RNase P-mediated inhibition of cytomegalovirus protease expression and viral DNA encapsidation by oligonucleotide external guide sequences

Abstract

External guide sequences (EGSs) are oligonucleotides that consist of a sequence complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. In this study, DNA-based EGS molecules were chemically synthesized to target the mRNA coding for the protease of human cytomegalovirus (HCMV). The EGS molecules efficiently directed human RNase P to cleave the target mRNA sequence in vitro. When EGSs were exogenously administered into HCMV-infected human foreskin fibroblasts, a reduction of about 80 -90% in the expression level of the protease and a reduction of about 300-fold in HCMV growth were observed in the cells that were treated with a functional EGS, but not in cells that were not treated with the EGS or with a ''disabled'' EGS carrying nucleotide mutations that precluded RNase P recognition. Moreover, packaging of the viral DNA genome into the capsid was blocked in the cells treated with the functional EGS. These results indicate that HCMV protease is essential for viral DNA encapsidation. Moreover, our study provides direct evidence that exogenous administration of a DNAbased EGS can be used as a therapeutic approach for inhibiting gene expression and replication of a human virus.

Human Cytomegalovirus Primase UL70 Specifically Interacts with Cellular Factor Snapin

“…Cells that were grown on glass coverslips were cotransfected with DNAs of the different constructs with the aid of Lipofectamine (Invitrogen) (11). At 48 h posttransfection, cells were either mock infected or infected with HCMV at a multiplicity of infection (MOI) of 1 to 5.…”

“…Nuclear staining was performed with 4Ј,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Images were captured with a Nikon Eclipse TE300 microscope using a Spot RT Slider camera and imaging software (Diagnostic Instruments, Inc., Sterling Heights, MI) (11). The digital images were subsequently merged using the SimplePCI software program (Compix Inc., Sewickley, PA).…”

Cytomegalovirus Assemblin (pUL80a): Cleavage at Internal Site Not Essential for Virus Growth; Proteinase Absent from Virions

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Herpesviruses encode a proteinase that functions during assembly and maturation of the capsid and that is essential for the production of infectious virus (13,17,24,31,41,42,53,55). The human cytomegalovirus (HCMV) homolog is synthesized as an enzymatically active 74-kDa precursor that cleaves itself at two consensus sequences called the maturational (M) and release (R) sites (Fig.

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Modulation of the Cellular Distribution of Human Cytomegalovirus Helicase by Cellular Factor Snapin

“…Cells (grown on glass coverslips) were cotransfected with DNAs of the different constructs and at 48 h posttransfection were either mock infected or infected with HCMV at an MOI of 1 to 5 (34). At different time points postinfection, cells were fixed with 4% formaldehyde, blocked with 0.1% bovine serum albumin and 0.1% sodium azide in PBS, and stained with primary antibodies, followed by incubation with secondary antibody Alexa Fluor 555 antimouse IgG or Alexa Fluor 488 anti-rabbit IgG (Invitrogen).…”