Silencing the Morphogenesis of Rotavirus

Abstract

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...

The flexible C terminus of the rotavirus non-structural protein NSP4 is an important determinant of its biological properties

“…NSP4, encoded by genome segment 10, is a multifunctional protein (Fig. 1a) and is essential for virus morphogenesis and pathogenesis (Lopez et al, 2005;Silvestri et al, 2005). With the N terminus of the 175 aa protein anchored in the endoplasmic reticulum (ER), the C terminus attains a cytoplasmic orientation (Estes, 2001).…”

Association of rotavirus viroplasms with microtubules through NSP2 and NSP5

“…The third layer formed by VP4 and VP7 is acquired by budding of the DLP particles into the endoplasmic reticulum (ER), a process that requires assistance of the viral non-structural protein 4 (NSP4), whereby a transient envelope is acquired, that is finally lost within the ER along with NSP4. The fundamental role of NSP4 in this step is evident by the reduction in size of viroplasms that is observed when the NSP4 gene is silenced though RNA interference (Lopez et al 2005).…”

“…The third layer formed by VP4 and VP7 is acquired by budding of the DLP particles into the endoplasmic reticulum (ER), a process that requires assistance of the viral non-structural protein 4 (NSP4), whereby a transient envelope is acquired, that is finally lost within the ER along with NSP4. The fundamental role of NSP4 in this step is evident by the reduction in size of viroplasms that is observed when the NSP4 gene is silenced though RNA interference (Lopez et al 2005).Rotavirus infection changes the intracellular distribution of microtubule associated protein 2 (MAP2) and subviral particles normally located in viroplasms have been co-purified with MAP2 (Weclewicz et al 1993), suggesting a possible relationship of these particles, or the viroplasms where they are assembled, with microtubules.In addition, other members of the Reoviridae family replicate in association with the cytoskeleton. The prototype of the Reoviridae family is the genus Reovirus that requires an intact microtubule network for virus assembly (Dales 1963, Parker et al 2002.…”

How Viruses Use the Endoplasmic Reticulum for Entry, Replication, and Assembly

“…Thus, VP7 likely undergoes conformational rearrangements after budding, exposing its membrane-lytic portion to disrupt envelope integrity. Although NSP4 also has membrane lytic activity (Browne et al 2000), its function in removing the transient envelope is difficult to assess as NSP4 knockdown attenuates DLP assembly severely (Lopez et al 2005 Clarifying how the transient enveloped intermediate's membrane is removed to facilitate TLP formation is unquestionably the most crucial step in illuminating rotavirus assembly. What ER factors impart conformational changes on VP7 to render it membrane penetrationcompetent?…”

“…This process requires two steps that occur in or surrounding the ER. (Lopez et al 2005;Cuadras et al 2006). How might VP7 promote envelope shedding?…”