Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Found 1 result for PubMed for id: 407944004
Filters applied. Clear all
. 2012 Oct 9;109(41):16534-9.
doi: 10.1073/pnas.1210418109. Epub 2012 Sep 25.

Structural and functional insights into alphavirus polyprotein processing and pathogenesis

Gyehwa Shin  1 , Samantha A Yost, Matthew T Miller, Elizabeth J Elrod, Arash Grakoui, Joseph Marcotrigiano
Affiliations

Structural and functional insights into alphavirus polyprotein processing and pathogenesis

Gyehwa Shin et al. Proc Natl Acad Sci U S A. .

Abstract

Alphaviruses, a group of positive-sense RNA viruses, are globally distributed arboviruses capable of causing rash, arthritis, encephalitis, and death in humans. The viral replication machinery consists of four nonstructural proteins (nsP1-4) produced as a single polyprotein. Processing of the polyprotein occurs in a highly regulated manner, with cleavage at the P2/3 junction influencing RNA template use during genome replication. Here, we report the structure of P23 in a precleavage form. The proteins form an extensive interface and nsP3 creates a ring structure that encircles nsP2. The P2/3 cleavage site is located at the base of a narrow cleft and is not readily accessible, suggesting a highly regulated cleavage. The nsP2 protease active site is over 40 Å away from the P2/3 cleavage site, supporting a trans cleavage mechanism. nsP3 contains a previously uncharacterized protein fold with a zinc-coordination site. Known mutations in nsP2 that result in formation of noncytopathic viruses or a temperature sensitive phenotype cluster at the nsP2/nsP3 interface. Structure-based mutations in nsP3 opposite the location of the nsP2 noncytopathic mutations prevent efficient cleavage of P23, affect RNA infectivity, and alter viral RNA production levels, highlighting the importance of the nsP2/nsP3 interaction in pathogenesis. A potential RNA-binding surface, spanning both nsP2 and nsP3, is proposed based on the location of ion-binding sites and adaptive mutations. These results offer unexpected insights into viral protein processing and pathogenesis that may be applicable to other polyprotein-encoding viruses such as HIV, hepatitis C virus (HCV), and Dengue virus.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Overview of the alphavirus replication machinery and P23pro-zbd structure. (A) Schematic representation of the alphavirus replication machinery with emphasis on domain organization of nsP2 and nsP3. The crystallization construct consists of four domains: protease (blue) and methyltransferase-like (teal) domains of nsP2 and the macro (yellow) and zinc-binding (red) domain of nsP3, encompassing amino acids 1011–1675 of the P1234 polyprotein. Amino acid numbering provided from the amino terminus of nsP2 and nsP3 are also noted in parentheses. (B–D) Ribbon diagram of P23pro-zbd colored according to A. A gray sphere denotes the position of the zinc ion. The P2/3 cleavage site, nsP2 protease active site, and ADP ribose–binding site are labeled with an arrow, asterisk (*), and filled circle, respectively. (C) Ribbon diagram of P23 rotated 90° about a vertical axis from the view in B. (D) Ribbon diagram of P23 rotated 180° about a vertical axis from the view in B. Box shows location of the view in E. (E) Previously described nsP3 temperature-sensitive mutant ts7 (F312S) highlighted in green with the immediately surrounding amino acids located in the macro, ZBD, and MT-like domains. nsP2 residues are shown in italics. (F and G) The P2/3 cleavage site is located at the base of a narrow cleft formed by the MT-like and macro domains. Three amino acids on either side of the scissile bond (arrow) are shown in stick representation. The view in F is similar to that in B, whereas that in G is rotated 90° about a horizontal axis from F, looking directly into the cleft.
Fig. 2.
Fig. 2.
nsP2 and nsP3 interface and the location of nsP2 noncytopathic mutants. (A) Solvent-accessible surface of nsP3. (B and C) Surface of nsP3 (B) or nsP2 (C) colored for electrostatic potential at ±5 kT/e; blue (basic), red (acidic), and white (neutral) with ribbon diagram of nsP2 (B) or nsP3 (C). (D) Molecular surface of nsP2 highlighting (white) the location of nsP2 noncytopathic mutations. The numbering corresponds to SINV nsP2 sequence. (E) Location of P726 in nsP2, highlighting interactions with the nsP3 linker. A portion of the last helix in the nsP3 macro domain is shown in yellow. (F) Surface of nsP2 P726 and surrounding area colored for electrostatic potential at ±4 kT/e with nsP3 linker region in stick format. Residues labeled in italics are located in nsP2. Arrow indicates P2/3 cleavage site. Domain coloring in each panel is identical to Fig. 1.
Fig. 3.
Fig. 3.
In vivo effects of nsP3 linker region mutations. (A and B) Viral RNA infectivity [infectious units (IU)/μg RNA] as measured by infectious center assay (A) and viral titers [plaque-forming units (pfu)/mL] (B) 24 h after electroporation of BHK cells, as determined by plaque assay over two or more independent experiments. Error bars represent the SEM. (C and D) Cells were infected with wild-type or mutant SINV at a multiplicity of infection (MOI) of 10. At indicated time points postinfection, RNAs were labeled for 3 h in the presence (C) and absence (D) of actinomycin D. Total RNAs were harvested and analyzed by denaturing agarose gel electrophoresis. The position of genomic (G), subgenomic (SG), 28S, and 18S RNAs are labeled. M indicates mock-infected cells labeled for 3 h. (E and F) Analysis of P23 cleavage of nsP2 P726 and nsP3 L165 mutants in vivo. Total protein was harvested from wild-type and indicated mutant SINV-infected BHK cells at indicated hours postinfection and analyzed by Western blot using anti-nsP2 (E) and anti-nsP3 (F) antibodies.
Fig. 4.
Fig. 4.
(A and B) Superposition of the MT-like domains of P23pro-zbd (teal) and nsP2pro (gray) (12). The nsP3 linker (for simplicity only residues V162-W178 are shown) connecting the macro domain and ZBD is shown in red. The view in B is rotated 90° about a horizontal axis from A. (C and D) Potential RNA-binding surface of P23pro-zbd. The location of sulfate and MES ligands are represented by spheres. Surface of P23pro-zbd is colored for electrostatic potential at ±5 kT/e. D is a 180° rotation about the vertical axis from the view in panel C. (E) Ribbon diagram of P23pro-zbd in the identical orientation as in C. (F) ZBD of nsP3 showing the four zinc-coordinating cysteines, T286, a sulfate ion represented by spheres bound to Y267, R273, and R276, and surrounding residues. The close-up view corresponds to the box in E.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Griffin DE. Alphaviruses. In: Knipe DM, editor. Fields Virology. Vol 1. Philadelphia: Lippincott Williams & Wilkins; 2007. pp. 1023–1067.
    1. Thiboutot MM, et al. Chikungunya: A potentially emerging epidemic? PLoS Negl Trop Dis. 2010;4:e623. - PMC - PubMed
    1. Nkoghe D, et al. Clinical forms of chikungunya in Gabon, 2010. PLoS Negl Trop Dis. 2012;6:e1517. - PMC - PubMed
    1. de Groot RJ, Hardy WR, Shirako Y, Strauss JH. Cleavage-site preferences of Sindbis virus polyproteins containing the non-structural proteinase. Evidence for temporal regulation of polyprotein processing in vivo. EMBO J. 1990;9:2631–2638. - PMC - PubMed
    1. Vasiljeva L, et al. Regulation of the sequential processing of Semliki Forest virus replicase polyprotein. J Biol Chem. 2003;278:41636–41645. - PubMed

Publication types

MeSH terms

Associated data

LinkOut - more resources

-