Applications of protein microarray technology in the study of virus infections and host-virus interactions.

摘要

Virus infections are major topics for study by both biological and medical researchers due to their public health importance. The protein microarray technology has recently developed into an advanced biological and medical tool. My thesis projects explore the application of this novel technology in the context of virus infections.;Herpesviruses are a clinically important DNA virus family that maintains a lifelong latency in host cells. Herpesviruses encode protein kinases that are conserved across the three sub-families (EBV: BGLF4; KSHV: ORF36; CMV: UL97; HSV: UL13). The production of these kinases during the lytic cycle facilitates viral replication by phosphorylating both viral and host substrates. Using a recently fabricated Epstein-Barr virus (EBV) protein microarray, the viral protein substrates for the EBV-encoded serine/threonine protein kinase BGLF4 were systematically evaluated. Most of identified substrates were lytic proteins while one of the substrates was the latent protein, EBNA1. Further studies showed that BGLF4 not only phosphorylated but also interacted with EBNA1, and was recruited to the EBV latency replication origin oriP. Such recruitment of BGLF4 reduced EBV latent genome replication. I also screened the human protein microarrays to identify host proteins that are the substrates of BGLF4 and other homologs (ORF36, U197 and UL13).;Using yeast proteome microarrays, the yeast proteins preferentially binding to a small RNA hairpin that contains a clamped adenine motif (CAM) from Brome Mosaic Virus (BMV) were identified at a proteome-wide scale. BMV is an RNA plant virus that can replicate in yeast. Several hits were selected for further characterization in the plant Nicotiana benthamiana. Over-expression of host proteins, Pseudouridine Synthase 4 (Pus4) and the Actin Patch Protein 1 (App1), dramatically prevented systemic spread in the infected plants. Further studies showed that Pus4 prevented the encapsidation of BMV RNA in plants and the assembly of BMV virions in vitro. I also used the same strategy with the human protein microarrays to identify host proteins which bind to the EBV encoded small RNAs (EBERs).;In conclusion, protein microarrays containing either the host or the virus proteins serve as a useful platform to study host-virus interactions.