Interferons are secreted proteins that play an important role in innate immune defense. During viral infection, the antiviral function of IFNs operates through induction of antiviral proteins, such as OAS, PKR and MxA protein, via the JAK/STAT pathway . Activated PKR, formed by binding to dsRNA, phosphorylates the alpha subunit of eIF2α, resulting in inhibition of translation. Many DNA and RNA viruses, such as influenza virus NS1, reovirus σ3, rotavirus NSP3, vaccinia virus E3L and Epstein–Barr virus SM protein, code dsRNA binding proteins and PKR inhibitors . HSV-1 US11, a true late γ2 gene product, is also a PKR inhibitor and is known to inhibit activation of PKR by binding to dsRNA and PKR.
Herpes simplex virus type 1, a double-stranded DNA virus that belongs to the alphaherpesvirinae subfamily, is known to have little susceptibility to IFN . Three virus polypeptides, ICP0, γ34.5 and US11, are reportedly involved in this resistance to IFN. ICP0 suppresses the anti-viral activity of IFN by inhibiting IRF3 and IRF7 functions [4-7] and γ34.5 aids recovery from inhibition of protein synthesis by de-phosphorylating PKR-phosphorylated eIF2 [8-14]. Recently, researchers have reported that degradation of nuclear IFI16 by ICP0 inhibits IRF3 signaling, and that ICP0 interacts with PML and induces its degradation [15-17]. In addition, that US11 reportedly inhibits phosphorylation of PKR by binding to dsRNA, the protein activator of the interferon-induced protein kinase (PACT) and PKR [18-24]. Recently, researchers have reported that US11 can also competitively inhibit activation of OAS by binding to dsRNA .
Acyclovir is a safe and potent anti-herpesvirus drug that has been widely used in the treatment of HSV infection. The majority of ACV-resistant HSV mutants are thymidine kinase deficient (TK−) [26-30]. Leib et al. reported that the reduced replication of TK− mutants in the corneas of wild-type mice is significantly greater in those of IFN receptor knockout mice . Replication of TK− mutant viruses is also readily detectable in the trigeminal ganglia of IFN receptor knock-out mice, but not in those of wild-type mice. Furthermore, the HSV-1 VRTK− strain, isolated in our laboratory as an ACV-resistant mutant, is also a TK-deficient mutant with hyper-sensitivity to type 1 IFN [32-35]. Why the mutation in the enzyme for nucleoside metabolism influences sensitivity to IFN is unclear. Therefore, the mechanism for the hyper-sensitivity of the VRTK− strain to IFN was studied here, with a focus on the function of US11 in mutant-infected cells.
- Top of page
- 1 MATERIALS AND METHODS
- 2 RESULTS
- 3 DISCUSSION
- 4 DISCLOSURE
In this paper, we have shown that a HSV-1 TK-deficient mutant strain, VRTK−, is more sensitive to IFN-α when the host cells are pre-treated with IFN-α prior to infection, but is as sensitive as the VR3 parental strain without pretreatment (Fig. 1). IFN(s) plays important roles in the antiviral immune response and escape from the antiviral activity of IFN is essential for pathogenic viruses. In particular, HSV-1 possesses various strategies for counteracting IFN-induced antiviral responses, namely: (i) HSV-1 suppresses the signaling pathway of IFN through up-regulation of SOCS3 by an unknown pathway  and degradation of nuclear interferon inducible protein 16 (IFI16) by ICP0 inhibits IRF3 signaling [4-7, 15, 38]; and (ii) the PKR pathway plays a major role in the antiviral function of the IFN system. This pathway functions through the sequential phosphorylation of PKR and eIF2α. PKR phosphorylation is induced by binding with virus-induced dsRNA and regulates conversion of PKR from an inactive to an active form. Activated PKR then phosphorylates eIF2α, causing repression of protein synthesis. HSV-1 US11 inhibits the first step, PKR activation, by competitive binding of dsRNA and γ34.5 dephosphorylates phospho-eIF2α, releasing host cells from the antiviral state [11, 12, 19, 21, 45-47]. Based on this knowledge, we focused on US11 and ICP0 to clarify the mechanism for the hyper-sensitivity of the TK-deficient HSV mutant to IFN.
Compared with the wild-type strain, the VRTK, TK− mutant virus, expresses less US11 and has less US11 protein packaged into the tegument of the virus particles, preventing sufficient inhibition of PKR phosphorylation at the early stage of virus infection (Figs. 2, 4 and 5). When the VR3 parent strain is replicated in ACV-treated cells, although the virus expresses less US11, the virion contains a normal amount of US11 protein and these viruses have the same susceptibility to IFN as do viruses replicated in the absence of ACV (Fig. 6). Proteins packaged into the tegument, many of which are late gene products, are thought to be important for control of host cells during the early stage of the virus replication cycle. Therefore, the functions of the tegument proteins should be divided into two distinct phases: the functions that occur in the cells expressing them and those that occur in cells into which they are introduced by infected virions. In this study, we showed US11 derived from the tegument acts as the initial factor in the evasion of IFN in IFN-pretreated cells. γ34.5 mutants with intact US11 are reportedly unable to overcome PKR-mediated shut-off of protein synthesis in cells infected at an MOI of 100 pfu/cell ; however, that study was performed without IFN pretreatment. On the other hand, a γ34.5 recombinant mutant expressing US11 as an immediate-early protein has been found to reverse inhibition of protein synthesis due to IFN pretreatment . This indicates that US11 is required at the early stage of viral replication in IFN-pretreated cells and supports the findings of our study. Virion US11 appears to play an important role in escape from antiviral functions in IFN-pretreated cells. However, it is not important in IFN-untreated cells because HSV up-regulates a cellular factor, SOCS3, which blocks the IFN signal pathway at the early stage of infection. The results of this study, together with those of previous reports, suggest that US11 plays an important role in secondary HSV-1-infected cells that have been exposed to type 1 IFN secreted from primary infected cells or macrophages activated by the HSV-1 antigen (Fig. 8).
