Role of specific innate immune responses in herpes simplex virus infection of the central nervous system

Herpes simplex virus 1 (HSV-1) causes a spectrum of disease, including herpes labialis, herpes keratitis, and herpes encephalitis, which can be lethal. Viral recognition by pattern recognition receptors plays a central role in cytokine production and in the generation of antiviral immunity. The relative contributions of different Toll-like receptors (TLRs) in the innate immune response during central nervous system infection with HSV-1 have not been fully characterized. In this study, we investigate the roles of TLR2, TLR9, UNC93B1, and the type I interferon (IFN) receptor in a murine model of HSV-1 encephalitis. TLR2 is responsible for detrimental inflammatory cytokine production following intracranial infection with HSV-1, and the absence of TLR2 expression leads to increased survival in mice. We prove that inflammatory cytokine production by microglial cells, astrocytes, neutrophils, and monocytes is mediated predominantly by TLR2. We also demonstrate that type I IFNs are absolutely required for survival following intracranial HSV-1 infection, as mice lacking the type I IFN receptor succumb rapidly following infection and have high levels of HSV in the brain. However, the absence of TLR9 does not impact survival, type I IFN levels, or viral replication in the brain following infection. The absence of UNC93B1 leads to a survival disadvantage but does not impact viral replication or type I IFN levels in the brain in HSV-1-infected mice. These results illustrate the complex but important roles that innate immune receptors play in host responses to HSV-1 during infection of the central nervous system.     

Norovirus evolution in immunodeficient mice reveals potentiated pathogenicity via a single nucleotide change in the viral capsid

Interferons (IFNs) are key controllers of viral replication, with intact IFN responses suppressing virus growth and spread. Using the murine norovirus (MNoV) system, we show that IFNs exert selective pressure to limit the pathogenic evolutionary potential of this enteric virus. In animals lacking type I IFN signaling, the nonlethal MNoV strain CR6 rapidly acquired enhanced virulence via conversion of a single nucleotide. This nucleotide change resulted in amino acid substitution F514I in the viral capsid, which led to >10,000-fold higher replication in systemic organs including the brain. Pathogenicity was mediated by enhanced recruitment and infection of intestinal myeloid cells and increased extraintestinal dissemination of virus. Interestingly, the trade-off for this mutation was reduced fitness in an IFN-competent host, in which CR6 bearing F514I exhibited decreased intestinal replication and shedding. In an immunodeficient context, a spontaneous amino acid change can thus convert a relatively avirulent viral strain into a lethal pathogen.     

NK cells require type I IFN receptor for antiviral responses during genital HSV-2 infection

Type I interferon (IFN) signalling, NK cells and NK cell-derived IFN-γ are critical in the early control of genital HSV-2 infection. We have recently reported that NK cells are the source of early IFN-γ in the genital tract in response to HSV-2. However, the response of NK cells to genital HSV-2 infection is not well defined in the context of type I IFN signalling. Here we show that HSV-2 replication was significantly higher in mice deficient in the type I IFN receptor or NK cells compared to wild type controls. There was no detectable IFN-γ production in the genital washes from IFN-α/βR^−/− mice or NK cell depleted mice in response to HSV-2 infection compared to control mice. Absence of the type I IFN receptor does not alter homing of NK cells to the genital mucosa. Moreover, the absence of IL-12 had no significant effect on NK cell-derived IFN-γ. Surprisingly, IFN-α/βR^−/− mice had more IL-15 positive cells in the genital mucosa in response to HSV-2 infection compared to control mice. We then examined the expression of IL-15 receptors on NK cells. There was no significant differences in the levels of IL-15 receptor expression on NK cells from IFN-α/βR^−/− or control mice. Our data clearly suggest that type I IFN receptor signalling is essential for NK cell activation in response to genital HSV-2 infection, and propose that NK cell activation by IL-15 may involve type I IFNs.     

Type I interferon promotes cell‐to‐cell spread of Listeria monocytogenes

Type I interferons (IFNs) play a critical role in antiviral immune responses, but can be deleterious to the host during some bacterial infections. Listeria monocytogenes (Lm) induces a type I IFN response by activating cytosolic antiviral surveillance pathways. This is beneficial to the bacteria as mice lacking the type I IFN receptor (IFNAR1^?/?) are resistant to systemic infection by Lm. The mechanisms by which type I IFNs promote Lm infection are unclear. Here, we show that IFNAR1 is required for dissemination of Lm within infection foci in livers of infected mice and for efficient cell‐to‐cell spread in vitro in macrophages. IFNAR1 promotes ActA polarization and actin‐based motility in the cytosol of host cells. Our studies suggest type I IFNs directly impact the intracellular life cycle of Lm and provide new insight into the mechanisms used by bacterial pathogens to exploit the type I IFN response.     

