A guide to viral inclusions, membrane rearrangements, factories, and viroplasm produced during virus replication

Abstract

Virus replication can cause extensive rearrangement of host cell cytoskeletal and membrane compartments leading to the "cytopathic effect" that has been the hallmark of virus infection in tissue culture for many years. Recent studies are beginning to redefine these signs of viral infection in terms of specific effects of viruses on cellular processes. In this chapter, these concepts have been illustrated by describing the replication sites produced by many different viruses. In many cases, the cellular rearrangements caused during virus infection lead to the construction of sophisticated platforms in the cell that concentrate replicase proteins, virus genomes, and host proteins required for replication, and thereby increase the efficiency of replication. Interestingly, these same structures, called virus factories, virus inclusions, or virosomes, can recruit host components that are associated with cellular defences against infection and cell stress. It is possible that cellular defence pathways can be subverted by viruses to generate sites of replication. The recruitment of cellular membranes and cytoskeleton to generate virus replication sites can also benefit viruses in other ways. Disruption of cellular membranes can, for example, slow the transport of immunomodulatory proteins to the surface of infected cells and protect against innate and acquired immune responses, and rearrangements to cytoskeleton can facilitate virus release.

Figure 1

The replicase proteins of positive‐stranded RNA viruses are directed to membranes by NSP with membrane‐targeting information. (A) Picornavirus. The replication complex contains 3D, the RdRp (red), and 2C which has NTPase and helicase motifs (purple). The 3D polymerases do not have membrane‐targeting information but are synthesized as a 3ABCD precursor. 3ABCD is processed to 3AB by the 3C protease (red triangle) and a hydrophobic domain in 3A targets 3AB to the cytoplasmic face of ER membranes. 3AB binds directly to 3D and this targets the polymerase to the replication complex. The replication complex also requires 2BC and 2C proteins that are targeted to membranes via their own hydrophobic domains (black lines). (B) Flavivirues. The replication complex is encoded at the C‐terminus of a polyprotein that is processed by the NS2 protease (red triangle). NS5B is the RNA‐dependent polymerase (red), and NS3 acts as helicase (purple). NS4B is a polytopic membrane protein inserted into the ER cotranslationally. NS4A, 5A, and 5B have hydrophobic domains (gray lines) that allow posttranslational insertion into the cytoplasmic face of the ER membrane. NS3 is recruited into the complex by associating with NS4A. (C) Alphavirus. The NSP1234 polyprotein is processed by a protease activity in the C‐terminus of P2 (red triangle). The polyprotein is anchored to the cytoplasmic face of endosome and lysosome membranes by a hydrophobic region at the N‐terminus of P1. P1 also acts as the methyltransferase (yellow). P2 encodes the helicase (purple) and P4 is the RdRp (red). The P123 precursor associates with P4 and generates negative‐stranded RNA. Further processing produces a complex of separate P1, 2, 3, and 4 proteins that produce positive‐stranded RNA. (D) Nidoviruses. The Nidovirales order comprises the Arteriviridae, Coronaviridae, and Roniviridae families. The replicase gene is composed of two open reading frames termed ORF1a and ORF1b, both of which encode complex polyproteins. Arterivirus ORF1b encodes NSPs 9–12 including the RdRp (NSP9, red), helicase (NSP10, purple). The ORF1b reading frame lacks hydrophobic domains able to target the replicase to membranes. Proteins necessary for membrane targeting (brown and blue) are encoded by ORF1a (NSP2, 3, and 5). For the CoVs, for example, MHV and SARS‐CoV transmembrane domains are located in NSP3, 4, and 6, and helicase and polymerase proteins are NSP12 and 13, respectively. ORF1b also contains a methyltransferase (NSP16, yellow).

Figure 2

Protein trafficking in the early secretory pathway. 1. Anterograde transport from the ER to the ERGIC is mediated by COPII‐coated vesicles. Formation of COPII coats is regulated by the Sar1p‐GTPase. Binding of Sar1p to the ER requires binding of GTP and this is facilitated by the Sec12p‐GTP exchange protein. Sar1p‐GTP recruits the Sec23–Sec24p subcomplex (light blue) of the COPII coat and this recruits cargo proteins (light green) to ERES. The Sec23–Sec24p subcomplex then recruits the Sec13–Sec31p proteins (purple) that induce membrane curvature and formation of a vesicle. Hydrolysis of GTP on Sar1p by Sec23p results in coat disassembly. The vesicle docks with ERGIC membranes by binding tethering proteins and interactions between v‐SNAREs and t‐SNAREs results in vesicle fusion. 2. Retrograde transport from the ERGIC to the ER provides a pathway to retrieve proteins from the ERGIC and Golgi apparatus and is mediated by COPI‐coated vesicles. Formation of COPI coats is regulated by the Arf1‐GTPase. Binding of Arf1 to the ERGIC requires binding of GTP and this is facilitated by the GBF1 and BIG1/2 GTP exchange proteins. Arf1‐GTP recruits the COPI coat complex (dark blue), which induces membrane curvature and formation of a vesicle that returns to the ER.

