditions lead to morphogenesis at the site where newly synthesized genomes emerge from the replication complex.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Figure 1. The genomic organization of C-HEV RNAs, proteolytic processing of the polyprotein and PV 2CATPase motifs.
(A) The single-stranded genomic RNA is covalently linked to the viral-encoded protein VPg at the 5′ end of the NTR (5′NTR). The 5′NTR consists of the cloverleaf, and the IRES. The coding region (open box) depicts the structural (P1) and nonstructural (P2 and P3) regions. Within the P2 (2CATPase) coding region, the cis replication element (cre) is indicated. The 3′NTR contains a heteropolymeric region and is polyadenylated. The precursors and mature cleavage products of the P1, P2 and P3 domains along with their cleavage sites are shown. (B) Functional motifs in 2CATPase. Motifs A and B are homologous to those in NTP binding/hydrolyzing proteins, motif C shares homology to superfamily helicase III. Two RNA binding motifs, two amphipathic helixes, the N-terminal membrane binding motif, oligomerization motif and the C-terminal zinc-binding motif are indicated. Amino acid positions of each motif are numbered.
Figure 2. Chimeric virus C20PP containing the capsid-coding region of CAV20 is defective in encapsidation.
(A) Growth phenotypes of the parental and of chimeric viruses. The genomic structures of the parental and of chimeric viruses used are illustrated on the left. PVM sequences are shown with open boxes, CAV sequences are shown by black boxes. Nomenclature used for the chimeric viruses: the first letter refers to the origin of the P1 region, the 2nd and 3rd letters refer to the origins of the P2 and P3 regions, respectively. RNA transcripts were transfected into HeLa H1 cells and the viruses obtained at CPE, if any, were titered by a plaque assay (Materials and Methods). (B) C20PP is normal in translation and polyprotein processing. RNA transcripts (500 ng) derived from wt PVM, wt CAV20 and C20PP were translated in vitro in HeLa cell extracts at 34 degree Celsius ° overnight, as described in Material and Methods. The reaction mixtures were analyzed by SDS/PAGE with 12% acrylamide and the viral proteins were visualized by autoradiography. The positions of the precursor and mature proteins after processing are indicated. (C) The genomic structures of the parental and of chimeric luciferase reporter viruses used are illustrated. The R-Luc gene is shown in dotted box. (D) Comparison of RNA replication and encapsidation of the parental and of chimeric reporter viruses. To determine the level of RNA replication (black bar), RNA transcripts were transfected into HeLa H1 cells both in the absence and presence of 2 mM GnHCl. To determine encapsidation (grey bar), the transfected cell lysates were passaged into fresh HeLa H1 cells in the absence and presence of GnHCl. The luciferase activity in the absence (−GnHCl) and presence of 2 mM GnHCl (+GnHCl) was measured and the ratio of luciferase activity (–GnHCl/+GnHCl) was calculated to quantify the extent of replication (Materials and Methods). The luciferase data are the averages of at least three independent experiments.
Figure 3. Rescue of the C20PP virus by mutations in 2CATPase or/and in VP3.
(A) Growth phenotypes of the C20PP chimera and of its derivatives. The genomic structures of the C20PP virus and of its derivatives are illustrated on the left. For the nomenclature of the chimera see the legend to Figure 2A. CAV20 sequences are shown with black boxes, PVM sequences with open boxes. The amino acid changes in 2CATPase and in VP3 are shown in superscript. C20PP-DM represents the double mutant. RNA transcripts were transfected into HeLa H1 cells and the viruses present after complete CPE were titered by a plaque assay (Materials and Methods). (B) The genomic structures of the C20PP reporter chimeras, carrying the R-Luc gene fused N-terminal to P1, and of its derivatives. (C) Comparison of RNA replication and encapsidation of the C20PP virus and of its derivative reporter viruses. To determine the level of RNA replication (black bar), RNA transcripts were transfected into HeLa H1 cells both in the absence and presence of 2 mM GnHCl. To determine encapsidation (grey bar), the transfected cell lysates were passaged into fresh HeLa H1 cells in the absence and presence of GnHCl. Luciferase activity in the absence (−GnHCl) and presence of 2 mM GnHCl (+GnHCl) was measured and the ratio of luciferase activity (–GnHCl/+GnHCl) was calculated to quantify the extent of replication (Materials and Methods). The luciferase data are the averages of at least three independent experiments.
Figure 4. Rescue of the C20PP virus by a CAV20-like amino acid or CAV20 2CATPase.
