Dynamics of coronavirus replication-transcription complexes.
Coronaviruses induce in infected cells the formation of double-membrane vesicles (DMVs) in which the replication-transcription complexes (RTCs) are anchored. To study the dynamics of these coronavirus replicative structures, we generated recombinant murine hepatitis coronaviruses that express tagged versions of the nonstructural protein nsp2. We demonstrated by using immunofluorescence assays and electron microscopy that this protein is recruited to the DMV-anchored RTCs, for which its C terminus is essential. Live-cell imaging of infected cells demonstrated that small nsp2-positive structures move through the cytoplasm in a microtubule-dependent manner. In contrast, large fluorescent structures are rather immobile. Microtubule-mediated transport of DMVs, however, is not required for efficient replication. Biochemical analyses indicated that the nsp2 protein is associated with the cytoplasmic side of the DMVs. Yet, no recovery of fluorescence was observed when (part of) the nsp2-positive foci were bleached. This result was confirmed by the observation that preexisting RTCs did not exchange fluorescence after fusion of cells expressing either a green or a red fluorescent nsp2. Apparently, nsp2, once recruited to the RTCs, is not exchanged with nsp2 present in the cytoplasm or at other DMVs. Our data show a remarkable resemblance to results obtained recently by others with hepatitis C virus. The observations point to intriguing and as yet unrecognized similarities between the RTC dynamics of different plus-strand RNA viruses.
Hagemeijer
Marne C
MC
Virology Division, Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands.
Verheije
Monique H
MH
Ulasli
Mustafa
M
Shaltiël
Indra A
IA
de Vries
Lisa A
LA
Reggiori
Fulvio
F
Rottier
Peter J M
PJ
de Haan
Cornelis A M
CA
D000818:Animals; D001483:Base Sequence; D002415:Cats; D002460:Cell Line; D018352:Coronavirus Infections / Q000378:metabolism / Q000821:virology; D022162:Cytoplasmic Vesicles / Q000378:metabolism / Q000648:ultrastructure / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D046529:Microscopy, Electron, Transmission; D006517:Murine hepatitis virus / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D014779:Virus Replication / Q000235:genetics / Q000502:physiology
D017931:DNA Primers; D004279:DNA, Viral; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D049452:Green Fluorescent Proteins