Actin-Dependent Nonlytic Rotavirus Exit and Infectious Virus Morphogenetic Pathway in Nonpolarized Cells

Abstract

During the late stages of rotavirus morphogenesis the surface proteins VP4 and VP7 are assembled onto the previously structured double-layered virus particles to yield a triple-layered, mature infectious virus. The current model for the assembly of the outer capsid is that it occurs within the lumen of the endoplasmic reticulum. However, it has been shown that VP4 and infectious virus associate with lipid rafts, suggesting that the final assembly of the rotavirus spike protein VP4 involves a post-endoplasmic reticulum event. In this work, we found that the actin inhibitor jasplakinolide blocks the cell egress of rotavirus from non-polarized MA104 cells at early times of infection when there is still no evidence of cell lysis. These findings are in contrast with the traditional assumption that rotavirus is released from non-polarized cells by a non-specific mechanism when the cell integrity is lost. Inspection of the virus present in the extracellular media by density flotation gradients revealed that a fraction of the released virus is associated with low-density membranous structures. Furthermore, the intracellular localization of VP4, its interaction with lipid rafts and its targeting to the cell surface were shown to be prevented by jasplakinolide, implying a role for actin in these processes. Finally, the VP4 present at the plasma membrane was shown to be incorporated into the extracellular infectious virus, suggesting the existence of a novel pathway for the assembly of the rotavirus spike protein. Rotavirus is a major etiological agent of infantile acute severe diarrhea. It is a non-enveloped virus formed by three concentric layers of protein. The early stages of rotavirus replication, including cell attachment and entry, synthesis and translation of viral mRNAs, replication of the genomic dsRNA, and the assembly of double-layered viral particles, have been widely studied. However, the mechanism involved in the later stages of infection, i.e, viral particle maturation and cell exit, have been less characterized. It has been historically assumed that rotavirus exits non-polarized cells following cell lysis. In this work, we show that the virus exits cells by a non-lytic, actin-dependent mechanism and, most importantly, we describe that VP4, the spike protein of the virus, is present on the cell surface and is incorporated into mature, infectious virus, indicating a novel pathway for the assembly of this protein.

The C Terminus of Rotavirus VP4 Protein Contains an Actin Binding Domain Which Requires Cooperation with the Coiled-Coil Domain for Actin Remodeling

“…Such an interaction results in the formation of what we previously called actin bodies (14), which are focal clusters of F-actin fully colocalized with phalloidin. This mechanism explains how VP4 may induce a destabilization of the subcortical microfilaments, favoring the crossing of viral particles through the apical cell membrane and their release in the extracellular space without significant cell death (6,17). This mode of action of VP4 is in agreement with the modifications of the actin cytoskeleton previously observed during rotavirus infection of MA104, Cos-7, and Caco-2 cells, where microfilaments are progressively gathered in actin bodies (14,15).…”

“…Finally, we demonstrated that blocking the treadmilling by using jasplakinolide leads to a fully depolarized release of new virions (15). A similar process has been recently demonstrated by another group in MA104 cells (17), a nonpolarized cell line, indicating that our initial observation is not restricted to polarized enterocytes.…”

The actin cytoskeleton is important for rotavirus internalization and RNA genome replication

“…The data presented in this work demonstrate that the actin network structure and its dynamics are important for different stages of the virus life cycle. In this regard, actin has also been shown to be important for rotavirus cell exit (Trejo-Cerro et al, 2018). Understanding the detailed mechanisms through which these proteins facilitate rotavirus replication may reveal cross pathway connections and possible cellular proteins that could be targeted for antiviral developments.…”

“…Although considerable information about different stages of the rotavirus life cycle, like entry, transcription and translation, viroplasm formation, morphogenesis, genome replication, maturation and release has been obtained, the role of the actin cytoskeleton in rotavirus infection has been poorly characterized. A recent study described the T actin-binding protein Drebrin1 as a restriction factor for rotavirus infection (Li et al, 2017), and it has been reported that changes in microfilaments occur at early (Zambrano et al, 2012) and late (Gardet et al, 2006) times of rotavirus infection; it has also been shown that rotavirus release from polarized and non-polarized cells is sensitive to the action of drugs that disrupt actin filaments (Gardet et al, 2007;Trejo-Cerro et al, 2018). However, the potential role of microfilaments in virus entry and genome replication has not been explored.…”

Discrete virus factories form in the cytoplasm of cells co-infected with two strains of the segmented dsRNA virus, infectious bursal disease virus (IBDV), that subsequently coalesce

“…5 and 6B and Movie S4). Interestingly, VFs have also been observed co-localising with actin in cells infected with Fowlpox [34] and Bunyamwera virus [35], and actin is involved in the internalisation, replication, and non-lytic egress of rotavirus [36, 37]. IBDV internalisation into the cell is also dependent on an intact actin network [32], [38].…”