DISTINCT MECHANISMS OF INHIBITION OF VSV REPLICATION IN NEURONS MEDIATED BY TYPE I AND TYPE II IFN

Abstract

Acute viral infection of neurons presents a difficult problem to the host, since neurons are essential and not replaced, therefore cell-autonomous pathway(s) of suppressing viral replication are critical. We have examined the mechanisms by which neurons respond to exogenous interferons (IFNs) and observed that novel pathways inhibit acute vesicular stomatitis virus (VSV) replication. For both type I (IFN-β) and Type II (IFN-γ) interferons, post-translational modification of viral proteins contributed to the replication blockade, diminishing the efficiency of viral assembly and budding from the host neuron. IFN-γ treatment induces the accumulation of NOS-1 in the absence of an increase of mRNA encoding this enzyme; a NOS-1-inhibiting protein, PIN, is rapidly ubiquitinated and eliminated in the presence of IFN-γ. NOS-1 produces NO which combines with superoxide to form peroxynitrite (ONOO-), this binds tyrosines, cysteines, and serines; antagonism of NOS-1 with either non-specific or selective inhibitors block the antiviral effect of IFN-γ. VSV proteins are decorated with −NO₂ in IFN-γ-treated neurons, probably resulting in their diminished ability to interact properly and mature into budding virus. For IFN-β, protein phosphorylation of the Matrix protein (M) and Phosphoprotein (P) were altered in infected neurons, with hyperphosphorylation of M (but not hypophosphorylated P) found in released virions. Hyperphosphorylated M protein does not immunoprecipitate with the viral ribonucleoprotein complex in IFN-β-treated neurons. Thus both types of IFN interfere with viral assembly and release of infectious particles, but by distinct pathways.

INTRODUCTION

Interferons were discovered 50 years ago and have been characterized for their profound ability to protect cells from viral infections (Isaacs & Lindenmann 1957, Vilcek 2006). Although there are additional forms including λ and τ, principally, research has been carried out with Types I and II IFNs. Type I is produced by most nucleated cells, and includes multiple IFN-α forms and IFN-β, which share a receptor IFNAR (Pestka et al. 2004). Type II, includes just IFN-γ is produced by NK, CD4 Th1, and CD8 cells, and has a distinct receptor, IFNGR, which is ubiquitously expressed (Stetson & Medzhitov 2006). Multiple effects of IFNs on cell cycle progression, on innate and adaptive immune responses, and the regulation of IFN expression are beyond the scope of this communication.

We began to study the effect of IFNs on the viral infection of neurons because of the essential need to eliminate the pathogen in a cell autonomous way. Since neurons are post-mitotic and are generally considered to be resistant to the cytolytic effects of T cells and NK cells, and since viral infections are limited and cleared from neurons, IFNs seemed to be an important host cell response to investigate. Neurons and neuronal cell lines express functional surface receptors for both Type I and II IFNs. We found that IFN-γ treatment takes two days to maximally inhibit VSV replication, and that this is suppressed by about 10-100-fold (Komatsu et al. 1996, Chesler & Reiss 2002). In contrast, the ability of IFN-β to protect neurons comes on much more rapidly and is more profound, with 10³-10⁴-fold inhibition of viral replication (Trottier et al. 2005). Our findings are summarized in Table 1.

Table 1.

Comparison of mechanisms of IFN-β and IFN-γ inhibition of VSV replication in neurons

	IFN-β	IFN-γ
Time for optimal protection	4-8 hours	48-72 hours
Synthesis v-mRNA	3-10 fold inhibited	20% inhibited
Synthesis v-protein	6-12 fold inhibited	75% inhibited
Synthesis v-RNA	10-15 fold inhibited	No determined
Yield infectious virus	103-104 fold inhibited	102 fold inhibited
“bald particles”	No	No
Role for PKR	No	No
Role for NOS-1	No	Required
NO2-modified S, Y, C	No	Yes
Hypophosphorylated P in cells	Yes	Not tested
Hypophosphorylated P in virion	No	Not tested
Hyperphosphorylated M in cells	Yes	Not tested
Hyperphosphorylated M in virion	Yes	Not tested
Assembly	Compromised	Compromised

