Rotaviruses are the leading etiological agents of acute gastroenteritis in infants and young children worldwide. These nonenveloped viruses enter cells using different types of endocytosis and, depending on the virus strain, travel to different endosomal compartments before exiting to the cytosolic space. In this Gem, we review the viral and cellular factors involved in the different stages of a productive virus cell entry and share with the readers the journey that we have taken into the cell to learn about virus entry. R otaviruses (RVs) are nonenveloped viruses, members of the family Reoviridae, and the leading etiological agents of viral gastroenteritis. In vivo, these viruses infect primarily mature enterocytes in the intestinal epithelium. They are composed of a triple-layered protein capsid that surrounds the viral genome. The outermost layer is formed by VP7, which makes the smooth surface of the virus, and by the spike protein VP4, which functions as the virus attachment protein. VP4 is cleaved by trypsin into two subunits, VP8 and VP5, and this cleavage is required for the virus to enter the cell (1).The initial step in a viral infection is the attachment of the virus to specific receptors on the cell surface, an interaction that frequently triggers cellular signaling cascades that facilitate either virus entry or replication. In most instances, regardless of whether the virus is enveloped or not, virus internalization proceeds through an endocytic pathway that delivers the viral particle to early endosomes (EEs), characterized by the presence of the GTPase Rab5 and early endosomal antigen 1 (EEA1) (2). Some viruses then traffic from these EE compartments to late endosomes (LEs), which are enriched in the GTPase Rab7 (2). The switch from Rab5 to Rab7 occurs via formation of hybrid endosomes that carry both Rab GTPases in separate domains (2) and are known as maturing endosomes (MEs) (3). Some MEs contain intraluminal vesicles (ILVs) that are formed by the endosomal sorting complex required for transport (ESCRT) machinery (4).The study of RV entry and vesicular traffic has been challenging and its advancement slow because a robust reverse genetic system to manipulate the genome of the virus is lacking. This limitation has been partially overcome, however, by use of RNA interference (RNAi) technology to explore the function of individual viral and cellular genes involved in this process, as well as by determination of the three-dimensional structures of the RV surface proteins. ATTACHMENT AND POSTATTACHMENT INTERACTIONSRV cell entry is a multistep process involving cellular glycans for cell binding and several coreceptors during postattachment steps (5) (Fig. 1, step a). The VP8 domain of VP4 mediates the initial interaction of the virus with the cell surface, whereas VP5 and likely the surface glycoprotein VP7 interact with downstream coreceptors (5). Several glycans have been identified as receptors for rotavirus; RV strains were classified initially as neuraminidase (NA) sensitive or NA resistant, dep...

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.

Here we show that the ubiquitin-proteasome system is required for the efficient replication of rotavirus RRV in MA104 cells. The proteasome inhibitor MG132 decreased the yield of infectious virus under conditions where it severely reduces the synthesis of not only viral but also cellular proteins. Addition of nonessential amino acids to the cell medium restored both viral protein synthesis and cellular protein synthesis, but the production of progeny viruses was still inhibited. In medium supplemented with nonessential amino acids, we showed that MG132 does not affect rotavirus entry but inhibits the replication of the viral genome. It was also shown that it prevents the efficient incorporation into viroplasms of viral polymerase VP1 and the capsid proteins VP2 and VP6, which could explain the inhibitory effect of MG132 on genome replication and infectious virus yield. We also showed that ubiquitination is relevant for rotavirus replication since the yield of rotavirus progeny in cells carrying a temperature-sensitive mutation in the E1 ubiquitin-activating enzyme was reduced at the restrictive temperature. In addition, overexpression of ubiquitin in MG132-treated MA104 cells partially reversed the effect of the inhibitor on virus yield. Altogether, these data suggest that the ubiquitin-proteasome (UP) system has a very complex interaction with the rotavirus life cycle, with both the ubiquitination and proteolytic activities of the system being relevant for virus replication.

Rotaviruses (RVs) enter cells through different endocytic pathways. Bovine rotavirus (BRV) UK uses clathrin-mediated endocytosis, while rhesus rotavirus (RRV) employs an endocytic process independent of clathrin and caveolin. Given the differences in the cell internalization pathway used by these viruses, we tested if the intracellular trafficking of BRV UK was the same as that of RRV, which is known to reach maturing endosomes (MEs) to infect the cell. We found that BRV UK also reaches MEs, since its infectivity depends on the function of Rab5, the endosomal sorting complex required for transport (ESCRT), and the formation of endosomal intraluminal vesicles (ILVs). However, unlike RRV, the infectivity of BRV UK was inhibited by knocking down the expression of Rab7, indicating that it has to traffic to late endosomes (LEs) to infect the cell. The requirement for Rab7 was also shared by other RV strains of human and porcine origin. Of interest, most RV strains that reach LEs were also found to depend on the activities of Rab9, the cation-dependent mannose-6-phosphate receptor (CD-M6PR), and cathepsins B, L, and S, suggesting that cellular factors from the trans-Golgi network (TGN) need to be transported by the CD-M6PR to LEs to facilitate RV cell infection. Furthermore, using a collection of UK ؋ RRV reassortant viruses, we found that the dependence of BRV UK on Rab7, Rab9, and CD-M6PR is associated with the spike protein VP4. These findings illustrate the elaborate pathway of RV entry and reveal a new process (Rab9/CD-M6PR/cathepsins) that could be targeted for drug intervention. IMPORTANCERotavirus is an important etiological agent of severe gastroenteritis in children. In most instances, viruses enter cells through an endocytic pathway that delivers the viral particle to vesicular organelles known as early endosomes (EEs). Some viruses reach the cytoplasm from EEs, where they start to replicate their genome. However, other viruses go deeper into the cell, trafficking from EEs to late endosomes (LEs) to disassemble and reach the cytoplasm. In this work, we show that most RV strains have to traffic to LEs, and the transport of endolysosomal proteases from the Golgi complex to LEs, mediated by the mannose-6-phosphate receptor, is necessary for the virus to exit the vesicular compartment and efficiently start viral replication. We also show that this deep journey into the cell is associated with the virus spike protein VP4. These findings illustrate the elaborate pathway of RV entry that could be used for drug intervention.

