Short hairpin RNAs specific to human cytomegalovirus terminase subunit pUL89 prevent viral maturation

Abstract

Background

In order to define the role of the human cytomegalovirus (HCMV) small terminase subunit pUL89, analysis by RNA interference was applied.

Methods

Cell lines expressing pUL89-specific short hairpin RNAs (shRNAs) were constructed by transduction of shR-NAs via infection with retroviral vectors. These cell lines were infected with HCMV AD169 and were analysed for pUL89 expression, viral yield, plaque reduction, amount of viral DNA and particle formation.

Results

After infection of the cell lines with HCMV, the expression of pUL89 was reduced by up to 86% for shRNA_A and 84% for shRNA_B at the later time points of infection. Cell lines expressing shRNA_C and the control had no effect on the pUL89 expression level. In addition, the inhibitory effect corresponded to a decrease in viral growth kinetics, viral DNA and plaque formation. Analysis by electron microscopy demonstrated that infection of cells expressing pUL89-specific shRNA_A and shRNA_B resulted in a complete inhibition of viral particle formation.

Conclusions

HCMV is a serious life-threatening opportunistic pathogen in immunocompromised patients. Because of multiple problems caused by the current available drugs, development of new strategies are needed. Our data clearly demonstrate that pUL89-specific shRNAs mediated the inhibition of formation of replicative infectious particles and therefore represent a new promising mechanism for antiviral therapy against HCMV infection.