(A) Schematic outline of the MHV-GFP-N domain recombinant viruses (not drawn to scale). in infected cells the formation of double-membrane vesicles (DMVs) and convoluted membranes (CMs). These structures harbor the nonstructural proteins (nsp’s) (9,14,25,26,28) and are associated with viral RNA synthesis (1,9,20,22). The nsp’s, which jointly form the replication-transcription complexes (RTCs), presumably mediate the formation of these membranous structures by modifying endoplasmic reticulum-derived membranes and by recruiting cellular components to their need. In addition to the nsp’s, coronaviruses express several structural proteins, including at least DKFZp781H0392 the spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins (6). The N protein packages the viral genomic RNA to form the helical nucleocapsid that is incorporated into the budding particle but also fulfills additional roles during the viral contamination. It has been shown to function as an RNA chaperone (33) and to facilitate viral RNA synthesis (2,5,16). Not surprisingly, the N protein localizes to DMVs and CMs, the sites where the RTCs are concentrated, in addition to the virion assembly sites (3,7,23,28,29). Furthermore, the nucleocapsid protein contributes to the perturbation of several host cellular processes (reviewed in reference27). Recently, we exhibited that nsp2, once recruited to the RTCs, is not exchanged for nsp2 molecules present in the cytoplasm and in other DMVs/CMs. That is, no recovery of fluorescence was observed when (a part of) the nsp2-positive foci were photobleached (10). Whether the other nsp’s or the N protein pool associated with the RTCs also lacks mobility at these sites remains unfamiliar. Of particular interest are the dynamics of the N protein, as it is usually involved in different, spatially and temporally separated actions of the viral life cycle. We hypothesized that this N protein is not permanently bound to the RTCs but rather possesses a manifest intracellular mobility, as it is probably not involved only in viral RNA synthesis but also in its transport from the site of synthesis to the virion assembly sites, where it participates in virion assembly. To test our hypothesis, we analyzed the dynamics of the N protein localized at the RTCs by live-cell imaging. To this end, we generated a recombinant mouse hepatitis coronavirus (MHV) expressing an additional copy of the N protein C-terminally fused to green fluorescent protein (N-GFP). The coding sequence for N-GFP was launched into the viral genome as an additional expression cassette between genes 2a and S by targeted RNA recombination as previously explained (18), thereby replacing the nonfunctional hemagglutinin-esterase gene (Fig.1A). The resulting recombinant computer virus, MHV-N-GFP, was viable; however, it was rapidly outcompeted by viruses that had lost expression of the N fusion protein. As we were unable to demonstrate incorporation of N-GFP into progeny virions, we speculated that this fusion protein acts as a dominant unfavorable during virion assembly. Of passage 2, approximately 10 to 20% of the computer virus population expressed detectable levels of N-GFP (data not shown), which was sufficient for our experimental goal. == FIG. 1. == Recruitment and localization of the N protein to the RTCs and DMVs. (A) Schematic summarize of the MHV-N-GFP recombinant computer virus (not drawn to level). UTR, untranslated region. (B) LR7 cells inoculated with MHV or MHV-N-GFP were fixed at 6 h p.i. and stained with antibodies directed against nsp2/3 (D4 [24]; kind gift of S. Baker) or nsp8 (anti-p22 antibody [15]; kind gift of M. Valifenalate Denison). Production of newly synthesized viral RNA was visualized by using Click-It detection of RNA. To this end, infected cells were fed with 5-ethynyl uridine from 5.5 to 6.5 h p.i., Valifenalate after which the cells were fixed. (C) HeLa-CEACAM1a cells infected with MHV-N-GFP and control cells (mock) were fixed at 6 h p.i. and processed for immunoelectron microscopy using antibodies against GFP. Arrowheads show colocalization sites between N-GFP and either RTC protein markers (B) or Valifenalate DMVs (C). To determine whether the N-GFP fusion protein, when expressed from your viral genome, was recruited to the RTCs, LR7 cells were inoculated with MHV-N-GFP, fixed at 6 h postinfection (p.i.), and subsequently processed for immunofluorescence analysis. The results show that this N-GFP was present throughout the cytoplasm at a low level and was concentrated in cytoplasmic foci that were colocalizing with the RTC protein markers nsp2/3 (antibody D4) and nsp8 (anti-p22.
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