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.     

Enhanced pathogenesis of an attenuated herpes simplex virus for mice lacking Stat1

Mice lacking the Stat1 interferon signaling gene were infected with herpes simplex virus type 1 (HSV-1) or an attenuated recombinant lacking virion host shutoff (Delta vhs). Delta vhs virus-infected Stat1(-/-) mice showed levels of replication equivalent to that of the wild-type virus-infected control mice but reduced relative to wild-type virus-infected Stat1(-/-) mice. Stat1 deficiency relieves the immunomodulatory deficiency of Delta vhs virus, but not its inherent growth defect. Also Vhs is dispensable for reactivation.     

Efficient reactivation of latent herpes simplex virus from mouse central nervous system tissues

For decades, numerous ex vivo studies have documented that latent herpes simplex virus (HSV) reactivates efficiently from ganglia, but rarely from the central nervous systems (CNS), of mice when assayed by mincing tissues before explant culture, despite the presence of viral genomes in both sites. Here we show that 88% of mouse brain stems reactivated latent virus when they were dissociated into cell suspensions before ex vivo explant culture. The efficient reactivation of HSV from the mouse CNS was demonstrated with more than one viral strain, viral serotype, and mouse strain, further indicating that the CNS can be an authentic latency site for HSV with the potential to cause recurrent disease.     

Selective spread of herpes simplex virus in the central nervous system after ocular inoculation.

The spread of herpes simplex virus (HSV) was studied in the mouse central nervous system (CNS) after ocular inoculation. Sites of active viral replication in the CNS were identified by autoradiographic localization of neuronal uptake of tritiated thymidine. Labeled neurons were first noted in the CNS at 4 days postinoculation in the Edinger-Westphal nucleus, ipsilateral spinal trigeminal nucleus, pars caudalis, pars interpolaris, and ipsilateral dorsal horn of the rostral cervical spinal cord. By 5 days postinoculation, additional sites of labeling included the seventh nerve nucleus, nucleus locus coeruleus, and the nuclei raphe magnus and raphe pallidus. None of these sites are contiguous to nuclei infected at 4 days, but all are synaptically related to these nuclei. By 7 days postinoculation, no new foci of labeled cells were noted in the brain stem, but labeled neurons were noted in the amygdala, hippocampus, and somatosensory cortex. Neurons in both the amygdala and hippocampus receive axonal projections from the locus coeruleus. On the basis of these findings, we conclude that the spread of HSV in the CNS after intracameral inoculation is not diffuse but is restricted to a small number of noncontiguous foci in the brain stem and cortex which become infected in a sequential fashion. Since these regions are synaptically related, the principal route of the spread of HSV in the CNS after ocular infection appears to be along axons, presumably via axonal transport rather than by local spread.     

Molecular biology of herpes simplex virus type 1 latency in the nervous system

Herpes simplex virus (HSV) is one of the best studied examples of viral ability to remain latent in the human nervous system and to cause recurrent disease by reactivation. Intensive effort was directed in recent years to unveil the molecular viral mechanisms and the virus-host interactions associated with latent HSV infection. The discovery of the state of the latent viral DNA in nervous tissues and of the presence of latency-associated gene expression during latent infection, both differing from the situation during viral replication, provided important clues relevant to the pathogenesis of latent HSV infection. This review summarizes the current state of knowledge on the site of latent infection, the molecular phenomena of latency, and the mechanisms of the various stages of latency: acute infection, establishment and maintenance of latency, and reactivation. This information paved the way to recent trials aiming to use herpes viruses as vectors to deliver genes into the nervous system, an issue that is also addressed in this review.     

Involvement of apolipoprotein E in the hematogenous route of herpes simplex virus type 1 to the central nervous system

Apolipoprotein E (ApoE), a constituent of the lipoproteins, may be relevant in herpes simplex virus type 1 (HSV-1) infection of the central nervous system (CNS), since HSV-1 binds to human serum ApoE lipoproteins. This study demonstrates the involvement of ApoE in the hematogenous route of HSV-1 to the CNS.     

Inflammatory infiltration of the trigeminal ganglion after herpes simplex virus type 1 corneal infection.

