A central paradigm within virology is that each viral particle largely behaves as an independent infectious unit. Here, we demonstrate that clusters of enteroviral particles are packaged within phosphatidylserine (PS) lipid-enriched vesicles that are non-lytically released from cells and provide greater infection efficiency than free single viral particles. We show that vesicular PS lipids are co-factors to the relevant enterovirus receptors in mediating subsequent infectivity and transmission, in particular to primary human macrophages. We demonstrate that clustered packaging of viral particles within vesicles enables multiple viral RNA genomes to be collectively transferred into single cells. This study reveals a novel mode of viral transmission, where enteroviral genomes are transmitted from cell-to-cell en bloc in membrane-bound PS vesicles instead of as single independent genomes. This has implications for facilitating genetic cooperativity among viral quasispecies as well as enhancing viral replication.

Coronaviruses express two very large replicase polyproteins, the 16 autoproteolytic cleavage products of which collectively form the membrane-anchored replication complexes. How these structures are assembled is still largely unknown, but it is likely that the membrane-spanning members of these nonstructural proteins (nsps) are responsible for the induction of the double-membrane vesicles and for anchoring the replication complexes to these membranes. For 3 of the 16 coronavirus nsps-nsp3, nsp4, and nsp6-multiple transmembrane domains are predicted. Previously we showed that, consistent with predictions, nsp4 occurs in membranes with both of its termini exposed in the cytoplasm (M. Oostra et al., J. Virol. 81:12323-12336, 2007). Strikingly, however, for both nsp3 and nsp6, predictions based on a multiple alignment of 27 coronavirus genome sequences indicate an uneven number of transmembrane domains. As a consequence, the proteinase domains present in nsp3 and nsp5 would be separated from their target sequences by the lipid bilayer. To look into this incongruity, we studied the membrane disposition of nsp3 and nsp6 of the severe acute respiratory syndrome coronavirus and murine hepatitis virus by analyzing tagged forms of the proteins expressed in cultured cells. Contrary to the predictions, in both viruses, both proteins had their amino terminus, as well as their carboxy terminus, exposed in the cytoplasm. We established that two of the three hydrophobic domains in nsp3 and six of the seven in nsp6 are membrane spanning. Subsequently, we verified that in nsp4, all four hydrophobic domains span the lipid bilayer. The occurrence of conserved non-membrane-spanning hydrophobic domains in nsp3 and nsp6 suggests an important function for these domains in coronavirus replication.Positive-strand RNA viruses induce the formation of cytoplasmic membrane structures in their host cells to accomplish the efficient replication of their genomes. These structures probably facilitate the orchestration of the replication process and the recruitment of the components required for RNA synthesis and may shield the RNA intermediates from recognition by the host cell's defense mechanisms. The membranes of these structures can be acquired from different cellular compartments. In many virus families, such as Picorna-, Flavi-, and Bromoviridae, the RNA replication complex is associated with membranes derived from the endoplasmic reticulum (ER). However, endosomes and lysosomes (Togaviridae), peroxisomes and chloroplasts (Tombusviridae), and mitochondria (Nodaviridae) are also used as membrane donors (for a review, see reference 44). In general, viral nonstructural proteins (nsps) are responsible for the assembly of the replication complex at these specific cellular organelles and for the observed membrane rearrangements.Coronaviruses are enveloped, plus-strand RNA viruses belonging to the family Coronaviridae that, together with the Arteri-and Roniviridae, belong to the order Nidovirales. With sizes ranging between 27 and 32 kb, coronavi...

Coronaviruses replicate their genomes in association with rearranged cellular membranes. The coronavirus nonstructural integral membrane proteins (nsps) 3, 4 and 6, are key players in the formation of the rearranged membranes. Previously, we demonstrated that nsp3 and nsp4 interact and that their co-expression results in the relocalization of these proteins from the endoplasmic reticulum (ER) into discrete perinuclear foci. We now show that these foci correspond to areas of rearranged ER-derived membranes, which display increased membrane curvature. These structures, which were able to recruit other nsps, were only detected when nsp3 and nsp4 were derived from the same coronavirus species. We propose, based on the analysis of a large number of nsp3 and nsp4 mutants, that interaction between the large luminal loops of these proteins drives the formation of membrane rearrangements, onto which the coronavirus replication-transcription complexes assemble in infected cells.