Noninvasive bioluminescence imaging of herpes simplex virus type 1 infection and therapy in living mice

Mouse models of herpes simplex virus type 1 (HSV-1) infection provide significant insights into viral and host genes that regulate disease pathogenesis, but conventional methods to determine the full extent of viral spread and replication typically require the sacrifice of infected animals. To develop a noninvasive method for detecting HSV-1 in living mice, we used a strain KOS HSV-1 recombinant that expresses firefly (Photinus pyralis) and Renilla (Renilla reniformis) luciferase reporter proteins and monitored infection with a cooled charge-coupled device camera. Viral infection in mouse footpads, peritoneal cavity, brain, and eyes could be detected by bioluminescence imaging of firefly luciferase. The activity of Renilla luciferase could be imaged after direct administration of substrate to infected eyes but not following the systemic delivery of substrate. The magnitude of bioluminescence from firefly luciferase measured in vivo correlated directly with input titers of recombinant virus used for infection. Treatment of infected mice with valacyclovir, a potent inhibitor of HSV-1 replication, produced dose-dependent decreases in firefly luciferase activity that correlated with changes in viral titers. These data demonstrate that bioluminescence imaging can be used for noninvasive, real-time monitoring of HSV-1 infection and therapy in living mice.     

Type I IFN protects against murine lupus

Both the type I (IFN-alpha beta) and type II (IFN-gamma) IFNs have been heavily implicated in the pathogenesis of systemic lupus erythematosus. To test the relative roles of these systems, congenic lupus-prone MRL/CD95(lpr/lpr) (MRL/lpr) mice lacking the type I IFN receptor (IFN-RI), type II IFN receptor (IFN-RII), or both, were derived. As expected, deficiency for IFN-RII protected MRL/lpr mice from the development of significant autoimmune-associated lymphadenopathy, autoantibodies, and renal disease. However, deficiency for the IFN-RI surprisingly worsened lymphoproliferation, autoantibody production, and end organ disease; animals doubly deficient for IFN-RI and IFN-RII developed an autoimmune phenotype intermediate between wild-type and IFN-RII-deficient animals, all correlating with an ability of type I IFN to suppress MRL B cell activation. Thus, type I IFNs protect against both the humoral and end organ autoimmune syndrome of MRL/lpr mice, independent of IFN-gamma. These findings warrant caution in the use of type I IFN antagonists in the treatment of autoimmune diseases and suggest further investigation into the interplay between the types I and II IFNs during the ontogeny of pathogenic autoantibodies.     

Human primary immunodeficiencies of type I interferons

Type I interferons (IFN-alpha/beta and related molecules) are essential for protective immunity to experimental infection by numerous viruses in the mouse model. In recent years, human primary immunodeficiencies affecting either the production of (UNC-93B deficiency) or the response to (STAT1 and TYK2 deficiencies) these IFNs have been reported. Affected patients are highly susceptible to certain viruses. Patients with STAT1 or TYK2 deficiency are susceptible to multiple viruses, including herpes simplex virus-1 (HSV-1), whereas UNC-93B-deficient patients present isolated HSV-1 encephalitis. However, these immunological defects are not limited to type I IFN-mediated immunity. Impaired type II IFN (IFN-gamma)-mediated immunity plays no more than a minor role in the pathogenesis of viral diseases in these patients, but the contribution of impaired type III IFN (IFN-lambda)-mediated immunity remains to be determined. These novel inherited disorders strongly suggest that type I IFN-mediated immunity is essential for protection against natural infections caused by several viruses in humans.     

Interleukin-12- and gamma interferon-dependent innate immunity are essential and sufficient for long-term survival of passively immunized mice infected with herpes simplex virus type 1

Interferon (IFN) type I (alpha/beta IFN [IFN-alpha/beta]) is very important in directly controlling herpes simplex virus type I (HSV-1) replication as well as in guiding and upregulating specific immunity against this virus. By contrast, the roles of IFN type II (IFN-gamma) and antibodies in the defense against HSV-1 are not clear. Mice without a functional IFN system and no mature B and T cells (AGR mice) did not survive HSV-1 infection in the presence or absence of neutralizing antibodies to the virus. Mice without a functional IFN type I system and with no mature B and T cells (AR129 mice) were unable to control infection with as little as 10 PFU of HSV-1 strain F. By contrast, in the presence of passively administered neutralizing murine antibodies to HSV-1, some AR129 mice survived infection with up to 10(4) PFU of HSV-1. This acute immune response was dependent on the presence of interleukin-12 (IL-12) p75. Interestingly, some virus-infected mice stayed healthy for several months, at which time antibody to HSV-1 was no longer detectable. Treatment of these virus-exposed mice with dexamethasone led to death in approximately 40% of the mice. HSV-1 was found in brains of mice that did not survive dexamethasone treatment, whereas HSV-1 was absent in those that survived the treatment. We conclude that in the presence of passively administered HSV-1-specific antibodies, the IL-12-induced IFN-gamma-dependent innate immune response is able to control low doses of virus infection. Surprisingly, in a significant proportion of these mice, HSV-1 appears to persist in the absence of antibodies and specific immunity.     