Following herpes simplex virus type 1 (HSV-1) infection of the cornea, the virus is transmitted to the trigeminal ganglion, where a brief period of virus replication is followed by establishment of a latent infection in neurons. A possible role of the immune system in regulating virus replication and maintaining latency in the sensory neurons has been suggested. We have investigated the phenotype and cytokine pattern of cells that infiltrate the A/J mouse trigeminal ganglion at various times after HSV-1 corneal infection. HSV antigen expression in the trigeminal ganglion (indicative of the viral lytic cycle) increased until day 3 postinfection (p.i.) and then diminished to undetectable levels by day 7 p.i. The period of declining HSV antigen expression. was associated with a marked increase in Mac-1+ cells. These cells did not appear to coexpress the F4/80+ (macrophage) or the CD8+ (T cell) markers, and none showed polymorphonuclear leukocyte morphology, suggesting a possible early infiltration of natural killer cells. There was also a significant increase in the trigeminal ganglion of cells expressing the gamma delta T-cell receptor, and these cells were found almost exclusively in very close association with neurons. This period was also characterized by a rapid and equivalent increase in cells expressing gamma interferon and interleukin-4. The density of the inflammatory infiltrate in the trigeminal ganglion increased until days 12 to 21 p.i., when it was predominated by CD8+, Mac-1+, and tumor necrosis factor-expressing cells, which surrounded many neurons. By day 92 p.i., the inflammatory infiltrate diminished but was heaviest in mice with active periocular skin disease. Our data are consistent with the notion that gamma interferon produced by natural killer cells and/or gamma delta T cells may play an important role in limiting HSV-1 replication in the trigeminal ganglion during the acute stage of infection. In addition, tumor necrosis factor produced by CD8+ T cells and macrophages may function to maintain the virus in a latent state.     

Development and migration of protective CD8+ T cells into the nervous system following ocular herpes simplex virus-1 infection

After infection of epithelial surfaces, HSV-1 elicits a multifaceted antiviral response that controls the virus and limits it to latency in sensory ganglia. That response encompasses the CD8(+) T cells, whose precise role(s) is still being defined; immune surveillance in the ganglia and control of viral spread to the brain were proposed as the key roles. We tracked the kinetics of the CD8(+) T cell response across lymphoid and extralymphoid tissues after ocular infection. HSV-1-specific CD8(+) T cells first appeared in the draining (submandibular) lymph node on day 5 and were detectable in both nondraining lymphoid and extralymphoid tissues starting on day 6. However, although lymphoid organs contained both resting (CD43(low)CFSE(high)) and virus-specific cells at different stages of proliferation and activation, extralymphoid sites (eye, trigeminal ganglion, and brain) contained only activated cells that underwent more than eight proliferations (CD43(high)CFSE(neg)) and promptly secreted IFN-gamma upon contact with viral Ags. Regardless of the state of activation, these cells appeared too late to prevent HSV-1 spread, which was seen in the eye (from day 1), trigeminal ganglia (from day 2), and brain (from day 3) well before the onset of a detectable CD8(+) T cell response. However, CD8(+) T cells were critical in reducing viral replication starting on day 6 and for its abrogation between days 8 and 10; CD8-deficient animals failed to control the virus, exhibited persisting high viral titers in the brain after day 6, and died of viral encephalitis between days 7 and 12. Thus, CD8(+) T cells do not control HSV-1 spread from primary to tertiary tissues, but, rather, attack the virus in infected organs and control its replication in situ.     

The immune response to herpes simplex virus type 1 infection in susceptible mice is a major cause of central nervous system pathology resulting in fatal encephalitis

This study was undertaken to investigate possible immune mechanisms in fatal herpes simplex virus type 1 (HSV-1) encephalitis (HSE) after HSV-1 corneal inoculation. Susceptible 129S6 (129) but not resistant C57BL/6 (B6) mice developed intense focal inflammatory brain stem lesions of primarily F4/80(+) macrophages and Gr-1(+) neutrophils detectable by magnetic resonance imaging as early as day 6 postinfection (p.i.). Depletion of macrophages and neutrophils significantly enhanced the survival of infected 129 mice. Immunodeficient B6 (IL-7R(-/-) Kit(w41/w41)) mice lacking adaptive cells (B6-E mice) and transplanted with 129 bone marrow showed significantly accelerated fatal HSE compared to B6-E mice transplanted with B6 marrow or control nontransplanted B6-E mice. In contrast, there was no difference in ocular viral shedding in B6-E mice transplanted with 129 or B6 bone marrow. Acyclovir treatment of 129 mice beginning on day 4 p.i. (24 h after HSV-1 first reaches the brain stem) reduced nervous system viral titers to undetectable levels but did not alter brain stem inflammation or mortality. We conclude that fatal HSE in 129 mice results from widespread damage in the brain stem caused by destructive inflammatory responses initiated early in infection by massive infiltration of innate cells.     

A functional role for TorsinA in herpes simplex virus 1 nuclear egress

Herpes simplex virus 1 (HSV-1) capsids leave the nucleus by a process of envelopment and de-envelopment at the nuclear envelope (NE) that is accompanied by structural alterations of the NE. As capsids translocate across the NE, transient primary enveloped virions form in the perinuclear space. Here, we provide evidence that torsinA (TA), a ubiquitously expressed ATPase, has a role in HSV-1 nuclear egress. TA resides within the lumen of the endoplasmic reticulum (ER)/NE and functions in maintaining normal NE architecture. We show that perturbation of TA normal function by overexpressing torsinA wild type (TAwt) inhibits HSV-1 production. Ultrastructural analysis of infected cells overexpressing TAwt revealed reduced levels of surface virions in addition to accumulation of novel, double-membrane structures called virus-like vesicles (VLVs). Although mainly found in the cytoplasm, VLVs resemble primary virions in their size, by the appearance of the inner membrane, and by the presence of pUL34, a structural component of primary virions. Collectively, our data suggest a model in which interference of TA normal function by overexpression impairs de-envelopment of the primary virions leading to their accumulation in a cytoplasmic membrane compartment. This implies novel functions for TA at the NE.     

