Identification of residues in West Nile virus pre-membrane protein that influence viral particle secretion and virulence.

The pre-membrane protein (prM) of West Nile virus (WNV) functions as a chaperone for correct folding of the envelope (E) protein, and prevents premature fusion during virus egress. However, little is known about its role in virulence. To investigate this, we compared the amino acid sequences of prM between a highly virulent North American strain (WNV(NY99)) and a weakly virulent Australian subtype (WNV(KUN)). Five amino acid differences occur in WNV(NY99) compared with WNV(KUN) (I22V, H43Y, L72S, S105A and A156V). When expressed in mammalian cells, recombinant WNV(NY99) prM retained native antigenic structure, and was partially exported to the cell surface. In contrast, WNV(KUN) prM (in the absence of the E protein) failed to express a conserved conformational epitope and was mostly retained at the pre-Golgi stage. Substitutions in residues 22 (Ile to Val) and 72 (Leu to Ser) restored the antigenic structure and cell surface expression of WNV(KUN) prM to the same level as that of WNV(NY99), and enhanced the secretion of WNV(KUN) prME particles when expressed in the presence of E. Introduction of the prM substitutions into a WNV(KUN) infectious clone (FLSDX) enhanced the secretion of infectious particles in Vero cells, and enhanced virulence in mice. These findings highlight the role of prM in viral particle secretion and virulence, and suggest the involvement of the L72S and I22V substitutions in modulating these activities.

Effector cytolotic function but not IFN-gamma production in cytotoxic T cells triggered by virus-infected target cells in vitro.

Cytolysis and interferon(IFN)-gamma production are two independent effector functions of activated cytotoxic T (Tc) cells. We have used the Tc-cell response against the flavivirus, Murray Valley encephalitis virus (MVE), to investigate the requirements for inducing these two functions with regard to antigen-concentration and CD8 coreceptor involvement. Cognate peptide-pulsed target cells triggered cytolysis by primary ex vivo MVE-immune as well as in vitro peptide-restimulated splenocytes at lower peptide concentrations than IFNgamma-production (100-fold lower in the case of primary ex vivo effectors). Little difference was observed in CD8 dependency. Importantly, neither of the effector populations were triggered to produce IFN-gamma by virus-infected target cells, although cytolysis occurred. This result raises the possibility that the levels of presentation of cognate antigen on virus-infected cells in vivo may be below the threshold required for the IFN-gamma production.

Immunogenicity of two peptide determinants in the cytolytic T-cell response to flavivirus infection: inverse correlation between peptide affinity for MHC class I and T-cell precursor frequency.

We used the CD8+ cytotoxic T (Tc) cell immune response against the flavivirus, Murray Valley encephalitis virus (MVE), restricted by the H-2Kk major histocompatibility complex (MHC) class I molecule, to investigate immunodominance. Split-clone limiting dilution analysis revealed almost exclusive recognition of two peptides, MVE1785 and MVE1971, derived from the viral NS3 protein. The precursor frequency of MVE-reactive Tc cells was determined by limiting dilution analysis for cytotoxic function and intracellular staining for interferon-gamma; the latter gave a 100-fold higher estimate of MVE-reactive Tc cell precursors. MHC class I cell surface stabilization assays revealed that affinity for H-2Kk as well as halflives of the peptide-H-2Kk-complexes were markedly different for the two peptides. However, a kinetic study of antigen presentation showed that both peptides are presented for recognition by Tc cells with a comparable kinetics during the latent period of virus infection. Nevertheless, the lower affinity peptide MVE1785 elicited roughly twofold more Tc cell clones than the high-affinity peptide MVE1971. While the cytolytic activity against both determinants was similar after in vitro restimulation at the peak of the primary response, the smaller pool of memory anti-MVE1971 Tc cells correlated with an impaired memory response against that determinant, suggesting that the available T-cell repertoire is a major factor influencing the establishment of T-cell memory.

Modulation of transporter associated with antigen processing (TAP)-mediated peptide import into the endoplasmic reticulum by flavivirus infection.