The coronavirus nucleocapsid (N) protein is a virion structural protein. It also functions, however, in an unknown way in viral replication and localizes to the viral replication-transcription complexes (RTCs). Here we investigated, using recombinant murine coronaviruses expressing green fluorescent protein (GFP)-tagged versions of the N protein, the dynamics of its interactions with the RTCs and the domain(s) involved. Using fluorescent recovery after photobleaching, we showed that the N protein, unlike the nonstructural protein 2, is dynamically associated with the RTCs. Recruitment of the N protein to the RTCs requires the C-terminal N2b domain, which interacts with other N proteins in an RNA-independent manner.

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. Microtubulemediated 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.Viruses have evolved elaborate strategies to manipulate and exploit host cellular components and pathways to facilitate various steps of their replication cycle. One common feature among plus-strand RNA viruses is the assembly of their replication-transcription complexes (RTCs) in association with cytoplasmic membranes (reviewed in references 41, 44, and 54). The induction and modification of replicative vesicles seem to be beneficial to the virus (i) in orchestrating the recruitment of all cellular and viral constituents required for viral RNA synthesis and (ii) in providing a protective microenvironment against virus-elicited host defensive (immune) mechanisms.The enveloped coronaviruses (CoVs) possess impressively large plus-strand RNA genomes, with sizes ranging from ϳ27 to 32 kb (22). The coronavirus polycistronic genome can roughly be divided into two regions: the first two-thirds of the genome contains the large replicase gene that encodes the proteins collectively responsible for viral RNA replication and transcription while the remaining 3Ј-terminal part of the genome encodes the structural proteins and some accessory proteins that are expressed from a nested set of subgenomic mRNAs (sgmRNAs) (55).Almost all of the constituents of the coronavirus RTCs are encoded by the large replicase gene that is comprised of two partly overlapping open reading frames (ORFs), ORF1a and ORF1b. Translation of these ORFs results in two very large polyproteins, pp1a and pp1ab, the latter of which is prod...

Coronaviruses induce in infected cells the formation of replicative structures, consisting of double-membrane vesicles (DMVs) and convoluted membranes, where viral RNA synthesis supposedly takes place and to which the nonstructural proteins (nsp's) localize. Double-stranded RNA (dsRNA), the presumed intermediate in RNA synthesis, is localized to the DMV interior. However, as pores connecting the DMV interior with the cytoplasm have not been detected, it is unclear whether RNA synthesis occurs at these same sites. Here, we studied coronavirus RNA synthesis by feeding cells with a uridine analogue, after which nascent RNAs were detected using click chemistry. Early in infection, nascent viral RNA and nsp's colocalized with or occurred adjacent to dsRNA foci. Late in infection, the correlation between dsRNA dots, then found dispersed throughout the cytoplasm, and nsp's and nascent RNAs was less obvious. However, foci of nascent RNAs were always found to colocalize with the nsp12-encoded RNA-dependent RNA polymerase. These results demonstrate the feasibility of detecting viral RNA synthesis by using click chemistry and indicate that dsRNA dots do not necessarily correspond with sites of active viral RNA synthesis. Rather, late in infection many DMVs may harbor dsRNA molecules that are no longer functioning as intermediates in RNA synthesis.

Green fluorescent protein (GFP)-tagged mouse hepatitis coronavirus nonstructural protein 4 (nsp4) was shown to localize to the endoplasmic reticulum (ER) and to be recruited to the coronavirus replicative structures. Fluorescence loss in photobleaching and fluorescence recovery after photobleaching experiments demonstrated that while the membranes of the ER are continuous with those harboring the replicative structures, the mobility of nsp4 at the latter structures is relatively restricted. In agreement with that observation, nsp4 was shown to be engaged in homotypic and heterotypic interactions, the latter with nsp3 and nsp6. In addition, the coexpression of nsp4 with nsp3 affected the subcellular localization of the two proteins.

Coronaviruses are positive-strand RNA viruses that are important infectious agents of both animals and humans. A common feature among positive-strand RNA viruses is their assembly of replication-transcription complexes in association with cytoplasmic membranes. Upon infection, coronaviruses extensively rearrange cellular membranes into organelle-like replicative structures that consist of double-membrane vesicles and convoluted membranes to which the nonstructural proteins involved in RNA synthesis localize. Double-stranded RNA, presumably functioning as replicative intermediate during viral RNA synthesis, has been detected at the double-membrane vesicle interior. Recent studies have provided new insights into the assembly and functioning of the coronavirus replicative structures. This review will summarize the current knowledge on the biogenesis of the replicative structures, the membrane anchoring of the replication-transcription complexes, and the location of viral RNA synthesis, with particular focus on the dynamics of the coronavirus replicative structures and individual replication-associated proteins.