RNase P ribozyme inhibits cytomegalovirus replication by blocking the expression of viral capsid proteins.
By linking a guide sequence to the catalytic RNA subunit of RNase P (M1 RNA), we constructed a functional ribozyme (M1GS RNA) that targets the overlapping mRNA region of two human cytomegalovirus (HCMV) capsid proteins, the capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. The ribozyme efficiently cleaved the target mRNA sequence in vitro. Moreover, a reduction of >85% in the expression of CSP and assemblin and a reduction of 4000-fold in viral growth were observed in the HCMV-infected cells that expressed the functional ribozyme. In contrast, there was no significant reduction in viral gene expression and growth in virus-infected cells that either did not express the ribozyme or produced a 'disabled' ribozyme carrying mutations that abolished its catalytic activity. Characterization of the effects of the ribozyme on the HCMV lytic replication cycle further indicates that the expression of the functional ribozyme specifically inhibits the expression of CSP and assemblin, and consequently blocks viral capsid formation and growth. Our results provide the direct evidence that RNase P ribozymes can be used as an effective gene-targeting agent for antiviral applications, including abolishing HCMV growth by blocking the expression of the virus-encoded capsid proteins.
Kim
Kihoon
K
Program in Infectious Diseases and Immunity, Program in Comparative Biochemistry, School of Public Health, 140 Warren Hall, University of California, Berkeley, CA 94720, USA.
Trang
Phong
P
Umamoto
Sean
S
Hai
Rong
R
Liu
Fenyong
F
D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D002213:Capsid / Q000502:physiology; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003587:Cytomegalovirus / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D043262:Ribonuclease P / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects
D000998:Antiviral Agents; D036022:Capsid Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D043262:Ribonuclease P; D012697:Serine Endopeptidases; C071910:assemblin