Antiviral effect of an oligo(nucleoside methylphosphonate) complementary to the splice junction of herpes simplex virus type 1 immediate early pre-mRNAs 4 and 5.

Abstract

Selective inhibition of regulatory immediate early (IE) genes of herpes simplex virus type 1 (HSV-1) should inhibit virus growth. Treatment of HSV-1-infected cells with the oligo(nucleoside methylphosphonate) d(TpCCTCCTG) (deoxynucleoside methylphosphonate residues in italic), which is complementary to the acceptor splice junction of HSV-1 IE pre-mRNA 4 and 5, before (1-24 hr) or at the time of infection caused a dose-dependent inhibition in virus replication. Virus titers were decreased 50% and 90% in cells treated with 25 ILM and 75 ,uM oligomer, respectively; at 300 tsM, a 99% reduction in virus production was observed. Viral DNA synthesis was reduced 70-75% and there was a 90% reduction in synthesis of viral proteins, including other LE species and viral functional (130-kDa major DNA-binding) and structural (glycoprotein gB) proteins. In the same concentration range, d(TpCCTC-CTG) caused a minimal reduction (0-30%) in protein synthesis and growth rates (<40%) of uninfected cells. The data suggest that oligo(nucleoside methylphosphonate)s may be effective in antiviral chemotherapy.Numerous nucleoside or nucleotide analogues have been screened for antiviral activity (1). Their mode of action exploits differences in the specificity of viral versus host enzymes, thus affecting differentially the rate of viral as compared to host-cell nucleic acid synthesis. However, with rare exceptions, most of them are not effective in experimentally infected animals (2).An alternative approach to antiviral chemotherapy is the use of sequence-specific oligonucleotides or their analogues to selectively inhibit viral gene expression at the level of mRNA processing or translation. For this purpose we have developed sequence-specific nonionic nucleic acid analogues that contain a 3'-5' methylphosphonate group in place of the negatively charged phosphodiester group normally found in oligonucleotides (3). These analogues are resistant to nuclease hydrolysis and penetrate mammalian cells in culture (4). An oligo(deoxyribonucleoside methylphosphonate) complementary to the Shine-Dalgarno sequence of 16S ribosomal RNA inhibits protein synthesis in Escherichia coli, but not in mammalian cells (5). Oligomers complementary to the initiation codon regions ofrabbit globin mRNA inhibit translation in a cell-free system and in rabbit reticulocytes (6), while oligomers complementary to the initiation codon regions of vesicular stomatitis virus mRNAs inhibit viral but not cellular protein synthesis in infected mouse L cells (7).To explore the possibility that control of gene expression by methylphosphonates could be an effective antiviral modality, we synthesized an oligo(nucleoside methylphosphonate) [d(TpCCTCCTG); deoxynucleoside methylphosphonate residues in italic] that is complementary to the acceptor splice junction of herpes simplex virus type 1 (HSV-1) immediate early (IE) mRNAs 4 and 5 (8). The rationale for this choice is based on previous findings indicating that (i) HSV proteins form at least three kinetic groups [IE(a); earl...

UV-induced early-domain binding factor as the limiting component of simian virus 40 DNA amplification in rodent cells.

“…The uptake of single-and double-stranded oligodeoxyribonucleotides by cells has been shown previously. For instance, probes complementary to the splice junction of herpes simplex virus (HSV) immediate-early pre-mRNAs specifically inhibited HSV protein synthesis, replication, and viral growth in Vero cells (37). Antisense oligonucleotides to the common miniexon structure of trypanosome mRNAs killed cultured trypanosomes (44).…”

Partial protection of oncogene, anti-sense oligodeoxynucleotides against serum nuclease degradation using terminal methylphosphonate groups

“…In the present work we have determined the relative extent to which 3'phosphodiesterase, 5'-phosphodiesterase and endonucleases present in fetal calf serum contribute to breakdown of oligodeoxynucleotides by using preparations of an N-ras anti-sense sequence (Figure 1) blocked at both the 5' and 3' ends or just the 3' terminus with two methylphosphonate diester linkages (Smith et al, 1986). In addition, piperidine catalysed hydrolysis of the parent chimeric molecules (Miller et al, 1983;Murakami et al, 1985) provided a convenient source of oligodeoxynucleotides with which to evaluate the protective effects of methylphosphonate monoester end groups (Figure 1).…”

5′-,3′-Inverted Thymidine-modified Antisense Oligodeoxynucleotide Targeting Midkine

“…PS-modified ODNs are considerably more stable in vivo (6,37). Any modified ODN that would be useful as an antisense agent should show reasonable stability against nucleases as well as acceptable hybridization with the target mRNA.…”