Mutations Inactivating Herpes Simplex Virus 1 MicroRNA miR-H2 Do Not Detectably Increase ICP0 Gene Expression in Infected Cultured Cells or Mouse Trigeminal Ganglia

Abstract

Herpes simplex virus 1 (HSV-1) latency entails the repression of productive ("lytic") gene expression. An attractive hypothesis to explain some of this repression involves inhibition of the expression of ICP0, a lytic gene activator, by a viral microRNA, miR-H2, which is completely complementary to ICP0 mRNA. To test this hypothesis, we engineered mutations that disrupt miR-H2 without affecting ICP0 in HSV-1. The mutant virus exhibited drastically reduced expression of miR-H2 but showed wild-type levels of infectious virus production and no increase in ICP0 expression in lytically infected cells, which is consistent with the weak expression of miR-H2 relative to the level of ICP0 mRNA in that setting. Following corneal inoculation of mice, the mutant was not significantly different from wild-type virus in terms of infectious virus production in the trigeminal ganglia during acute infection, mouse mortality, or the rate of reactivation from explanted latently infected ganglia. Critically, the mutant was indistinguishable from wild-type virus for the expression of ICP0 and other lytic genes in acutely and latently infected mouse trigeminal ganglia. The latter result may be related to miR-H2 being less effective in inhibiting ICP0 expression in transfection assays than a host microRNA, miR-138, which has previously been shown to inhibit lytic gene expression in infected ganglia by targeting ICP0 mRNA. Additionally, transfected miR-138 reduced lytic gene expression in infected cells more effectively than miR-H2. While this study provides little support for the hypothesis that miR-H2 promotes latency by inhibiting ICP0 expression, the possibility remains that miR-H2 might target other genes during latency. IMPORTANCE Herpes simplex virus 1 (HSV-1), which causes a variety of diseases, can establish lifelong latent infections from which virus can reactivate to cause recurrent disease. Latency is the most biologically interesting and clinically vexing feature of the virus. Ever since miR-H2's discovery as a viral microRNA bearing complete sequence complementarity to the mRNA for the important viral gene activator ICP0, inhibition of ICP0 expression by miR-H2 has been a major hypothesis to help explain the repression of lytic gene expression during latency. However, this hypothesis remained untested in latently infected animals. Using a miR-H2-deficient mutant virus, we found no evidence that miR-H2 represses the expression of ICP0 or other lytic genes in cells or mice infected with HSV-1. Although miR-H2 can repress ICP0 expression in transfection assays, such repression is weak. The results suggest that other mechanisms for miR-H2 activity and for the repression of lytic gene expression during latency deserve investigation.

Herpes Simplex Virus 1 Deregulation of Host MicroRNAs

“…Similarly, miR-H3 and H4 are antisense to ICP34.5, the main neurovirulence factor. However, although the ICP0 transcript is an obvious target of miR-H2, and although several lines of evidence indicate their interplay (e.g., confirmed specific targeting [44] and interaction identified by ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) [52]), virus mutants of the HSV-1 strain KOS lacking the miR-H2 expression did not show a significant difference in the expression of ICP0 or replication, compared to wild-type (wt) in cultured cells [52,53] or in a mouse model [53]. Then again, a mutant virus with disrupted expression of miR-H2 in the virulent wt strain (McKrae) showed an increased expression of ICP0 during productive infection and increased virulence and rate of reactivation [54,55].…”

“…It is important to note, as described above, that the ICP0 transcript also contains two binding sites for the virus-encoded miR-H2, and that mutations of miR-H2 also showed an increased neurovirulence in strain McKrae [54,55], but the phenotype was not statistically significant in strain KOS [53]. It would be interesting to learn if mutations in both miR-H2 and binding sites for miR-138 would additionally accentuate the observed phenotype.…”

Toll‐like receptors as novel therapeutic targets for herpes simplex virus infection

“…Epithelial or mucosal cells are the primary targets of initial infection after which the virus can form latent infection in sensory ganglia . There is a role for noncoding short RNAs, namely, microRNAs (miRNAs) in HSV latency, because lytic gene expression, IC50 is suppressed by miR‐H2, which is completely complementary to ICP0 mRNA . These small sequences are able to regulate the process of gene expression through direct binding of the coding mRNA sequences and further translational impairment …”

“…Earlier studies recognized numerous HSV‐encoded functions that impede antiviral host immunity including ICP0‐mediated suppression of cytokine/interferon response, nonspecific degradation of host mRNA by the virion host shut‐off (VHS) RNase, inhibition of PKR by US11 and γ34.5, inhibition of MHC‐I peptide loading by ICP47, and modulation of TLRs . In addition, recent data show that HSV1 incorporates a human protein, the DEAD‐box ATP‐dependent RNA helicase (DDX3X) to stimulate HSV1 gene expression and, consequently, virion assembly without inducing interferon production .…”

Viral Ubiquitin Ligase Stimulates Selective Host MicroRNA Expression by Targeting ZEB Transcriptional Repressors

“…In the case of the prototype α-herpesvirus, herpes simplex virus 1 (HSV-1, human alphaherpesvirus 1), several viral miRNAs are preferentially expressed in latency, a persistent but largely silent infection restricted to neurons, while others are preferentially expressed during productive (lytic) infection [ 8 , 9 , 10 , 11 ]. Interestingly, one HSV-1 miRNA, miR-H2, and host miRNA, miR-138, both target a key viral regulatory factor, ICP0, with the host miRNA being a more effective suppressor in both cultured cells and ganglia of infected mice [ 12 , 13 , 14 , 15 ]. Additional examples of cooperation between viral and host miRNAs to benefit viral replication or latency have been described for other herpesviruses, including human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV) [ 16 , 17 , 18 ].…”