In contrast to many other viruses that escape the cellular immune response by downregulating major histocompatibility complex (MHC) class I molecules, flavivirus infection can upregulate their cell surface expression. Previously we have presented evidence that during flavivirus infection, peptide supply to the endoplasmic reticulum is increased (A. Müllbacher and M. Lobigs, Immunity 3:207-214, 1995). Here we show that during the early phase of infection with different flaviviruses, the transport activity of the peptide transporter associated with antigen processing (TAP) is augmented by up to 50%. TAP expression is unaltered during infection, and viral but not host macromolecular synthesis is required for enhanced peptide transport. This study is the first demonstration of transient enhancement of TAP-dependent peptide import into the lumen of the endoplasmic reticulum as a consequence of a viral infection. We suggest that the increased supply of peptides for assembly with MHC class I molecules in flavivirus-infected cells accounts for the upregulation of MHC class I cell surface expression with the biological consequence of viral evasion of natural killer cell recognition.

Nonobese diabetic mice display elevated levels of class II-associated invariant chain peptide associated with I-Ag7 on the cell surface.

Peptide presentation by MHC class II molecules plays a pivotal role in determining the peripheral T cell repertoire as a result of both positive and negative selection in the thymus. Homozygous I-A(g7) expression imparts susceptibility to autoimmune diabetes in the nonobese diabetic mouse, and recently, it has been proposed that this arises from ineffectual peptide binding. Following biosynthesis, class II molecules are complexed with class II-associated invariant chain peptides (CLIP), which remain associated until displaced by Ag-derived peptides. If I-A(g7) is a poor peptide binder, then this may result in continued occupation by CLIP to the point of translocation to the cell surface. To test this hypothesis we generated affinity-purified polyclonal antisera that recognized murine CLIP bound to class II molecules in an allele-independent fashion. We have found abnormally high natural levels of cell surface class II occupancy by CLIP on nonobese diabetic splenic B cells. Experiments using I-A-transfected M12.C3 cells showed that I-A(g7) alone was associated with elevated levels of CLIP, suggesting that this was determined solely by the amino acid sequence of the class II molecule. These results indicated that an intrinsic property of I-A(g7) would affect both the quantity and the repertoire of self-peptides presented during thymic selection.

Antiviral cytotoxic T cells cross-reactively recognize disparate peptide determinants from related viruses but ignore more similar self- and foreign determinants.

We have investigated the reactivities of cytotoxic T (Tc) cells against the two immunodominant, H-2K(k)-restricted determinants from the FLAVIVIRUS: Murray Valley encephalitis virus (MVE), MVE(1785) (REHSGNEI) and MVE(1971) (DEGEGRVI). The respective Tc cell populations cross-reactively lysed target cells pulsed with determinants from the MVE(1785)- and MVE(1971)-corresponding positions of six other flaviviruses, despite low sequence homology in some cases. Notably, anti-MVE(1785) Tc cells recognized a determinant (TDGEERVI) that shares with the determinant used for stimulation only the carboxyl-terminal amino acid residue, one of two H-2K(k) anchor residues. These reactivity patterns were also observed in peptide-dependent IFN-gamma production and the requirements for in vitro restimulation of memory Tc cells. However, the broad cross-reactivity appeared to be limited to flavivirus-derived determinants, as none of a range of determinants from endogenous mouse-derived sequences, similar to the MVE-determinants, were recognized. Neither were cells infected with a number of unrelated viruses recognized. These results raise the paradox that virus-immune Tc cell responses, which are mostly directed against only a few "immunodominant" viral determinants, are remarkably peptide cross-reactive.

Cytotoxic T cells specifically induce Fas on target cells, thereby facilitating exocytosis-independent induction of apoptosis.

Cytotoxic T (Tc) cells deficient in perforin lyse Fas-negative targets after lengthy incubation periods. This process is independent of granzymes, and killing occurs via the Fas pathway for the following reasons. Interaction of perforin-deficient Tc cells with Fas-negative targets leads to an up-regulation of Fas that is dependent on Ag recognition, de novo synthesis, and transport of proteins to the target cell surface. Treatment of effectors with brefeldin A, but not with the exocytosis inhibitor concanamycin, inhibited this process. Lysis of targets is inhibited by anti-Fas Abs, soluble mouse Fas-Fc, and the caspase-cascade inhibitor, crm-A. Targets from Fas-mutant lpr mice are refractory to lysis, and Tc cells from mice deficient in Fas- and perforin-mediated lysis do not lyse Fas-negative targets. The possible relevance of this exocytosis-independent cytolytic process in the regulation of T cell activity and control of pathogens is discussed.

Substitutions at the putative receptor-binding site of an encephalitic flavivirus alter virulence and host cell tropism and reveal a role for glycosaminoglycans in entry.

