In this work, we have identified the heat shock cognate protein (hsc70) as a receptor candidate for rotaviruses. hsc70 was shown to be present on the surface of MA104 cells, and antibodies to this protein blocked rotavirus infectivity, while not affecting the infectivity of reovirus and poliovirus. Preincubation of the hsc70 protein with the viruses also inhibited their infectivity. Triple-layered particles (mature virions), but not double-layered particles, bound hsc70 in a solid-phase assay, and this interaction was blocked by monoclonal antibodies to the virus surface proteins VP4 and VP7. Rotaviruses were shown to interact with hsc70 at a postattachment step, since antibodies to hsc70 and the protein itself did not inhibit the virus attachment to cells. We propose that the functional rotavirus receptor is a complex of several cell surface molecules that include, among others, hsc70.

The morphogenesis of rotaviruses follows a unique pathway in which immature double-layered particles (DLPs) assembled in the cytoplasm bud across the membrane of the endoplasmic reticulum (ER), acquiring during this process a transient lipid membrane which is modified with the ER resident viral glycoproteins NSP4 and VP7; these enveloped particles also contain VP4. As the particles move towards the interior of the ER cisternae, the transient lipid membrane and the nonstructural protein NSP4 are lost, while the virus surface proteins VP4 and VP7 rearrange to form the outermost virus protein layer, yielding mature infectious triple-layered particles (TLPs). In this work, we have characterized the role of NSP4 and VP7 in rotavirus morphogenesis by silencing the expression of both glycoproteins through RNA interference. Silencing the expression of either NSP4 or VP7 reduced the yield of viral progeny by 75 to 80%, although the underlying mechanism of this reduction was different in each case. Blocking the synthesis of NSP4 affected the intracellular accumulation and the cellular distribution of several viral proteins, and little or no virus particles (neither DLPs nor TLPs) were assembled. VP7 silencing, in contrast, did not affect the expression or distribution of other viral proteins, but in its absence, enveloped particles accumulated within the lumen of the ER, and no mature infectious virus was produced. Altogether, these results indicate that during a viral infection, NSP4 serves as a receptor for DLPs on the ER membrane and drives the budding of these particles into the ER lumen, while VP7 is required for removing the lipid envelope during the final step of virus morphogenesis.Rotaviruses are nonenveloped icosahedral viruses whose capsid is formed by three concentric layers of protein. The innermost layer is formed by 60 dimers of VP2 that surrounds the viral genome composed of 11 segments of double-stranded RNA and 12 copies of each VP1, the virus polymerase, and VP3, the virus capping enzyme. The second layer of protein is formed by 280 trimers of VP6, which sits on top of VP2 to form double-layered particles (DLPs). Finally, the addition of 280 trimers of glycoprotein VP7 which constitute the outermost layer of the virus and 60 dimeric spikes of the VP4 protein to DLPs form triple-layered particles (TLPs) that represent the mature infectious virus (13).Rotavirus morphogenesis occurs by an unusual process where DLPs, which are thought to assemble in cytoplasmic inclusions termed viroplasms, bud across the membrane of the endoplasmic reticulum (ER). During this process, the DLPs acquire a transient lipid envelope which is subsequently lost to yield the mature infectious TLPs (33). The ER membrane through which DLPs bud is modified by two viral proteins, the virion surface protein VP7 and the nonstructural polypeptide NSP4 (5, 7, 21). NSP4 has a large cytosolic domain that interacts with DLPs, and it has been proposed that this interaction drives the translocation of the double-layered particles into the lume...

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While recently we have learned much about the viral and cellular proteins involved in the initial attachment of rotaviruses to MA104 cells, the mechanism by which these viruses reach the interior of the cell is poorly understood. For this study, we observed the effects of drugs and of dominant-negative mutants, known to impair clathrin-mediated endocytosis and endocytosis mediated by caveolae, on rotavirus cell infection. Rotaviruses were able to enter cells in the presence of compounds that inhibit clathrin-mediated endocytosis as well as cells overexpressing a dominant-negative form of Eps15, a protein crucial for the assembly of clathrin coats. We also found that rotaviruses infected cells in which caveolar uptake was blocked; treatment with the cholesterol binding agents nystatin and filipin, as well as transfection of cells with dominant-negative caveolin-1 and caveolin-3 mutants, had no effect on rotavirus infection. Interestingly, cells treated with methyl-␤-cyclodextrin, a drug that sequesters cholesterol from membranes, and cells expressing a dominant-negative mutant of the large GTPase dynamin, which is known to function in several membrane scission events, were not infected by rotaviruses, indicating that cholesterol and dynamin play a role in the entry of rotaviruses.The initial steps in a viral infection involve the specific attachment of the viral particle to a receptor(s) on the cell surface, followed by internalization of the virus into the cell and the subsequent uncoating of the virion to release the active transcription complex. These events are essential for the suc-

Rotavirus RRV gene 11 encodes two non-structural proteins, NSP5 and NSP6. NSP5 is a phosphorylated non-structural protein that binds single-and double-stranded RNA in a non-specific manner. Transient expression of this protein in uninfected cells has provided evidence for its participation in the formation of electron-dense cytoplasmic structures, known as viroplasms, which are thought to be key structures for the replication of the virus. NSP6 is a protein of unknown function that seems not to be essential for virus replication in cell culture. To study the function of NSP5 in the context of a viral infection, the expression of RRV gene 11 was silenced by RNA interference. Reduction in the synthesis of NSP5, as shown by immunoblot and immunofluorescence assays, correlated with a reduction in the number and size of viroplasms and with an altered intracellular distribution of other viroplasm-associated proteins. Silencing of gene 11 also resulted in a reduced synthesis of viral RNA(+) and double-stranded RNA and of all viral proteins, as well as in a decreased production of infectious virus. A similar phenotype was observed when the NSP5 coding gene of the lapine rotavirus strain Alabama was silenced. The fact that the NSP5 gene of rotavirus Alabama lacks the AUG initiator codon for a complete NSP6 protein, suggests that the described phenotype in gene 11-silenced cells is mostly due to the absence of NSP5. The data presented in this work suggest that NSP5 is a key protein during the replication cycle of rotaviruses.