Ultrastructural characterization of membranous torovirus replication factories

Abstract

Plus-stranded RNA viruses replicate in the cytosol of infected cells, in membrane-bound replication complexes containing the replicase proteins, the viral RNA and host proteins. The formation of the replication and transcription complexes (RTCs) through the rearrangement of cellular membranes is currently being actively studied for viruses belonging to different viral families. In this work, we identified double-membrane vesicles (DMVs) in the cytoplasm of cells infected with the equine torovirus Berne virus (BEV), the prototype member of the Torovirus genus (Coronaviridae family, Nidovirales order). Using confocal microscopy and transmission electron microscopy, we observed a close relationship between the RTCs and the DMVs of BEV. The examination of BEV-infected cells revealed that the replicase proteins colocalize with each other and with newly synthesized RNA and are associated to the membrane rearrangement induced by BEV. However, the double-stranded RNA, an intermediate of viral replication, is exclusively limited to the interior of DMVs. Our results with BEV resemble those obtained with other related viruses in the Nidovirales order, thus providing new evidence to support the idea that nidoviruses share a common replicative structure based on the DMV arranged clusters.

Antiviral activity of K22 against members of the order Nidovirales

“…Similar virus-induced membrane re-arrangements were also described for other arteriviruses including porcine reproductive and respiratory syndrome virus (PRRSV) and simian hemorrhagic fever virus (SHFV) (Metwally et al, 2010;Sun et al, 2012;Wood et al, 1970). The host-derived membrane structures induced by a torovirus infection were described for the first time in a recent study (Avila-Perez et al, 2016). Using cells infected with the equine torovirus (EToV) prototype, Avila-Pérez and colleagues described the presence of DMVs of heterogeneous size in the cytoplasm of infected cells, located frequently in the perinuclear area and surrounded by mitochondria and ER membranes.…”

“…These specialized microenvironments are presumed to facilitate the assembly and function of the viral RNA-synthesizing machinery, and may also function to delay or prevent the detection of double-stranded RNA (dsRNA) intermediates of viral replication by innate immune sensors (Ahlquist et al, 2003;Angelini et al, 2014;den Boon and Ahlquist, 2010;Neufeldt et al, 2016;Salonen et al, 2005;Schwartz et al, 2004). Following the emergence of new three-dimensional imaging techniques, such as electron cryo-tomography, the ultrastructural organization of these host-derived replication organelles have been intensively studied for a number of positive-strand RNA viruses including coronaviruses, toroviruses and arteriviruses (Avila-Perez et al, 2016;Knoops et al, 2012;Knoops et al, 2008;Maier et al, 2013;Romero-Brey and Bartenschlager, 2015;van der Hoeven et al, 2016). Coronavirus replication occurs within the cytoplasm of infected cells where mainly endoplasmic reticulum (ER) membranes appear to be recruited for the formation of replication organelles.…”

“…Unlike coronaviruses infected cells, no CM or spherules were identified. For the nidoviruses mentioned above (corona-, arteriand toroviruses), the use of confocal and transmission electron microscopy showed the association of key replicative enzymes of the replication transcription complex (RTC) with the modified host membrane structures (Pedersen et al, 1999;Posthuma et al, 2008;Snijder et al, 2006;van Hemert et al, 2008) and the accumulation of doublestranded RNA in the DMV interior (Avila-Perez et al, 2016;Knoops et al, 2012;Knoops et al, 2008).…”

Activation of the autophagy pathway by Torovirus infection is irrelevant for virus replication

“…In fact, the presence of double membrane vesicles (DMVs) in the cytoplasm of cells infected with different nidoviruses has been associated with the formation of their replication and transcription complexes (RTCs) [29][30][31][32][33][34][35]. In this regard, we recently described the presence of DMVs in the cytoplasm of BEV-infected cells, providing new evidence supporting the notion that nidoviruses share a common replicative structure based on DMV arranged clusters [15]. However, the mechanism and host factors required for the generation of these structures remain unknown.…”

“…The first two-thirds of the genome carry two large overlapping open reading frames, ORF1a and ORF1b, directly translated from the viral genome, rendering two large polyproteins (pp1a and pp1ab), which are proteolytically processed by virus-encoded proteases (the papain-like proteinase [PLP] and the main proteinase [M pro ]) to release replication and transcriptional machinery virus components (e.g. the RNAdependent RNA polymerase [RdRp] and the helicase [Hel] amongst others) [1,10,14,15]. The rest of the viral genome harbors ORFs encoding structural proteins: spike (S) [16,17], membrane (M) [18,19], hemagglutinin-esterase (HE) [20][21][22], and nucleocapsid (N) [14,23], which are individually expressed from a nested set of 3'-coterminal subgenomic mRNAs [11,24,25].Accumulating evidence from different positive-strand RNA viruses indicates that in this heterogeneous group of viruses, genome replication and transcription invariably occur in close association with highly modified cellular membranes of diverse origins [26][27][28].…”