The flavivirus receptor-binding domain has been putatively assigned to a hydrophilic region (FG loop) in the envelope (E) protein. In some flaviviruses this domain harbors the integrin-binding motif Arg-Gly-Asp (RGD). One of us has shown earlier that host cell adaptation of Murray Valley encephalitis virus (MVE) can result in the selection of attenuated variants altered at E protein residue Asp(390), which is part of an RGD motif. Here, a full-length, infectious cDNA clone of MVE was constructed and employed to systematically investigate the impact of single amino acid changes at Asp(390) on cell tropism, virus entry, and virulence. Each of 10 different E protein 390 mutants was viable. Three mutants (Gly(390), Ala(390), and His(390)) showed pronounced differences from an infectious clone-derived control virus in growth in mammalian and mosquito cells. The altered cell tropism correlated with (i) a difference in entry kinetics, (ii) an increased dependence on glycosaminoglycans (determined by inhibition of virus infectivity by heparin) for attachment of the three mutants to different mammalian cells, and (iii) the loss of virulence in mice. These results confirm a functional role of the FG loop in the flavivirus E protein in virus entry and suggest that encephalitic flaviviruses can enter cells via attachment to glycosaminoglycans. However, it appears that additional cell surface molecules are also used as receptors by natural isolates of MVE and that the increased dependence on glycosaminoglycans for entry results in the loss of neuroinvasiveness.

Proteolytic processing of peptides in the lumen of the endoplasmic reticulum for antigen presentation by major histocompatibility class I.

We have tested the hypothesis that MHC class I molecules are actively involved as protease in the production of natural MHC class I ligands. First, the structure of a class I molecule was analyzed for homology with catalytic sites of known proteases. While several clusters of amino acids in the restriction element resembled protease active sites, structural discrepancies and the influence of nearby residues suggest that these sites are unlikely to have protease activity. Second, we have tested the presentation of viral cytotoxic T cell determinants with affinity for the same restriction element (H-2K(d) or K(k)), when targeted as tandem peptides into the endoplasmic reticulum. Peptide transporter-defective cells were used to exclude cleavage of the tandem peptides by cytosolic proteases. Cleavage by signal peptidase of the tandem peptides was ascertained. The C-terminal peptides in the tandem arrays were almost exclusively presented, suggesting that an aminopeptidase in the endoplasmic reticulum degraded the N-terminally positioned peptides. This result is inconsistent with an MHC class I-catalyzed cleavage following binding of longer peptides in the cleft of the restriction elements. Finally, we conclusively show that an aminopeptidase in the endoplasmic reticulum is also involved in antigen presentation in cells with a functional peptide transporter.

Interferon-inducible chemokines and immunity to poxvirus infections.

The biological roles of two interferon-inducible chemokines, monokine induced by gamma interferon (Mig) and cytokine responsive gene (Crg-2), in the immune response against vaccinia virus (VV) and ectromelia virus (EV) infections are discussed. To investigate their antiviral effects in vivo, the expression profiles of these chemokines during the course of VV or EV infections were first established. Mig and Crg-2 were induced in multiple organs at high levels early after infection with VV. Both chemokines were rapidly induced in popliteal lymph nodes of C57BL/6 mice but not in BALB/c mice following infection with EV. Secondly, recombinant vaccinia viruses (rVV) encoding Mig or Crg-2 were constructed to investigate the immunobiology of infection in athymic, nude and euthymic, normal mice. Finally, the EV model in combination with recombinant Mig and Crg-2 proteins was used to test their effects on viral replication and immune responses in vivo. The results of these investigations demonstrate that the mechanisms of Mig- and Crg-2-induced viral clearance involve natural killer cells and interferons.

Mutagenesis of the signal sequence of yellow fever virus prM protein: enhancement of signalase cleavage In vitro is lethal for virus production.

Proteolytic processing at the C-prM junction in the flavivirus polyprotein involves coordinated cleavages at the cytoplasmic and luminal sides of an internal signal sequence. We have introduced at the COOH terminus of the yellow fever virus (YFV) prM signal sequence amino acid substitutions (VPQAQA mutation) which uncoupled efficient signal peptidase cleavage of the prM protein from its dependence on prior cleavage in the cytoplasm of the C protein mediated by the viral NS2B-3 protease. Infectivity assays with full-length YFV RNA transcripts showed that the VPQAQA mutation, which enhanced signal peptidase cleavage in vitro, was lethal for infectious virus production. Revertants or second-site mutants were recovered from cells transfected with VPQAQA RNA. Analysis of these viruses revealed that single amino acid substitutions in different domains of the prM signal sequence could restore viability. These variants had growth properties in vertebrate cells which differed only slightly from those of the parent virus, despite efficient signal peptidase cleavage of prM in cell-free expression assays. However, the neurovirulence in mice of the VPQAQA variants was significantly attenuated. This study demonstrates that substitutions in the prM signal sequence which disrupt coordinated cleavages at the C-prM junction can impinge on the biological properties of the mutant viruses. Factors other than the rate of production of prM are vitally controlled by regulated cleavages at this site.

