| Summary: | 博士 === 國防醫學院 === 生命科學研究所 === 94 === We investigate the restrictive stage of vaccinia virus in HeLa cells using a vaccinia mutant virus (VV-hr) that contains a deletion of 18Kb genome sequences resulting in no growth in HeLa cells. Insertion of CP77 gene into VV-hr generated a recombinant virus (VV-36hr) that multiplied well in HeLa cells. Both viruses could enter cells, initiate viral DNA replication and intermediate gene transcription. However, translation of viral intermediate gene was only detected in cells infected with VV-36hr, indicating that CP77 relieves host restriction at the intermediate gene translation stage in HeLa cells. Apoptosis and phosphorylation of eIF2 were observed in HeLa cells infected with VV-hr. Apoptosis and phosphorylation of eIF2 were reduced in HeLa cells infected with VV-36hr, indicating that CP77 acts upstream of caspase activation and phosphorylation of eIF2. Suppression of apoptosis with the caspase inhibitor and phosphorylation of endogenous eIF2 by expression of a mutant eIF2S51A partially restored viral translation and moderately increased virus growth in HeLa cells.
In yeast two hybrid analyses, CP77 bound to cellular proteins, HMG20A and hRrp42. GST pulldown analyses showed that residues 1-234 of CP77 were sufficient for CP77-HMG20A interaction. After VV-hr virus infection, HMG20A was translocated from the nucleus to viral factories and bound to the viral genome via the HMG box region. In control VV-hr-infected CHO-K1 cells, binding of HMG20A to the viral genome persisted from 2 to 8h p.i.; in contrast, when CP77 was expressed, association of HMG20A with viral genome was transient, with little HMG20A remaining bound at 8 h p.i.. Finally, in cells expressing a CP77 deletion protein ANK5 that did not support vaccinia virus growth and did not contain the HMG20A binding site HMG20A remained bound to viral DNA, demonstrating that the binding of CP77 to HMG20A is essential for its host range function. In summary, our data revealed a novel cellular protein, HMG20A, the dissociation of which from viral DNA is regulated by CP77, providing the first cellular target regulated by viral hr CP77 protein. .
hRrp42, a component of cellular exosomes for RNA degradation, was another CP77 interacting cellular protein. Interaction of CP77 and hRrp42 was detected in vitro and in the infected HeLa cells in vivo. Mutational studies revealed that ankyrin 2/3 domains mediate CP77 binding to hRrp42, an essential feature for its host range activity in HeLa cells. CP77 caused redistribution of hRrp42 to viral transcripts at the proximity of virosomes within cytoplasm and RNAi knockdown of hRrp42 in HeLa cells reduced viral intermediate gene expression with concomitant accumulation of intermediate viral RNA in HeLa cells. Our results suggest that CP77 recruits cellular exosomes to regulate viral intermediate RNA stability in controlling cells permissiveness to vaccinia virus.
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