Chapter 7 : Cell Biology of Nidovirus Replication Complexes

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Viruses are obligate intracellular parasites that modify the host cell to generate an environment for optimal production of progeny virus. Positive-strand RNA viruses modify intracellular membranes to generate “factories” for viral RNA synthesis. These factories are made up of viral replicase proteins and host cell membranes that assemble to form novel structures, which can be visualized by electron microscopy (EM). For example, the replication complex of brome mosaic virus, a positive-strand RNA virus of plants, induces invaginations and spherule formation in the endoplasmic reticulum (ER). These spherules sequester the viral genomic RNA and polymerase together and allow for the efficient synthesis of viral genomic and subgenomic mRNAs. The replication complexes of hepatitis C virus form a membranous web in the cytoplasm of hepatoma cells. This membranous web may provide an environment for persistence of viral RNA during chronic infection. For nidoviruses, a striking feature is that viral replicase proteins induce the formation of double-membrane vesicles (DMVs), which are the sites of viral RNA synthesis. This chapter reviews the current literature on the visualization and assembly of nidovirus DMVs, and describes recent studies that provide insight into the possible host cell pathways subverted by the viral replication complexes to help generate these factories for nidovirus RNA synthesis.

Citation: Baker S, Denison M. 2008. Cell Biology of Nidovirus Replication Complexes, p 103-113. In Perlman S, Gallagher T, Snijder E (ed), Nidoviruses. ASM Press, Washington, DC. doi: 10.1128/9781555815790.ch7
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Image of Figure 1.
Figure 1.

Schematic diagram of nidovirus replicase domains illustrating the conservation of proteolytic processing and enzymatic activity in arteri-, corona-, toro-, and ronivirus families. Connected arrows indicate confirmed cleavage sites processed by the indicated protease. Arrowheads indicate predicted sites for proteolytic processing. Abbreviations: PCP/P1/P2/PL, papain-like cysteine proteases; CP, cysteine protease; A, ADP-ribose- 1” -phosphatase; SP, serine protease; 3CL, 3C-like protease (also termed M); RdRp, RNA-dependent RNA polymerase; Z, zinc-binding domain; Hel, helicase; NendoU, nidovirus uridylate-specific endoribonuclease; ExoN, exonuclease; MT, methyltransferase.

Citation: Baker S, Denison M. 2008. Cell Biology of Nidovirus Replication Complexes, p 103-113. In Perlman S, Gallagher T, Snijder E (ed), Nidoviruses. ASM Press, Washington, DC. doi: 10.1128/9781555815790.ch7
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Image of Figure 2.
Figure 2.

Cascade of proteolytic processing for murine coronavirus replicase polyprotein. Processing by papain-like proteases (PLP1 and PLP2) and 3CL is indicated by the arrows. Intermediates nsp2-3 ( ) and nsp4-10 ( ) have been detected in pulse-chase studies. nsp4-16 is proposed based on a protein band of >450 kDa. Ne-U, nidovirus uridylate-specific endoribonuclease; for other abbreviations, see legend to Fig. 1 .

Citation: Baker S, Denison M. 2008. Cell Biology of Nidovirus Replication Complexes, p 103-113. In Perlman S, Gallagher T, Snijder E (ed), Nidoviruses. ASM Press, Washington, DC. doi: 10.1128/9781555815790.ch7
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Image of Figure 3.
Figure 3.

Transmission EM analysis of membrane alterations in nidovirus-infected cells. (A and B) DMV formation in EAV-infected BHK-21 cells at 4 postinfection. Bar, 100 nm. (C) Formation of DMVs from paired ER membranes upon EAV nsp2-3 expression in BHK-21 cells at 8 h posttransfection. Bar, 100 nm. (D) DMVs seen in MHV-A59-infected 17cl-1 cells at 7 postinfection. The double membrane is fused into a trilayer (arrowheads). Bar, 100 nm. (E) Analysis of SARS-CoV-infected Vero-E6 cells cryofixed by high-speed plunge freezing in liquid ethane, a step followed by freeze substitution with 1% osmium tetroxide and 0.5% uranyl acetate in acetone and embedment in epoxy LX-112 resin. The arrow indicates apparent continuity between the other membrane of a DMV and a mitochondrion (M), which was occasionally observed. Bar, 250 nm. (F) Ultrastructural characteristics of a broncho alveolar lavage specimen from a patient with SARS. DMVs (arrow) are shown to contain diffuse, granular material. Bar, 1 μm. Reproduced with permission from Pedersen et al., 1999 (A); Snijder et al., 2001 (B and C); Gosert et al., 2002 (D); Snijder et al., 2006 (E); and Goldsmith et al., 2004 (public access) (F).

Citation: Baker S, Denison M. 2008. Cell Biology of Nidovirus Replication Complexes, p 103-113. In Perlman S, Gallagher T, Snijder E (ed), Nidoviruses. ASM Press, Washington, DC. doi: 10.1128/9781555815790.ch7
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Image of Figure 4.
Figure 4.

Models of nidovirus replicase proteins driving formation of DMVs and replication of viral RNA. Nidovirus replicase complexes are depicted as gray circles; viral positive-strand RNA is depicted as a solid line, and negative-strand RNA is depicted as a dotted line. (A) DMV formation sequesters viral genomic RNA for transcription and replication. (B) DMV formation generates a catalytic surface area competent for transcription and replication of viral RNA.

Citation: Baker S, Denison M. 2008. Cell Biology of Nidovirus Replication Complexes, p 103-113. In Perlman S, Gallagher T, Snijder E (ed), Nidoviruses. ASM Press, Washington, DC. doi: 10.1128/9781555815790.ch7
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