Genome-wide RNAi screen reveals a role for the ESCRT complex in rotavirus cell entry

Abstract

Rotavirus (RV) is the major cause of childhood gastroenteritis worldwide. This study presents a functional genome-scale analysis of cellular proteins and pathways relevant for RV infection using RNAi. Among the 522 proteins selected in the screen for their ability to affect viral infectivity, an enriched group that participates in endocytic processes was identified. Within these proteins, subunits of the vacuolar ATPase, small GTPases, actinin 4, and, of special interest, components of the endosomal sorting complex required for transport (ESCRT) machinery were found. Here we provide evidence for a role of the ESCRT complex in the entry of simian and human RV strains in both monkey and human epithelial cells. In addition, the ESCRT-associated ATPase VPS4A and phospholipid lysobisphosphatidic acid, both crucial for the formation of intralumenal vesicles in multivesicular bodies, were also found to be required for cell entry. Interestingly, it seems that regardless of the molecules that rhesus RV and human RV strains use for cell-surface attachment and the distinct endocytic pathway used, all these viruses converge in early endosomes and use multivesicular bodies for cell entry. Furthermore, the small GTPases RHOA and CDC42, which regulate different types of clathrin-independent endocytosis, as well as early endosomal antigen 1 (EEA1), were found to be involved in this process. This work reports the direct involvement of the ESCRT machinery in the life cycle of a nonenveloped virus and highlights the complex mechanism that these viruses use to enter cells. It also illustrates the efficiency of high-throughput RNAi screenings as genetic tools for comprehensively studying the interaction between viruses and their host cells.

Rho’ing in and out of cells

“…86 For several viruses, involvement of Rho GTPase signaling in virus entry was reported, although the specific uptake routes are still unclear. For example, Japanese encephalitis virus (JEV), 87 poliovirus, 88 rotavirus, 89,90 and baculovirus 91 all appear to depend on RhoA activation for their entry. Porcine circovirus 2 (PCV-2) infection also appears to rely on small Rho GTPase activity for infection, as inhibition with Clostridium difficile toxin B reduced PCV-2 infection in different cell types.…”

Drebrin restricts rotavirus entry by inhibiting dynamin-mediated endocytosis

“…S1A). TSG101 and NSP4 served as positive controls because these proteins were previously shown to be important for promoting RV infection (11,16). Interestingly, depletion of TMOD3 led to a decrease in VP7 expression comparable to that induced by the positive controls (>20%), suggesting that TMOD3 might facilitate simian RV RRV strain infection.…”

“…After VP4 binds to its cognate receptors on cellular surfaces, it undergoes a marked conformational change that allows the RV particles to be taken up by the host cells via endocytosis. Multiple studies including two recent genome-wide siRNA screens suggest that RV enters via a dynamin-2-dependent endocytosis (10,11). RV infection of polarized IECs from the apical side is also shown to depend on clathrin (12).…”

Antiviral drug discovery: broad-spectrum drugs from nature

“…However, as viruses are bound to utilize the host cellular machinery to propagate, they are critically dependent on cellular factors that are up-or downregulated as needed. Examples are the down-regulation of membrane receptors [48][49][50][51][52][53] , the up-regulation of the lipid metabolism 54 , the use of the mRNA processing machinery 55 or the hijacking of components of the endosomal-sorting complex (ESCRT) required for virus export from infected cells [56][57][58][59][60][61][62][63] (see 2.4 for details). Moreover, as the life cycle of different viruses share common cellular factors and pathways, it is feasible that these could be used as targets for the design of broad-spectrum antivirals.…”

“…siRNA downregulation of components of the ESCRT and associated factors such as ALIX has been shown to block cell entry of VSV, LFV, and LCMV, and cell exit of HIV and hepatitis A virus (HAV) 56,58,61,62,87 . However, a non-toxic chemical compound has not yet been released for clinical use.…”