Virucidal activity of a GT-rich oligonucleotide against herpes simplex virus mediated by glycoprotein B.
We have investigated the antiviral mechanism of a phosphorothioate oligonucleotide, ISIS 5652, which has activity against herpes simplex virus (HSV) in the low micromolar range in plaque reduction assays. We isolated a mutant that is resistant to this compound. Marker rescue and sequencing experiments showed that resistance was due to at least one of three mutations in the UL27 gene which result in amino acid changes in glycoprotein B (gB). Because gB has a role in attachment and entry of HSV, we tested the effects of ISIS 5652 at these stages of infection. The oligonucleotide potently inhibited attachment of virus to cells at 4 degrees C; however, the resistant mutant did not exhibit resistance at this stage. Moreover, a different oligonucleotide with little activity in plaque reduction assays was as potent as ISIS 5652 in inhibiting attachment. Similarly, ISIS 5652 was able to inhibit entry of pre-attached virions into cells at 37 degrees C, but the mutant did not exhibit resistance in this assay. The mutant did not attach to or enter cells more quickly than did wild-type virus. Strikingly, incubation of wild-type virus with 1 to 2 microM ISIS 5652 at 37 degrees C led to a time-dependent, irreversible loss of infectivity (virucidal activity). No virucidal activity was detected at 4 degrees C or with an unrelated oligonucleotide at 37 degrees C. The resistant mutant and a marker-rescued derivative containing its gB mutations exhibited substantial resistance to this virucidal activity of ISIS 5652. We hypothesize that the GT-rich oligonucleotide induces a conformational change in gB that results in inactivation of infectivity.
Shogan
Benjamin
B
Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Kruse
Lori
L
Mulamba
Gilbert B
GB
Hu
André
A
Coen
Donald M
DM
D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology*; D002522:Chlorocebus aethiops; D018900:Dinucleotide Repeats; D024882:Drug Resistance, Viral / Q000235:genetics; D006147:Guanine / Q000378:metabolism; D009154:Mutation; D009841:Oligonucleotides / Q000378:metabolism* / Q000494:pharmacology*; D018139:Simplexvirus / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development; D013873:Thionucleotides / Q000378:metabolism / Q000494:pharmacology; D013941:Thymine / Q000378:metabolism; D013997:Time Factors; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics / Q000502:physiology*
D000998:Antiviral Agents; D009841:Oligonucleotides; D013873:Thionucleotides; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus; D006147:Guanine; D013941:Thymine