B7 costimulation plays an important role in protection from herpes simplex virus type 2-mediated pathology

We have used mice lacking both B7-1 and B7-2 costimulation molecules (B7KO) to investigate the effects of B7 costimulation on herpes simplex virus type 2 (HSV-2) pathogenesis. B7KO mice infected intravaginally with virulent HSV-2 showed more severe genital and neurologic disease and higher mortality rates than their wild-type counterparts. These results suggest that B7 costimulation molecules play an important role in the development of primary immune responses protective against HSV-2.     

The range and distribution of murine central nervous system cells infected with the gamma(1)34.5- mutant of herpes simplex virus 1.

Wild-type herpes simplex virus 1 (HSV-1) multiplies, spreads, and rapidly destroys cells of the murine central nervous system (CNS). In contrast, mutants lacking both copies of the gamma(1)34.5- gene have been shown to be virtually lacking in virulence even after direct inoculation of high-titered virus into the CNS of susceptible mice (J. Chou, E. R. Kern, R. J. Whitley, and B. Roizman, Science 250:1262-1266, 1990). To investigate the host range and distribution of infected cells in the CNS of mice, 4- to 5-week-old mice were inoculated stereotaxically into the caudate/putamen with 3 x 10(5) PFU of the gamma(1)34.5- virus R3616. Four-micrometer-thick sections of mouse brains removed on day 3, 5, or 7 after infection were reacted with a polyclonal antibody directed primarily to structural proteins of the virus and with antibodies specific for neurons, astrocytes, or oligodendrocytes. This report shows the following: (i) most of the tissue damage caused by R3616 was at the site of injection, (ii) the virus spread by retrograde transport from the site of infection to neuronal cell nuclei at distant sites and to ependymal cells by cerebrospinal fluid, (iii) the virus infected neurons, astrocytes, oligodendrocytes, and ependymal cells and hence did not discriminate among CNS cells, (iv) viral replication in some neurons could be deduced from the observation of infected astrocytes and oligodendrocytes at distant sites, and (v) infected cells were being efficiently cleared from the nervous system by day 7 after infection. We conclude that the gamma(1)34.5- attenuation phenotype is reflected in a gross reduction in the ability of the virus to replicate and spread from cell to cell and is not due to a restricted host range. The block in viral replication appears to be a late event in viral replication.     

Specific activation of B-cell clones within the central nervous system in course of herpes simplex encephalitis

Total and virus-specific immunoglobulin (Ig) G oligoclonal bands were studied in paired serum and cerebrospinal fluid (CSF) of four patients with herpes simplex virus type 1 (HSV-1) encephalitis. We used the isoelectric focusing in agarose gel, a sensitive technique for protein separation, followed by passive transfer of proteins on nitrocellulose paper and specific immunostaining. Oligoclonal bands were observed in serum and CSF of all patients. HSV-1-specific oligoclonal IgG bands were present in the CSF only during a limited period of the disease, having their counterpart in serum during the remaining periods. Our findings contribute to tackle the issue of B-cell activation within central nervous system and peripheral blood compartments in course of HSV-1 encephalitis.     

An important role for syndecan-1 in herpes simplex virus type-1 induced cell-to-cell fusion and virus spread

Herpes simplex virus type-1 (HSV-1) is a common human pathogen that relies heavily on cell-to-cell spread for establishing a lifelong latent infection. Molecular aspects of HSV-1 entry into host cells have been well studied; however, the molecular details of the spread of the virus from cell-to-cell remain poorly understood. In the past, the role of heparan sulfate proteoglycans (HSPG) during HSV-1 infection has focused solely on the role of HS chains as an attachment receptor for the virus, while the core protein has been assumed to perform a passive role of only carrying the HS chains. Likewise, very little is known about the involvement of any specific HSPGs in HSV-1 lifecycle. Here we demonstrate that a HSPG, syndecan-1, plays an important role in HSV-1 induced membrane fusion and cell-to-cell spread. Interestingly, the functions of syndecan-1 in fusion and spread are independent of the presence of HS on the core protein. Using a mutant CHO-K1 cell line that lacks all glycosaminoglycans (GAGs) on its surface (CHO-745) we demonstrate that the core protein of syndecan-1 possesses the ability to modulate membrane fusion and viral spread. Altogether, we identify a new role for syndecan-1 in HSV-1 pathogenesis and demonstrate HS-independent functions of its core protein in viral spread.