Morphogenesis of Pestiviruses: New Insights from Ultrastructural Studies of Strain Giraffe-1

Abstract

Knowledge on the morphogenesis of pestiviruses is limited due to low virus production in infected cells. In order to localize virion morphogenesis and replication sites of pestiviruses and to examine intracellular virion transport, a cell culture model was established to facilitate ultrastructural studies. Based on results of virus growth kinetic analysis and quantification of viral RNA, pestivirus strain Giraffe-1 turned out to be a suitable candidate for studies on virion generation and export from culture cells. Using conventional transmission electron microscopy and single-tilt electron tomography, we found virions located predominately in the lumen of the endoplasmic reticulum (ER) in infected cells and were able to depict the budding process of virions at ER membranes. Colocalization of the viral core protein and the envelope glycoprotein E2 with the ER marker protein disulfide isomerase (PDI) was demonstrated by immunogold labeling of cryosections. Moreover, pestivirions could be shown in transport vesicles and the Golgi complex and during exocytosis. Interestingly, viral capsid protein and double-stranded RNA (dsRNA) were detected in multivesicular bodies (MVBs), which implies that the endosomal compartment plays a role in pestiviral replication. Significant cellular membrane alterations such as those described for members of the Flavivirus and Hepacivirus genera were not found. Based on the gained morphological data, we present a consistent model of pestivirus morphogenesis.

The Molecular Biology of Pestiviruses

“…Viral particles could also be detected in Golgi stacks and outside the infected cells. These data confirm the ER as the pestiviral budding site at which the core-RNA complex is packaged in envelopes that carry the envelope proteins (Schmeiser, Mast, Thiel, & K€ onig, 2014). Viral particles are then exported from the cell via the secretory pathway.…”

“…In contrast to other members of the Flaviviridae, an elaborate rearrangement of intracellular membranes was not found in pestivirusinfected cells (Schmeiser et al, 2014). Nevertheless, replication occurs in close association with membranes of the ER.…”

Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence

“…It was described that BVDV NS4B alone can rearrange the host membrane (Weiskircher et al, 2009) as well as HCV NS4B (Egger et al, 2002), suggesting that pestivirus NS4B might act as a trigger for building replication complex. So far, however, pestivirus replication is considered not to remodel cytoplasmic host cell membranes (Schmeiser et al, 2014) as opposed to HCV, which induces organellelike structures designated the membranous web where the formation of the viral replication complex is triggered (Romero-Brey et al, 2012), and to other positive-strand RNA viruses (Knoops et al, 2008;Kopek et al, 2007;Spuul et al, 2007;Welsch et al, 2009). Therefore, we hypothesize that the role of pestivirus NS4B may be partially different from that in other members of the Flaviviridae.…”

What can pestiviral endonucleases teach us about innate immunotolerance?

“…Thus, pestiviral replication involves the formation of dsRNA intermediates in the cytosol of infected cells as seen with a variety of RNA and DNA viruses [18]. This could be confirmed in BVDV-and CSFV-infected cultured cells by immunofluorescence or immunoelectron microscopy and flow cytometry using a dsRNA-specific monoclonal antibody [19][20][21]. In line with the rather unrestrained replication of the cp biotype of pestiviruses [22], the amount of plus-and minus-strand viral RNA and, thus, also of dsRNA is up to two order of magnitude higher in cells infected with cp than with ncp viruses ( [19,20,23], and references therein).…”

“…But the fact that pestiviral replication was purported to be even enhanced by autophagy in PK-15 or MDBK cells [40,41] argues against the latter possibility. Finally, virus replication complexes might be already formed in membranous vesicles as described, e. g., for corona-or hepatitis C viruses [42,43], but such large membrane arrangement were not observed in pestivirus infected cells [21]. Notwithstanding, dsRNA was localized inside the lumen and outer membranes of multivesicular bodies (MVBs) in MDBK cells infected with the pestivirus strain Giraffe-1, but whether these vesicles represent autophagosomes that hide the dsRNA from detection by the innate immune system followed by disposal in lysosomes, or whether they are true sites for viral replication is currently unknown [21].…”