Host protein CD63 enhances viral RNA replication by interacting with human astrovirus nonstructural protein nsP1a/4

wei zhao; Nian liu; Xiao Li Tao; Chun hong Zheng; Xiang yu Li; Man Yu; Yong gang Li

The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/331835 doi: bioRxiv preprint nsP1a/3 (protease), and nsP1a/4; Fig 4B) by RT-PCR. We used the PCR 157 products to construct PEF-HA-1a/1, PEF-HA-1a/2, PEF-HA-1a/3, and HA-1a/4 158 plasmids, which we confirmed by sequencing. We expressed recombinant 159 nsP1a/4-GST fusion protein in bacteria and isolated and purified the fusion The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/331835 doi: bioRxiv preprint overlapped each other (Fig 5D) . That finding confirmed that nsP1a/4 protein 179 interacts with CD63-LEL in HEK293T cells. That result along with those of a 180 previous Y2H screen and GSTpull-down and co-immunoprecipitation assays 181 suggested that nsP1a/4 interacts with the CD63LEL domain. HAstV-1 (MOI 10). We collected lysates from mock-infected (control) and 186 HAstV-infected cells 24h and 48h after infection. We isolated total cellar RNA 187 and quantified the expression of CD63 mRNA using real-time RT-PCR (Fig. 6A ). 188 We also performed western blot to detect CD63 protein expression in the 189 cellular lysates (Fig 6B) . The results showed that the levels of CD63 mRNA and The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/331835 doi: bioRxiv preprint Although the function of HAstVnsP1a remains unclear, nsP1a and nsP1a/4 223 have been suggested to be involved in many processes, including genome 224 replication, apoptosis induction, and capsid maturation (8, 11). To better 225 understand the role of nsP1a in viral replication, we analyzed the interaction of 226 nsP1a with host proteins. A Y2H screen of aCaco-2 cDNA library showed that 227 14 independent proteins, including CD63, interacted with nsP1a.