Herpes simplex virus type 2 growth and latency reactivation by cocultivation are inhibited with antisense oligonucleotides complementary to the translation initiation site of the large subunit of ribonucleotide reductase (RR1)

Antisense oligonucleotides complementary to the translation initiation site of the herpes simplex virus type 2 (HSV-2) large subunit of ribonucleotide reductase (RR1) were studied for their ability to inhibit RR1 expression, HSV-2 growth, and its reactivation from latently infected ganglia. The oligomers caused a significant decrease (90%-97% inhibition) in HSV-2 RR1 expression and inhibited HSV-2 growth, with IC50 and IC90 values of 0.11 and 1.0 microM, respectively. The titers of HSV-2 mutants that are respectively deleted in the PK (ICP10deltaPK) or RR (ICP10deltaRR) domains of RR1 were also significantly (500-20,000-fold) decreased, indicating that the antisense oligomers interfere with the independent contributions of the two RR1 functions (PK and RR) toward virus growth. Inhibition was sequence specific, as evidenced by the failure of a two-base mutant (RR1TImu) to inhibit protein expression and HSV-2 growth. Furthermore, the antisense oligomers inhibited HSV-2 reactivation by cocultivation of latently infected ganglia (0/8). Virus was reactivated from ganglia cultured without oligomers, in the presence of unrelated oligomers (6/8), or in the presence of the two-base mutant RR1TImu (5/8) (p < 0.007 by two-tailed Fisher exact test). HSV-2 growth was not inhibited by antisense oligonucleotides complementary to the splice junction of HSV-2 immediate-early (IE) pre-mRNA 4 and 5 (IE4,5SA) or the translation initiation site of IE mRNA 4 (IE4TI), although the respective HSV-1-specific oligomers inhibit HSV-1 growth.     

The alpha protein ICP0 does not appear to play a major role in the regulation of herpes simplex virus gene expression during infection in tissue culture.

The herpes simplex virus type 1 (HSV-1) alpha protein ICP0 trans-activates HSV-1 early genes in transient expression assays. To investigate the function of ICP0 during HSV-1 infection, we have lowered the level of ICP0 by use of a recombinant plasmid that has been engineered to express the antisense message. Cell lines were constructed which stably carry the antisense plasmid. Total protein profiles from infected antisense cell lines showed that the level of ICP0 was reduced to less than 10% of the wild type level in two of the cell lines. However, reducing the level of ICP0 did not have a significant effect on the expression of HSV-1 early or late genes. The polypeptide patterns for the remaining infected cell polypeptides were similar in that no bands were absent although there were some quantitative differences. The level of two early proteins, glycoprotein B and glycoprotein D was reduced in one of the cell lines, however, levels were nearly equivalent to the control infection for two other cell lines tested. Virus yields were the same for the antisense cell lines and for parent cells. Decreased ICP0 levels did not lead to more restrictive phenotypes for an alpha 4 or alpha 27 mutant as protein patterns were similar for these mutants in antisense and parent cells. Therefore, while ICP0 has been demonstrated to be a strong inducer of gene expression in transient expression assays, it does not appear to have a major role as an activator during the productive infection of tissue culture cells.     

Host MOV10 is induced to restrict herpes simplex virus 1 lytic infection by promoting type I interferon response

Moloney leukemia virus 10 protein (MOV10) is an interferon (IFN)-inducible RNA helicase implicated in antiviral activity against RNA viruses, yet its role in herpesvirus infection has not been investigated. After corneal inoculation of mice with herpes simplex virus 1 (HSV-1), we observed strong upregulation of both MOV10 mRNA and protein in acutely infected mouse trigeminal ganglia. MOV10 suppressed HSV-1 replication in both neuronal and non-neuronal cells, and this suppression required the N-terminus, but not C-terminal helicase domain of MOV10. MOV10 repressed expression of the viral gene ICP0 in transfected cells, but suppressed HSV-1 replication independently of ICP0. MOV10 increased expression of type I IFN in HSV-1 infected cells with little effect on IFN downstream signaling. Treating the cells with IFN-α or an inhibitor of the IFN receptor eliminated MOV10 suppression of HSV-1 replication. MOV10 enhanced IFN production stimulated by cytoplasmic RNA rather than DNA. IKKε co-immunoprecipitated with MOV10 and was required for MOV10 restriction of HSV-1 replication. Mass spectrometry identified ICP27 as a viral protein interacting with MOV10. Co-immunoprecipitation results suggested that this interaction depended on the RGG box of ICP27 and both termini of MOV10. Overexpressed ICP27, but not its RGG-Box deletion mutant, rendered MOV10 unable to regulate HSV-1 replication and type I IFN production. In summary, MOV10 is induced to restrict HSV-1 lytic infection by promoting the type I IFN response through an IKKε-mediated RNA sensing pathway, and its activity is potentially antagonized by ICP27 in an RGG box dependent manner.