Figure 3

Subcellular location of Foot‐and‐mouth disease NSP encoded in the P2 region of the FMDV genome. Vero cells expressing FMDV 2B (top), 2BC (middle), or 2C (bottom) were fixed and permeabilized and processed for immunofluorescence. 2C and 2BC were located using antibodies specific for 2C (3F7) and 2B was located using an antibody raised against an epitope tag in 2B. Cells were counterstained using antibodies against ER luminal protein ERP57 (top and middle panels), or COPI protein β‐COP (bottom). Merged images are shown at higher magnification on the far left. See Moffat et al. (2005) for more details. Reprinted from Moffat et al. (2005) with permission from American Society for Microbiology.

Figure 4

Schematics of inclusions induced during virus infection. ASFV induces single large perinuclear factories surrounded by mitochondria. Vaccinia virus induces multiple factories derived from membrane‐enclosed replication complexes (RC) both of which are associated with mitochondria. Certain poxviruses also induces electron‐dense A‐type inclusions (A). Human herpesvirus 1 induces capsid assembly sites, or assemblons (As), replication compartments (RC), inclusions of tegument proteins VP13/14 and VP22 (VP), and electron‐dense bodies of UL11 and UL12 gene products (11 and 12) in the nucleus. Human herpesvirus 2 also induces nuclear inclusions of UL55 gene product (55) and human herpesvirus 6 induces nuclear tegusomes (T). Herpesviruses induce cytoplasmic assembly sites where envelopment and some tegument are acquired (Env) in human herpesvirus 5, these sites include electron‐dense bodies (DB). Iridoviruses induce multiple cytoplasmic virus factories (VF) and crystalline arrays (CA), both of which associate with mitochondria. Reoviruses also induce multiple cytoplasmic virus factories (VF) and crystalline arrays (CA) that are enclosed within lysosomal membranes.

Figure 5

(A) Electron micrograph of an ASFV factory showing partially assembled, empty and fully mature capsids as well as electron‐dense viroplasm accumulating around viral membranes. Image courtesy of P. Hawes, J. Simpson, and P. Monaghan, Bioimaging Group, IAH‐Pirbright. (B) Confocal micrograph of ASFV‐infected cells immunolabeled with antimajor capsid protein (green) and vimentin (red) and stained with a DNA dye (blue). Note vimentin cages enclosing ASFV factories. Reprinted from Monaghan et al. (2003) with permission from Blackwell Publishing, Inc.

Figure 6

(A) Electron micrograph of A‐type inclusions from cowpox‐infected cells, showing intracellular mature virus in electron‐dense inclusions (A) surrounded by polyribosomes (arrows). Reprinted from Ichihashi et al. (1971) with permission from Elsevier. (B and C) Electron micrographs of factories of recombinant Vaccinia virus encoding the A15L gene under the control of the lac operon under nonpermissive (B) and permissive (C) conditions. Note empty immature virus particles (IV), viral crescents in an electron‐lucent environment, and a separate homogenous viroplasm (VP) in panel B and compare to wild‐type like conditions in panel C, which include immature virus with electron‐dense centers and particles containing nucleoids (n). Reprinted from Szajner et al. (2004a) with permission from Elsevier.

Figure 7

(A) Confocal micrograph of frog virus 3‐infected cell showing relationship between the major capsid protein (red), vimentin (green), and DNA (blue). Note multiple viral inclusions in the cytoplasm, each associated with an individual vimentin cage. Authors own. (B) Electron micrograph of a frog virus 3‐infected cell showing two crystalline arrays that appear to induce a kidney‐shaped nucleus (N). Reprinted from Darlington et al. (1966) with permission from Elsevier.

Figure 8

Schematic representing interaction of herpesvirus foci with ND10 bodies. (A) Cell expressing PML‐ECFP (green) and infected with human herpesvirus‐1‐encoding ICP4‐EYFP (red) 115‐min postinfection. Boxes show zoomed sections demonstrating juxtaposition of ND10 and ICP4 bodies early during virus infection. Reprinted from Everett et al. (2003) with permission from American Society for Microbiology. (B) Cell infected with human herpesvirus 2 showing assemblons immunolabeled with ICP35 (red) and UL55 inclusions (green). Note juxtaposition of the two compartments. Reprinted from Yamada et al. (1998) with permission from Society for General Microbiology. (C) Electron micrograph of human herpesvirus 5‐infected cell showing a section of a cytoplasmic assembly site. Note complete virus particle within a vacuole in bottom left‐hand corner, dense bodies in center of image, including one budding into a membrane. Reprinted from Craighead et al. (1972) with permission from American Society for Microbiology. (D) Electron micrograph of a tegusome within a nucleus of a human herpesvirus‐6‐infected cell, note apparent continuity between tegusome and cytoplasm (arrowed). Reprinted from Roffman et al. (1990) with permission from American Society for Microbiology.

Figure 9

(A and B) Confocal images of orthoreovirus type 3 Dearing (A)‐ and type 1 Lang (B)‐infected cells labeled with showing difference between globular and filamentous types of viral factories. (C) Confocal image of an infected cell immunolabeled with (red) and α‐tubulin (green) showing relationship between filamentous factories and microtubules. Reprinted from Parker et al. (2002) with permission from American Society for Microbiology. (D) Electron micrograph showing rotavirus viroplasm (V) next to TLP within membranes derived from the ER (arrow). Reprinted from Petrie et al. (1984) with permission from Elsevier. (E) Doughnut‐shaped rotavirus factory labeled with anti‐NSP2 antibody showing electron‐lucent center with electron‐dense core (arrow) surrounded by viroplasm (V). Reprinted from Altenburg et al. (1980) with permission from Society for General Microbiology.