Dissection of a type I interferon pathway in controlling bacterial intracellular infection in mice

Defence mechanisms against intracellular bacterial pathogens are incompletely understood. Our study characterizes a type I IFN-dependent cell-autonomous defence pathway directed against Legionella pneumophila, an intracellular model organism and frequent cause of pneumonia. We show that macrophages infected with L. pneumophila produced IFNβ in a STING- and IRF3- dependent manner. Paracrine type I IFNs stimulated upregulation of IFN-stimulated genes and a cell-autonomous defence pathway acting on replicating and non-replicating Legionella within their specialized vacuole. Our infection experiments in mice lacking receptors for type I and/or II IFNs show that type I IFNs contribute to expression of IFN-stimulated genes and to bacterial clearance as well as resistance in L. pneumophila pneumonia in addition to type II IFN. Overall, our study shows that paracrine type I IFNs mediate defence against L. pneumophila, and demonstrates a protective role of type I IFNs in in vivo infections with intracellular bacteria.     

Type I interferon response in the central nervous system

This review is dedicated to the influence of type I IFNs (also called IFN-alpha/beta) in the central nervous system (CNS). Studies in mice with type I IFN receptor or IFN-beta gene deficiency have highlighted the importance of the type I IFN system against CNS viral infections and non-viral autoimmune disorders. Direct antiviral effects of type I IFNs appear to be crucial in limiting early spread of a number of viruses in CNS tissues. Type I IFNs have also proved to be beneficial in autoimmune disorders like multiple sclerosis or experimental autoimmune encephalitis, probably through immunomodulatory effects. Increasing efforts are done to characterize IFN expression and response in the CNS: to identify type I IFN producing cells, to decipher pathways leading to type I IFN expression in those cells, and to identify responding cells. However, reversible and irreversible damages consecutive to chronic exposure of the CNS to type I IFNs underline the importance of a tightly regulated type I IFN homeostasis in this organ.     

Selective ablation of virion host shutoff protein RNase activity attenuates herpes simplex virus 2 in mice

The virion host shutoff (vhs) protein of herpes simplex virus (HSV) has endoribonuclease activity and rapidly reduces protein synthesis in infected cells through mRNA degradation. Herpes simplex virus 1 (HSV-1) and HSV-2 vhs mutants are highly attenuated in vivo, but replication and virulence are largely restored to HSV-2 vhs mutants in the absence of a type I interferon (IFN) response. The role of vhs in pathogenesis and the hindrance of the type I IFN response have classically been examined with viruses that completely lack vhs or express a truncated vhs protein. To determine whether RNase activity is the principal mechanism of vhs-mediated type I IFN resistance and virulence, we constructed a HSV-2 point mutant that synthesizes full-length vhs protein lacking RNase activity (RNase(-) virus). Wild-type and mutant HSV-2 vhs proteins coimmunoprecipitated with VP16 and VP22. vhs protein bearing the point mutation was packaged into the virion as efficiently as the wild-type vhs protein. Like a mutant encoding truncated vhs, the RNase(-) virus showed IFN-dependent replication that was restricted compared with that of the wild-type virus. The RNase(-) virus was highly attenuated in wild-type mice infected intravaginally, with reduced mucosal replication, disease severity, and spread to the nervous system comparable to those of the vhs truncation mutant. Surprisingly, in alpha/beta interferon (IFN-alpha/beta) receptor knockout mice, the vhs RNase mutant was more attenuated than the vhs truncation mutant in terms of disease severity and virus titer in vaginal swabs and central nervous system samples, suggesting that non-enzymatically active vhs protein interferes with efficient virus replication. Our results indicate that vhs enzymatic activity plays a complex role in vhs-mediated type I IFN resistance during HSV-2 infection.     