Several molecules have been identified as receptors or coreceptors for rotavirus infection, including glycans, integrins, and hsc70. In this work we report that the tight junction proteins JAM-A, occludin, and ZO-1 play an important role during rotavirus entry into MA104 cells. JAM-A was found to function as coreceptor for rotavirus strains RRV, Wa, and UK, but not for rotavirus YM. Reassortant viruses derived from rotaviruses RRV and YM showed that the virus spike protein VP4 determines the use of JAM-A as coreceptor.

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.

Rotavirus genome replication and assembly take place in cytoplasmic electron dense inclusions termed viroplasms (VPs). Previous conventional optical microscopy studies observing the intracellular distribution of rotavirus proteins and their organization in VPs have lacked molecular-scale spatial resolution, due to inherent spatial resolution constraints. In this work we employed super-resolution microscopy to reveal the nanometric-scale organization of VPs formed during rotavirus infection, and quantitatively describe the structural organization of seven viral proteins within and around the VPs. The observed viral components are spatially organized as five concentric layers, in which NSP5 localizes at the center of the VPs, surrounded by a layer of NSP2 and NSP4 proteins, followed by an intermediate zone comprised of the VP1, VP2, VP6. In the outermost zone, we observed a ring of VP4 and finally a layer of VP7. These findings show that rotavirus VPs are highly organized organelles.

19Cellular and viral factors participate in the replication cycle of rotavirus. We report that the 20 guanine nucleotide exchange factor GBF1, which activates the small GTPase Arf1 to induce 21 COPI transport processes, is required for rotavirus replication since knocking down GBF1 22 3 IMPORTANCE 40 Rotavirus, a member of the family Reoviridae, is the major cause of severe diarrhea in 41 children and young animals worldwide. Despite the significant advances in the 42 characterization of the biology of this virus, the mechanisms involved in morphogenesis of 43 the virus particle are still poorly understood. In this work, we show that the guanine 44 nucleotide exchange factor GBF1, relevant for the COPI/Arf1-mediated cellular vesicular 45 transport, participates in the replication cycle of the virus, influencing the correct processing 46 of viral glycoproteins VP7 and NSP4, and the assembly of the virus surface proteins VP7 47 59 Rotaviruses, members of the family Reoviridae, are non-enveloped particles formed by three 60 concentric layers of proteins that surround the eleven genome segments of double-stranded 61 RNA (dsRNA). The innermost layer is composed of the core-shell proteinVP2 that encloses 62 the replication intermediates, composed of the RNA dependent RNA polymerase VP1, and 63 the guanylyl-methyl transferase, VP3. The intermediate layer is formed by VP6 that 64 surrounds the VP2 layer to form double-layered particles (DLPs). Finally, the addition of the 65 glycoprotein VP7 and the spike protein VP4 onto the DLPs forms the infectious triple-layered 66 particles (TLPs) (1, 2). 67 The replication of rotavirus occurs in cytoplasmic non-membranous electron-dense 68 inclusions termed viroplasms composed of NSP2, NSP5, VP1, VP2, VP6 and host 69 components (1, 3). The replication and packaging of the viral genome into newly synthesized 70DLPs take place in these inclusions (4), which then bud into the lumen of the endoplasmic 71 reticulum (ER) through membrane sites modified by the presence of NSP4 (5, 6). NSP4 is a 72 transmembrane ER glycoprotein with two N-linked high mannose glycosylated chains (7) 73 that play a crucial role in the last steps of rotavirus assembly. It has been shown that the 74 cytoplasm oriented-terminus of NSP4 associates with VP4 (8), and binds the VP6 on DLPs 75 acting as a receptor for these particles to mediate their budding into the ER (9, 10). Moreover, 76 NSP4 has been shown to also interact with VP7 through its N-terminus oriented to the ER 77 lumen (11, 12). It has been proposed that these interactions drive the incorporation of the 78 outer layer proteins into the transitory lipid envelope that DLPs acquire during ER membrane 79 budding; this envelope is removed in the lumen of the ER by an unknown process in which 80 NSP4 is eliminated while VP4 and VP7 are assembled to produce the final infectious TLPs 81 All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.110...