To determine how viral replication was inhibited we examined the stages of viral replication in IFN-treated cells starting at mRNA expression and ending with release of progeny. This could potentially indicate a well-characterized antiviral pathway such as 2′5′Oligoadenylate Synthase-RNAseL if viral RNA were degraded or Protein Kinase induced by RNA (PKR; which phosphorylates and inhibits the activity of eIF2α, an initiation factor) if viral protein synthesis were halted. Our findings showed minimal effects on these pathways (Chesler et al. 2003, Trottier et al. 2005), when viral RNA and protein expression were compared in mock-treated or IFN-treated neuronal cells.

In a parallel set of early experiments, we asked the question: does NO inhibit VSV replication? Carl Nathan had just showed that in macrophages, where the inducible isoform of the enzyme Nitric Oxide Synthase (NOS-2) is rapidly expressed following inflammatory cytokine treatment, that the growth of DNA viruses was inhibited (Karupiah et al. 1993, Nathan & Xie 1994). We found that NO donor treatment of neurons did inhibit VSV replication, and IFN-γ, TNF-α, or IL-12 treatment resulted in both induction of NOS-1 and inhibition of VSV replication (Bi & Reiss 1995, Komatsu et al. 1996, Komatsu et al. 1997). We then observed that the IFN-γ-induced antiviral activity could be blocked by the addition of either non-specific enzyme inhibitors or the isoform-selective drug, 7-nitroindazole. This blocked the effect of IFN-γ in vitro and also resulted in increased viral replication in vivo. Mice deficient in NOS-1 (expressed by neurons and striated muscle cells at the dystrophin complex) were more susceptible to VSV encephalitis than wild type (WT) or knockouts of NOS-2 (expressed in microglia, macrophages) or NOS-3 (expressed by endothelial cells and some astrocytes) (Barna et al. 1996, Komatsu et al. 1999, Reiss & Komatsu 1998).

In addition, we found no evidence for a role of PKR in the IFN-γ-mediated antiviral effect in neurons; neurons expressing the NS-1 protein from influenza, which selectively blocks PKR, were equally sensitive to IFN-γ (Chesler et al. 2003). STAT-1 was also essential, but not the ERK signal transduction pathways (Chesler et al. 2004a).

NOS-1 is a constitutively expressed enzyme (Bredt & Snyder 1994). We found that although NOS-1 and the production of NO tripled in IFN-γ-treated neurons, the mRNA for NOS-1 did not change (Komatsu et al. 1996, Chesler et al. 2004b). This was due to several factors including increased half-life of the protein and depletion of a critical inhibitor, Protein inhibitor of NOS-1 (PIN) (Chesler et al. 2004b, Jaffrey & Snyder 1996, Yang et al. 2007). In fact, NOS-1 was not degraded by the proteasome in IFN-γ-treated neurons, but PIN was rapidly lost (Yang et al. 2006). When we over-expressed PIN or suppressed its expression using RNAi, we found suppression or enhancement of the effect of IFN-γ on VSV replication, respectively (Yang et al. 2007).

NOS-1 is activated by calcium release either from the extracellular environment or endoplasmic reticulum (Bredt & Snyder 1994). When ligands specific for the CB₁ receptor (either endocannabinoids or drugs such as WIN-55) engage the receptor, calcium mobilization is impaired in neurons, and IFN-γ is unable to suppress VSV replication (Herrera et al. 2008).