Induction of an Antiviral State and Attenuated Coxsackievirus Replication in Type III Interferon-Treated Primary Human Pancreatic Islets

Type III interferons (IFNs), also called lambda interferons (IFN-λ), comprise three isoforms, IFN-λ1 (interleukin-29 [IL-29]), IFN-λ2 (IL-28A), and IFN-λ3 (IL-28B). Only limited information is available on their expression and biological functions in humans. Type I and type II IFNs protect human pancreatic islets against coxsackievirus infection, and this is important since such viruses have been proposed to play a role in the development of human type 1 diabetes. Here we investigated whether type III IFN is expressed during infection of human islet cells with coxsackievirus and if type III IFN regulates permissiveness to such infections. We show that human islets respond to a coxsackievirus serotype B3 (CVB3) infection by inducing the expression of type III IFNs. We also demonstrate that islet endocrine cells from nondiabetic individuals express the type III IFN receptor subunits IFN-λR1 and IL-10R2. Pancreatic alpha cells express both receptor subunits, while pancreatic beta cells express only IL-10R2. Type III IFN stimulation elicited a biological response in human islets as indicated by the upregulated expression of antiviral genes as well as pattern recognition receptors. We also show that type III IFN significantly reduces CVB3 replication. Our studies reveal that type III IFNs are expressed during CVB3 infection and that the expression of the type III IFN receptor by the human pancreatic islet allows this group of IFNs to regulate the islets'' permissiveness to infection. Our novel observations suggest that type III IFNs may regulate viral replication and thereby contribute to reduced tissue damage and promote islet cell survival during coxsackievirus infection.     

Non-invasive bioluminescence imaging for monitoring herpes simplex virus type 1 hematogenous infection

Traditional studies on viral neuroinvasiveness and pathogenesis have generally relied on murine models that require the sacrifice of infected animals to determine viral distributions and titers. The present paper reports the use of in vivo bioluminescence imaging to monitor the replication and tropism of KOS strain HSV-1 viruses expressing the firefly luciferase reporter protein in hematogenously infected mice. Following intraperitoneal injection, a comparison was made between real-time PCR determinations of HSV-1 DNA concentrations (requiring the sacrifice of the experimental animals) and in vivo bioluminescence emissions in living animals. For further comparison, in vitro light emission was also measured in the ovaries and adrenal glands of sacrificed mice. After infection, HSV-1 spread preferentially to the ovaries and adrenal glands (these organs showed the highest virus levels). Both the PCR and bioluminescence methods detected low viral loads in the nervous system, where the virus was restricted to the spinal cord. The concentrations of viral DNA measured correlated with the magnitude of bioluminescence in vivo, and with the photon flux determined by the in vitro luciferase enzyme assay. The results show that bioluminescence imaging can be used for non-invasive, real-time monitoring of HSV-1 hematogenous infection in living mice, but that coupling this methodology with conventional techniques aids in the characterization of the infection.     

Poxviruses: Interfering with Interferon

Interferon (IFN) is an important innate defense against virus infection and many viruses have consequently evolved ways to interfere with the action of IFN. The poxviruses are an excellent example and devote at least four proteins to this task. Two function within the infected cell to block the action of IFN-induced antiviral proteins, and two are secreted to capture type I and type II IFNs before they can bind to cellular IFN receptors. The vaccinia virus IFN receptors have a surprisingly broad species specificity that may aid virus replication in several species and provide clues about the enigmatic origin of vaccinia virus.     

Oligoadenylate synthetase/protein kinase R pathways and alphabeta TCR+ T cells are required for adenovirus vector: IFN-gamma inhibition of herpes simplex virus-1 in cornea

An adenoviral (Ad) vector containing the murine IFN-gamma transgene (Ad:IFN-gamma) was evaluated for its capacity to inhibit HSV-1. To measure effectiveness, viral titers were analyzed in cornea and trigeminal ganglia (TG) during acute ocular HSV-1 infection. Ad:IFN-gamma potently suppressed HSV-1 replication in a dose-dependent fashion, requiring IFN-gamma receptor. Moreover, Ad:IFN-gamma was effective when delivered -72 and -24 h before infection as well as 24 h postinfection. Associated with antiviral opposition, TG from Ad:IFN-gamma-transduced mice harbored fewer T cells. Also related to T cell involvement, Ad:IFN-gamma was effective but attenuated in TG from alphabeta TCR-deficient mice. In corneas, alphabeta TCR(+) T cells were obligatory for protection against viral multiplication. Type I IFN involvement amid antiviral efficacy of Ad:IFN-gamma was further investigated because types I and II IFN pathways have synergistic anti-HSV-1 activity. Ad:IFN-gamma inhibited viral reproduction in corneas and TG from alphabeta IFNR-deficient (CD118(-/-)) mice, although viral titers were 2- to 3-fold higher in cornea and TG compared with wild-type mice. The absence of IFN-stimulated antiviral proteins, 2'-5' oligoadenylate synthetase/RNase L, and dsRNA-dependent protein kinase R completely eliminated the antiviral effectiveness of Ad:IFN-gamma. Collectively, the results demonstrate the following: 1) nonexistence of type I IFN receptor does not abolish defense of Ad:IFN-gamma against HSV-1; 2) antiviral pathways oligoadenylate synthetase-RNase L and protein kinase R are mandatory; and 3) alphabeta TCR(+) T cells are compulsory for Ad:IFN-gamma effectiveness against HSV-1 in cornea but not in TG.     

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.