In vivo interactions among rotavirus nonstructural proteins

The NSP5 protein is required for viroplasm formation during rotavirus infection and is hyperphosphorylated into 32-to 35-kDa isoforms. Earlier studies reported that NSP5 is not hyperphosphorylated without NSP2 coexpression or deleting the NSP5 N terminus and that serine 67 is essential for NSP5 hyperphosphorylation. In this report, we show that full-length NSP5 is hyperphosphorylated in the absence of NSP2 or serine 67 and demonstrate that hyperphosphorylated NSP5 is predominantly present in previously unrecognized cellular fractions that are insoluble in 0.2% sodium dodecyl sulfate. The last 68 residues of NSP5 are sufficient to direct green fluorescent protein into insoluble fractions and cause green fluorescent protein localization into viroplasm-like structures; however, NSP5 insolubility was intrinsic and did not require NSP5 hyperphosphorylation. When we mutated serine 67 to alanine we found that the NSP5 mutant was both hyperphosphorylated and insoluble, identical to unmodified NSP5, and as a result serine 67 is not required for NSP5 phosphorylation. Interestingly, treating cells with the phosphatase inhibitor calyculin A permitted the accumulation of soluble hyperphosphorylated NSP5 isoforms. This suggests that soluble NSP5 is constitutively dephosphorylated by cellular phosphatases and demonstrates that hyperphosphorylation does not direct NSP5 insolubility. Collectively these findings indicate that NSP5 hyperphosphorylation and insolubility are completely independent parameters and that analyzing insoluble NSP5 is essential for studies assessing NSP5 phosphorylation. Our results also demonstrate the involvement of cellular phosphatases in regulating NSP5 phosphorylation and indicate that in the absence of other rotavirus proteins, domains on soluble and insoluble NSP5 recruit cellular kinases and phosphatases that coordinate NSP5 hyperphosphorylation.Rotavirus is an icosahedral virus belonging to the family Reoviridae and has a genome composed of 11 double-stranded RNA segments (21). One characteristic feature of rotavirus infection is the formation of punctate perinuclear structures called viroplasms 2 to 3 h into the infectious cycle (36). Viroplasms are sites of viral RNA replication and packaging of genome segments into progeny virions. Several rotavirus proteins (VP1, VP2, VP3, VP6, NSP2, NSP5, and NSP6) have been found in viroplasms during infection (25,47). Expression of NSP2 and NSP5 is reportedly required and sufficient for viroplasm formation (19,22). However, it has also been shown that expression of N-terminally tagged NSP5 alone results in the formation of viroplasm-like structures (32).NSP5 contains 198 amino acids with a predicted molecular mass of approximately 21 kDa. NSP5 is highly phosphorylated in infected cells resulting in a series of posttranslationally modified isoforms that range from 26 to 35 kDa (2). The initial modification that results in the shift from 21 to 26 kDa is unknown, but the appearance of 28-and 32-to 35-kDa isoforms from a 26-kDa precursor has been ascribed to O...

Section: Vol 80 2006 Hyperphosphorylation Of Rotavirus Nsp5 Proteinmentioning

confidence: 99%

Hyperphosphorylation of the Rotavirus NSP5 Protein Is Independent of Serine 67 or NSP2, and the Intrinsic Insolubility of NSP5 Is Regulated by Cellular Phosphatases

Agresti

Mackow

2006

“…MAb HS2 directed to VP4 (21), the rabbit antirotavirus polyclonal serum raised against purified RRV TLPs, the rabbit anti-VP1, and antivimentin serum were produced in our laboratory. Rabbit polyclonal antisera to NSP2 and NSP3 have been described previously (9). Fab fragments to digoxigenin (DIG)-peroxidase were purchased from Roche, Inc. Alexa Fluor 488-and 647-conjugated secondary antibodies were purchased from Molecular Probes (Eugene, OR).…”

Section: Cells and Virusesmentioning

confidence: 99%

Analysis of the Kinetics of Transcription and Replication of the Rotavirus Genome by RNA Interference

Ayala-Bretón¹,

Arias²,

Espinosa³

et al. 2009

Rotaviruses have a genome composed of 11 segments of double-stranded RNA (dsRNA) surrounded by three protein layers. The virus contains an RNA-dependent RNA polymerase that synthesizes RNA transcripts corresponding to all segments of the viral genome. These transcripts direct the synthesis of the viral proteins and also serve as templates for the synthesis of the complementary strand to form the dsRNA genome. In this work, we analyzed the kinetics of transcription and replication of the viral genome throughout the replication cycle of the virus using quantitative reverse transcription-PCR. The role of the proteins that form doublelayered particles ([DLPs] VP1, VP2, VP3, and VP6) in replication and transcription of the viral genome was analyzed by silencing their expression in rotavirus-infected cells. All of them were shown to be essential for the replication of the dsRNA genome since in their absence there was little synthesis of viral mRNA and dsRNA. The characterization of the kinetics of RNA transcription and replication of the viral genome under conditions where these proteins were silenced provided direct evidence for a second round of transcription during the replication of the virus. Interestingly, despite the decrease in mRNA accumulation when any of the four proteins was silenced, the synthesis of viral proteins decreased when VP2 and VP6 were knocked down, whereas the absence of VP1 and VP3 did not have a severe impact on viral protein synthesis. Characterization of viral particle assembly in the absence of VP1 and VP3 showed that while the formation of triple-layered particles and DLPs was decreased, the amount of assembled lower-density particles, often referred to as empty particles, was not different from the amount in control-infected cells, suggesting that viral particles can assemble in the absence of either VP1 or VP3.The family Reoviridae includes viruses that have a genome composed of 9 to 12 segments of double-stranded RNA (dsRNA). Rotaviruses belong to the most medically significant genus of the family since they are the main cause of infantile gastroenteritis, causing approximately 500,000 deaths per year in children less than 5 years of age (23). These viruses have a genome composed of 11 segments of dsRNA, which is enclosed in a capsid formed by three concentric layers of protein (33). The innermost layer, formed by VP2, contains the viral genome and 12 copies each of the virus RNA-dependent RNA polymerase (RdRP; VP1) and the guanylyltransferase and methylase enzyme (VP3); these viral elements constitute the core of the virus. The addition of VP6 on top of the VP2 layer produces double-layered particles (DLPs). The outermost layer, characteristic of infectious, triple-layered particles (TLPs), is composed of two proteins, VP4 and VP7.During or shortly after cell entry, the infecting TLP uncoats, loosing the two proteins of the outer layer and yielding a DLP, which is transcriptionally active (14). The nascent transcripts are extruded into the cell's cytoplasm through channels located ne...

“…MAb HS2 directed to VP4 (25), the rabbit anti-rotavirus polyclonal serum raised against purified RRV TLPs, and the rabbit anti-vimentin sera were produced in our laboratory. Rabbit polyclonal sera to NSP2, NSP3, and NSP5 have been described previously (16). Alexa-488-and Alexa-568-conjugated secondary antibodies and phalloidin-Alexa-546 were purchased from Molecular Probes (Eugene, Oreg.).…”

Section: Cells and Virusesmentioning

confidence: 99%

Silencing the Morphogenesis of Rotavirus

López

Camacho

Zayas

et al. 2005

115

118

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...