Global quantitative proteomic analysis of human glioma cells profiled host protein expression in response to enterovirus type 71 infection.

Enterovirus 71 (EV71) is one of the leading causes of hand, foot and mouth disease with neurological complications in some cases. To study the pathogenesis of EV71 infection, large-scale analyses of EV71 infected cells have been performed. However, most of these studies employed rhabdomyosarcoma (RD) cells or used transcriptomic strategy. Here, we performed SILAC-based quantitative proteomic analysis of EV71-infected U251 cells, a human glioma cell line. A total of 3125 host proteins were quantified, in which 451 were differentially regulated as a result of EV71 infection at 8 or 20 hpi or both. Gene Ontology analysis indicates the regulated proteins were enriched in "metabolic process", "biological regulation" and "cellular process", implying that these biological processes were affected by EV71 infection. Furthermore, functional study indicated that TRAF2 and TRAF6 among the up-regulated proteins could inhibit the replication of EV71 at the early phase post infection, and the anti-EV71 function of both proteins was independent of interferon ß. Our study not only provided an overview of cellular response to EV71 infection in a human glioma cell line, but also found that TRAF2 and TRAF6 might be potential targets to inhibit the replication of EV71. All MS data have been deposited in the ProteomeXchange with identifier PXD002454 (http://proteomecentral.proteomexchange.org/dataset/PXD002454).

Identification and functional analysis of LsMNPV anti-apoptosis genes.

Three anti-apoptosis genes, Ls-iap2, iap3 and p49 were found in Leucania separata multiple nuclear polyhedrovirus. Amino acid sequence homology of Ls-IAP2 and Ls-IAP3 with Op-IAP2 and Op-IAP3 from Orgyia pseddotsugata MNPV were 20% and 42%, while that of Ls-P49 is 28% with Sl-P49 from Spodoptera littorolis MNPV. Ls-IAP2 contains one baculoviral IAP repeat (BIR) domain followed by a RING domain, while Ls-IAP3 contains two BIRs and a RING. Ls-P49 contains a reactive site loop, predicted cleavage site (KKLD(74) downward arrow G) that is different from Sl-P49 (TVID(94) downward arrow G). Expressed Ls-iap3 or Ls-p49 under presence of actinomycin D in SF9 cells, DNA ladder assay revealed that Ls- IAP3 or Ls-P49 could block the apoptosis of SF9 cells induced by actinomycin D. Replication of p35 deficient-mutant Autographa californica MNPV in SF9 cells was also rescued when Ls-iap3 or Ls-p49 was expressed transiently. No anti-apoptotic activity was observed for Ls-IAP2. The results showed that both of Ls-IAP3 and Ls-P49 were functional apoptotic suppressors in SF9 cells.

Interaction of Heliothis armigera nuclear polyhedrosis viral capsid protein with its host actin.

In order to find the cellular interaction factors of the Heliothis armigera nuclear polyhedrosis virus capsid protein VP39, a Heliothis armigera cell cDNA library was constructed. Then VP39 was used as bait. The host actin gene was isolated from the cDNA library with the yeast two-hybrid system. This demonstrated that VP39 could interact with its host actin in yeast. In order to corroborate this interaction in vivo, the vp39 gene was fused with the green fluorescent protein gene in plasmid pEGFP39. The fusion protein was expressed in the Hz-AM1 cells under the control of the Autographa californica multiple nucleopolyhedrovirus immediate early gene promoter. The host actin was labeled specifically by the red fluorescence substance, tetramethy rhodamine isothicyanete-phalloidin. Observation under a fluorescence microscopy showed that VP39, which was indicated by green fluorescence, began to appear in the cells 6 h after being transfected with pEGFP39. Red actin cables were also formed in the cytoplasm at the same time. Actin was aggregated in the nucleus 9 h after the transfection. The green and red fluorescence always appeared in the same location of the cells, which demonstrated that VP39 could combine with the host actin. Such a combination would result in the actin skeleton rearrangement.