Rotavirus NSP5 is a nonstructural protein that localizes in viroplasms of virus-infected cells. NSP5 interacts with NSP2 and undergoes a complex posttranslational hyperphosphorylation, generating species with reduced PAGE mobility. Here we show that NSP5 operates as an autoregulator of its own phosphorylation as a consequence of two distinct activities of the protein: substrate and activator. We developed an in vivo hyperphosphorylation assay in which two NSP5 mutant constructs are cotransfected. One of them, fused to an 11-aa tag, served as substrate whereas the other was used to map NSP5 domains required for activation. The activation and substrate activity could be uncoupled, demonstrating a hyperphosphorylation process in trans between the activator and substratum. This process involved dimerization of the two components through the 18-aa C-terminal tail. Phosphorylation of Ser-67 within the SDSAS motif (amino acids 63-67) was required to trigger hyperphosphorylation by promoting the activation function. We present evidence of casein kinase 1␣ being the protein kinase responsible for this key step as well as for the consecutive ones leading to NSP5 hyperphosphorylation.
The foot-and-mouth disease virus (FMDV) structural protein precursor, P1-2A, is cleaved by the virus-encoded 3C protease (3C pro ) into the capsid proteins VP0, VP1 and VP3 (and 2A). In some systems, it is difficult to produce large amounts of these processed capsid proteins since 3C pro can be toxic for cells. The expression level of 3C pro activity has now been reduced relative to the P1-2A, and the effect on the yield of processed capsid proteins and their assembly into empty capsid particles within mammalian cells has been determined. Using a vaccinia-virus-based transient expression system, P1-2A (from serotypes O and A) and 3C pro were expressed from monocistronic cDNA cassettes as P1-2A-3C, or from dicistronic cassettes with the 3C pro expression dependent on a mutant FMDV internal ribosome entry site (IRES) (designated P1-2A-mIRES-3C). The effects of using a mutant 3C pro with reduced catalytic activity or using two different mutant IRES elements (the wt GNRA tetraloop sequence GCGA converted, in the cDNA, to GAGA or GTTA) were analysed. For both serotypes, the P1-2A-mIRES-3C construct containing the inefficient GTTA mutant IRES produced the highest amount of processed capsid proteins. These products self-assembled to form FMDV empty capsid particles, which have a related, but distinct, morphology (as determined by electron microscopy and reconstruction) from that determined previously by X-ray crystallography. The assembled empty capsids bind, in a divalent cation-dependent manner, to the RGD-dependent integrin a v b 6, a cellular receptor for FMDV, and are recognized appropriately in serotype-specific antigen ELISAs.
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