(A) Comparison of the amino acid sequences of 2CATPase flanking residue 252 of PVM, CAV20 and C20PP-2CN252S. (B) The genomic structures of the C20PP derivatives. Note that the 2CN252G change is different from what was observed in the encapsidation adapted virus (2CN252S). N252S mutation in C20PP-2CN252S is shown by grey diamond whereas the N252G mutation is a black diamond. For nomenclatures see the legend to Figure 2A. The PVM sequences are shown with open boxes and the CAV sequences by black boxes. RNA transcripts were transfected into HeLa H1 cells and at the time of CPE the virus titers were determined by a plaque assay (Materials and Methods).
Figure 5. Chimeras of C-CAVs, C20C21C21 and C18C20C20, are defective in encapsidation.
(A) Growth phenotypes of parental and of chimeric viruses. The genomic structures of the parental and chimeric viruses are shown on the left. CAV20 sequences are shown by black boxes, CAV21 by gray boxes and CAV18 by hatched boxes. The nomenclature for the chimeric viruses is given in the legend to Figure 2. RNA transcripts were transfected into HeLa H1 cells and the viruses obtained at CPE, if any, were titered by plaque assay (Materials and Methods). (B) The genomic structures of the parental and chimeric luciferase reporter viruses. (C) Comparison of RNA replication and encapsidation of the parental C-CAVs and of chimeric C-CAVs reporter viruses. To determine the level of RNA replication (black bar), RNA transcripts were transfected into HeLa H1 cells both in the absence and presence of 2 mM GnHCl. To determine encapsidation (grey bar), the transfected cell lysates were passaged into fresh HeLa H1 cells in the absence and presence of GnHCl. The luciferase activity in the absence (−GnHCl) and presence of 2 mM GnHCl (+GnHCl) was measured and the luciferase activity ratio (–GnHCl/+GnHCl) was calculated to quantify the extent of replication (Materials and Methods). The luciferase data are averages of at least three independent experiments.
Figure 6. Rescue of lethal C-CAV chimeras by 2CATPase of the same origin as P1.
(A) Genomic structures of the chimeric viruses containing both the P1 and 2CATPase regions from the same origin. For the genomic structures of the chimeric viruses with only P1 exchanges see Figure 4A. CAV20 sequences are shown with a black box, CAV21 sequences with a gray box and CAV18 sequences with a hatched box. For the nomenclature of the chimeric viruses see the legend to Figure 2. (B) Comparison of growth phenotypes of the chimeric viruses with just P1 or with P1+2CATPase exchanges. RNA transcripts were transfected into HeLa H1 cells and the plaque phenotype of the virus, if any, was tested at the time of CPE (Materials and Methods). (C) Comparison of the amino acid sequences flanking residue 252 of 2CATPase among CAV20, CAV18 and CAV21.
Figure 7. Direct interaction between VP3 and 2CATPase demonstrated by biochemical assays.
(A) GST pull down assay. PV GST-2CATPase, PV VP3-His and GST were purified as described in Materials and Methods. Lane 1: GST was used to pull down PV VP3-His; Lane 2: PV GST-2CATPase was used to pull down PV VP3-His. (B) Co-immunoprecipitation (Co-IP) assays. RNA transcripts containing 2C and VP3 coding sequences of PV or CAV20, were co-translated in HeLa cell extracts in the presence of 35S-methionine. Lane 4: input CAV20 2CATPase & CAV20 VP3; Lane 5: input PV 2CATPase & CAV20 VP3; Lane 6: input PV 2CATPase (N252S) & CAV20 VP3 (E180G). The positions of 2CATPase and VP3 are indicated. The position of incomplete translation products of RNA transcripts of 2C coding sequence is indicated by asterisk. Using polyclonal antibody against PV 2CATPase, Co-IP assays were done as described in Materials & Methods. The co-immunoprecipitated protein products from co-translation reactions shown in lanes 4, 5 and 6 were loaded in Lanes 1, 2, 3, respectively, analyzed by SDS-PAGE, and detected by autoradiography. The extent of interactions was quantified and calculated as percentages of the levels observed in the CAV 2CATPase and CAV VP3 Co-IP reaction (shown below).
Figure 8. Model for C-HEVs morphogenesis involving the interaction between 2CATPase and capsid protein VP3.
Following its release from the polyprotein by 2Apro, P1 is first properly folded by chaperone Hsp90 before it becomes a substrate for 3CDpro, . Mature VP0, VP1 and VP3 assemble into a protomer, 5 of which subsequently form a pentamer. Some pentamers generate 75S empty capsids. Progeny viral RNA, released from the replication complexes, is associated with pentamers through their VP3 domains that interact with 2CATPase on the surface of membranous replication complexes. 12 pentamers assemble to enclose the viral RNA forming a provirion. Maturation cleavage of VP0 to VP2 and VP4 yields an infectious virion. The role of 3CDpro at this step is not yet understood .
References
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