We report on the properties and function of two herpes simplex virus-1 (HSV-1) microRNAs (miRNAs) designated “miR-H28” and “miR-H29.” Both miRNAs accumulate late in productive infection at a time when, for the most part, viral DNA and proteins have been made. Ectopic expression of miRNA mimics in human cells before infection reduced the accumulation of viral mRNAs and proteins, reduced plaque sizes, and at vey low multiplicities of infection reduced viral yields. The specificity of the miRNA mimics was tested in two ways. First, ectopic expression of mimics carrying mutations in the seed sequence was ineffective. Second, in similar tests two viral miRNAs made early in productive infection also had no effect. Both miR-H28 and miR-H29 are exported from infected cells in exosomes. A noteworthy finding is that both miR-H28 and miR-H29 were absent from murine ganglia harboring latent virus but accumulated in ganglia in which the virus was induced to reactivate. The significance of these findings rests on the principle that the transmission of HSV from person to person is by physical contact between the infected tissues of the donor and those of uninfected recipient. Diminished size of primary or recurrent lesions could be predicted to enhance person-to-person transmission. Reduction in the amount of reactivating latent virus would reduce the risk of retrograde transport to the CNS but would not interfere with anterograde transport to a site at or near the site of initial infection.

An earlier report showed that herpes simplex virus 1 (HSV-1) expresses two microRNAs (miRNAs), miR-H28 and miR-H29, late in the infectious cycle. The miRNAs are packed in exosomes and, in recipient cells, restrict the transmission of virus from infected cells to uninfected cells. We now report that (i) miR-H28 induced the synthesis of gamma interferon (IFN-γ) in both infected cells and cells transfected with miR-H28, (ii) IFN-γ accumulated concurrently with viral proteins in infected cells, (iii) IFN-γ was produced in HEp-2 cells derived from cancer tissue and in HEK293T cells derived from normal tissue, and (iv) HSV-1 replication was affected by exposure to IFN-γ before infection but not during or after infection. The results presented in this report support the growing body of evidence indicating that HSV-1 encodes functions designed to reduce the spread of infection from infected cells to uninfected cells, possibly in order to maximize the transmission of virus from infected individuals to uninfected individuals. IMPORTANCE In this report, we show that IFN-γ is produced by HSV-1 viral miR-H28 and viral replication is blocked in cells exposed to IFN-γ before infection but not during or after infection. The inevitable conclusion is that HSV-1 induces IFN-γ to curtail its spread from infected cells to uninfected cells. In essence, this report supports the hypothesis that HSV-1 encodes functions that restrict the transmission of virus from cell to cell.

miRNAs are potent tools that in principle can be used to control the replication of infectious agents. The objectives of the studies reported here were to design miRNAs that can block the replication of herpes simplex virus 1 and which could be delivered to infected cells via exosomes. We report the following: (1) We designed three miRNAs targeting the mRNA encoding ICP4, an essential viral regulatory protein. Of the three miRNAs, one miRNA401 effectively blocked ICP4 accumulation and viral replication on transfection into susceptible cells. (2) To facilitate packaging of the miRNA into exosomes, we incorporated into the sequence of miRNA401 an exosome-packaging motif. miRNA401 was shown to be packaged into exosomes and successfully delivered by exosomes to susceptible cells, where it remained stable for at least 72 hr. Finally, the results show that miRNA401 delivered to cells via exosomes effectively reduced virus yields in a miRNA401 dose-dependent fashion. The protocol described in this report can be applied to study viral gene functions without actually deleting or mutagenizing the gene.