An Inhibitory Function Of WW Domain-containing Ost Proteins In (+) RNA Virus Replication

As a model eukaryote, yeast has a small genome with few introns. Yeast also has thehighest percentage characterized genes. Because certain viruses can complete most of thesteps required for intracellular replication in yeast cells (Janda and Ahlquist1993; Naito et al.2007a; Panavas et al.2005b; Panavas and Nagy2003; Pantaleo et al.2003; Raghavan et al.2004), yeast has become a model host to study virus replication. The ww domains in yeastRsp5E3Ubiquitin ligase have been shown to interact with TBSV replication protein p92pol.Overexpression of Rsp5p inhibits TBSV replication through the degradation of p92polby itsww domains (Barajas et al.2009a). This dissertation focuses on the effect of host proteins on(+) RNA virus in yeast model host. Selective plasmids were transformed to yeast to expressthe replication proteins and replication RNAs of (+) RNA viruses Tomato bushy stunt virus(TBSV), Flock house virus (FHV) and Nodamura virus (NoV). The functions of five wwdomain-containing yeast proteins in the replication of TBSV, two insect viruses FHV andNoV were tested in yeast and in vitro. Four Arabidopsis thaliana ww domain-containingproteins were transformed to Nicotiana Benthamiana to test their function in Cucumbernecrosis virus (CNV) replication. Moreover, in vitro pulldown assay and membrane yeasttwo-hybrid (MYTH) assay were performed to test the interaction between wwdomain-containing host proteins and virus replication proteins. The binding domain was alsodetermined.Main results from this research are:(1) In addition to Rsp5p, three yeast ww domain-containing proteins inhibit TBSVreplication in yeast. They are Prp40p, Wwm1p and Ess1p. Overexpression of Rsp5p, Wwm1por Prp40p also inhibits the replication of FHV and NoV.(2) Overexpression of Prp40p or Ess1p inhibits the accumulation of TBSV p92pol.Overexpression of Rsp5p, Prp40p or Wwm1p inhibits the accumulation of FHV and NoVreplication proteins (Protein A, PrtA). They likely function in the same way as Rsp5p, whichinhibits (+) RNA replication by facilitating the degradation of the key replication protein(RNA-dependent RNA polymerase, RdRp).(3) Downregulation of Wwm1, two ww genes (Wwm1and Rsp5, Wwm1and Prp40,Wwm1and Ess1) or several genes (Wwm1, Rsp5and Prp40; Wwm1, Rsp5, Prp40and Ess1) increases TBSV replication and the accumulation of p33and p92pol. From the replicase assayin vitro, downregulation of Wwm1or all the gene combinations leads to higher TBSVreplication. These downregulation experiments suggest that Rsp5p and Wwm1p are importantregulators of TBSV replication, while the other ww domain proteins have lesser effects orthey have redundant function in TBSV replication in yeast.(4) Five yeast ww domain-containing proteins (Rsp5p, Prp40p, Wwm1p, Urn1p andEss1p) interact with TBSV p33and p92pol. The binding domain of Rsp5p and p33C is in theRNA binding domain (termed RPR domain). Moreover, all the five yeast ww proteins interactwith FHV and NoV replication protein PrtA.(5) More ww domains within Rsp5p, more inhibitory function on TBSV replication. Thehighest inhibition appears when there are three ww domains in Rsp5p.(6) Three Arabidopsis ww domain-containing proteins inhibit CNV replication in N.Benthamiana. They are AtDRH1,AtPrp40c (At3g19840) and AtFCA. All of them interactwith CNV replication protein p33.This dissertation studied the inhibitory functions of nine proteins from yeast model hostand Arabidopsis on four (+) RNA viruses. Results show that seven of them inhibit at least one(+) RNA virus replication. The number of ww domains in Rsp5p correlates with the inhibitoryfunction. Protein family members with same domains are studied here to test their effect on (+)RNA replication. This could be an efficient way to identify hose factors which were missed inthe genome-wide and proteomics-wide screens, as a supplement of the genome-wide andproteomics-wide studies, and broaden our understanding on (+) RNA virus replication.