Figure 8. Model for inhibition of PKR phosphorylation by US11 incorporated into the virion. In primary infected cells, virus infection and replication lead to activation of IRF3, resulting in IFN-α/β synthesis and release from primary infected cells. Released IFN binds to type I IFN receptors in both an autocrine and paracrine manner and induces PKR as an interferon-stimulating gene via the JACK/STAT pathway. The induced PKR binds to double-stranded RNA and changes to an active state, as shown by the “secondary infected cell” in the figure. US11, transported into the infected cell by the virion, binds to dsRNA and PKR, thus inhibiting PKR phosphorylation. dsRNA, double-stranded RNA; IFNAR, type I IFN receptor.
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Compared to that of VR3, ICP0 protein expression by the VRTK− strain is slightly reduced or delayed without IFN pretreatment; however, pretreatment with IFN leads to a marked reduction in ICP0 expression in the VRTK− strain (Fig. 3a). ICP0 reportedly inhibits expression of IFN-β by degradation of IFI16 and inhibition of IRF3-activation . The antiviral effects of exogenously added IFN are more pronounced in PML+/+ than in PML−/− . PML and Sp100 are both recruited to sites associated with their parental viral genomes and then repress HSV-1 gene expression [50, 51]. ICP0 interacts with PML and its E3 ubiquitin ligase activity induces PML degradation . In IFN-pretreated HSV-1-infected cells, ICP0 may mediate escape from PML-mediated antiviral activity. In this study, the reduction of ICP0 expression in IFN-pretreated VRTK−-infected cells may have been caused by insufficient inhibition of PKR phosphorylation by the reduced amount of virion-derived US11. Furthermore, there were differences between the VR3 and VRTK− strains in the copy numbers of ICP0 mRNA (Fig. 3b). Recently, Sanfilippo and Blaho reported that ICP0 mRNA can trigger the cell death cascade and induce apoptosis . The mechanism of this function is unclear, but the observation suggests a novel field of herpesvirus virology. The question of whether ICP0 mRNA contributes to the escape functions of HSV from the antiviral activity of interferon remains.
Thymidine kinase activities are not essential for virus replication in actively dividing cells in vitro, but are important for the expression of virulence [53, 54]. The low virulence of the TK− mutant has been explained simply by the lower yield caused by the lack of TTP necessary for DNA replication. Leib et al. reported that attenuation of TK− mutants in a mouse model depends on the IFN system . Our observations indicate that the VRTK− strain has a lower tolerance to IFN than does the VR3 parent virus, whereas ACV-treated VR3 virus has the same tolerance to IFN as the untreated virus. VRTK− and ACV-treated VR3 both express less US11, but only the former packages less US11 in virus particles and is hyper-sensitive to IFN. These results indicate that the lower tolerance of the VRTK− strain to IFN is caused by the reduced amount of US11 packaged in the virions. These results also raise questions as to why TK activity influences degree of protein expression by a mechanism other than reduction of DNA replication, and why the VRTK− strain shows abortive packaging of US11 into virus particles. It is probable that the VRTK− strain possesses mutations other than that in the TK gene. Indeed, ectopic viral TK expression does not restore US11 expression and ectopic TK and US11 expression does not improve the susceptibility of the VRTK strain to IFN (Fig. 7). For example, the ICP22 mutant has a reduced level of US11 expression, a smaller amount of US11 in the virion and hyper-sensitivity to IFN [7, 55]. Further studies on the expression of other true late genes, particularly gC and gE, which are involved in escape from the host defense response and in the mechanisms for regulation of tegument protein packaging, are required in TK− mutant-infected cells.
In this study, we showed that the amount of structural protein US11 in the virion controls virus susceptibility to type 1 IFN. Similarly, ICP22-deletion changes the ratio of structural proteins and confers complement-labile characteristics on the virions by lowering the copy number of glycoprotein C therein . Copy numbers of capsid proteins in the virion have been critically defined as 960 copies of VP5 incorporated into one virion [56, 57]. However, the amount of tegument proteins and (glyco)proteins inserted into the envelope per virion is uncertain. Differences in the degree of expression, localization and post-translational modification, such as phosphorylation, of viral proteins in infected cells may influence the balance between virion components, resulting in different virion phenotypes. To better understand the pathogenesis of HSV-1 in various diseases, the relationships between the characteristics of the progeny virus and their components replicated in various tissues should be studied in the future.