Viral proteins were modified at tyrosines, serines and cysteines by −NO₂ in IFN-γ-treated neurons (Komatsu et al. 1996). In another RNA virus infection, NO₂-modification of the viral protease blocked replication of a picornavirus by preventing the cleavage of the polyprotein to generate structural subunits (Saura et al. 1999). Many other viruses are also sensitive to NO-mediated inhibition (Reiss & Komatsu 1998, Akaike & Maeda 2000). We interpret our findings to be compatible with IFN-γ inducing enhanced NOS-1 activity, which leads to the decoration of viral proteins with −NO₂ to prevent necessary protein-protein interactions and thus lead to defective assembly and release of infectious progeny.

Neurons are exquisitely sensitive to the antiviral effects of IFN-β for inhibition of VSV replication. While neurons are able to make IFN-β in response to infection by other viruses, VSV M suppresses this capacity (Trottier et al. 2005). Ultimately this blockade shuts down host cell protein biosyntheses (Ahmed et al. 2003, Stojdl et al. 2003, von Kobbe et al. 2000, Gustin 2003). To examine which stage(s) of the virus life cycle were impaired in IFN-β-treated neurons, we compared the expression of mRNA, vRNA, viral proteins, and release of particles. The stages of viral production were compromised, at most 15-fold, but the release of progeny was inhibited 10³-10⁴-fold (Trottier et al. 2005). There was no evidence of NOS-1 involvement or PKR activity in IFN-β-mediated inhibition of VSV replication in neurons (Trottier et al. 2005).

In vivo, VSV infection of the CNS readily elicits a systemic IFN-β response produced by plasmacytoid dendritic cells in secondary lymphoid organs (Trottier et al. 2007), but not in the CNS (Ireland & Reiss 2006, Trottier et al. 2007). However, the peripherally-produced IFN is unable to cross the blood-brain barrier (Dafny & Yang 2005), and thus is incapable of protecting neurons from the local trans-synaptic spread.

When post-translational modification of VSV proteins was examined, we were surprised to find hypophosphorylation of the VSV P protein, an essential component of the RNA-dependent RNA polymerase complex (D'Agostino et al. 2009). However, the P which was incorporated into virions released from IFN-β-treated neurons was normally phosphorylated. Indeed, the pI of the protein was much more basic in P obtained from IFN-treated neurons when analyzed by two-dimensional gel electrophoresis, and was compatible with the observed changes in the ratios of ³²P to ³⁵S (D'Agostino et al. 2009). Work is in progress to determine if the alteration of P has modified its activity in the RNA-dependent RNA polymerase.

In contrast, the M protein was hyperphosphorylated in IFN-β-treated neurons and also in virus released from them (D'Agostino et al. 2009). M protein is essential to bridge the viral RNP and the glycoprotein, to seed the formation of new virions (Taylor et al. 2007, Craven et al. 1999). M protein has 3 late domains, which are proposed to interact with the intracellular macromolecular transport system which could bring nascent M from polysomes to the cytoplasmic face of the plasma membrane (Craven et al. 1999, Irie et al. 2004, Kaptur et al. 1995, Freed 2002). One of these Late domains, PPPY (a.a. 24-27) is potentially the site of phosphorylation (D'Agostino et al. 2009). Work is in progress to determine if this is one of the sites of hyperphosphorylation.

If M protein was to be modified at this site, it may be less well able to interact with the chaperone, and possibly, other viral or cellular proteins. We find no detectable protein-protein complexes of M with the viral RNP in IFN-β-treated neurons. We also examined the ubiquitination of VSV M protein in IFN-β-treated neurons, and found no difference in the ability of host cells to mono-ubiquitinate M (D'Agostino et al. 2009).

Work is in progress to map the sites of post-translational phosphorylation of both VSV M and P proteins, as well as the location of the mono-ubiquitination of M protein. Work is in progress to identify the host neuronal cell kinases and phosphatases which are critical to the IFN-β effects.

We interpret these data to indicate that, in contrast to the post-translational modifications seen in IFN-γ-treated neurons, IFN-β treatment leads to alterations in phosphorylation of two critical viral proteins which then are incapable of participating in protein-protein interactions to assemble new virions.