High peptide affinity for MHC class I does not correlate with immunodominance.

Cytotoxic T (Tc)-cell responses against influenza virus infection in BALB/c (H-2d) mice are dominated by Tc clones reactive to the viral nucleoprotein (NP). Here, we report investigations using recombinant vaccinia viruses (VV) encoding major histocompatibility complex (MHC) class I H-2Kd molecules differing by a single amino acid from glutamine (wild-type, Kdw) to histidine (mutant, Kdm) at position 114 located in the floor of the peptide-binding groove. Influenza-infected target cells expressing Kdw were strongly lysed by Kd-restricted Tc cells against A/WSN influenza virus or the immunodominant peptide of viral NP (NPP147-155), whereas infected Kdm-expressing targets gave little or no lysis, respectively, thus showing the immunodominance of NPP147-155. Kdm-expressing target cells saturated with synthetic NPP147-155 (10-5 M) were lysed similarly to Kdw-expressing targets by NPP147-155-specific Tc cells. Thus the defect in influenza-infected Kdm-expressing targets was quantitative; insufficient Kdm-peptide complexes were expressed. Tc-cell responses against four other viruses or alloantigens showed no effect of Kdm. When peptide transport-defective cells were infected with VV-Kdw or VV-Kdm and co-infected with a recombinant VV encoding an endoplasmic reticulum-targeted viral peptide, two influenza haemaglutinin peptides caused higher expression of Kdw than NPP147-155 indicating their higher affinity for Kdw. These results are inconsistent with the hypothesis that immunodominance in the anti-influenza response reflects high affinity of the immunodominant peptide, but are consistent with skewing of the Tc-cell receptor repertoire.

Antigen presentation in syrian hamster cells: substrate selectivity of TAP controlled by polymorphic residues in TAP1 and differential requirements for loading of H2 class I molecules.

Expression of mouse major histocompatibility complex (MHC) class I molecules in different cell lines derived from Syrian hamsters has revealed antigen presentation deficiencies of some H2 allelic products in two cell lines (BHK and NIL-2) which were overcome by transient expression of the rat transporter associated with antigen processing (TAP; Lobigs et al. 1995). Here we show that in both cell lines the endogenous MHC class I cell surface expression was completely down-regulated. Lymphokine treatment induced endogenous and recombinant mouse MHC class I cell surface expression to levels similar to that in other Syrian hamster cell lines competent for antigen presentation through transduced H2 molecules. Accordingly, constitutive downregulation of expression of accessory molecules of the MHC class I pathway can reveal differences between H2 class I alleles in antigen presentation not encountered when the expression levels are augmented. In addition to the differential expression of MHC class I pathway genes, two cell lines representing competent (FF) and defective (BHK) antigen presentation phenotypes for mouse class I MHC restriction elements demonstrated substantial sequence polymorphism in Tap1 but not Tap2. Cytokine-treated FF or BHK cells and human TAP-deficient T2 cells transfected with FF or BHK TAP1 in combination with FF TAP2 differed in their preference for C-terminal peptide residues, as shown by an in vitro peptide transport assay. Thus, polymorphic residues in TAP1 can influence the substrate selectivity of the Syrian hamster peptide transporter.

Alloreactive cytotoxic T-cell function, peptide nonspecific.

The recognition requirements necessary for murine alloreactive cytotoxic T-cells to carry out their effector function has been investigated using target cells that express a unique class I major histocompatibility complex (MHC)-peptide pair. The human cell line T2 and the murine cell line RMA-S are defective in peptide transport components needed to effectively express stable MHC class I molecules at the cell surface. When T2 cells were infected with a vaccinia virus that encoded the Kd gene and provided with a Kd-motif peptide from the nucleoprotein of influenza virus (NPP), these cells could be lysed by polyclonal allo Kd-reactive cytotoxic T-lymphocytes (CTL). Similar results were obtained with the murine RMA-S-Kd cell line, transfected with cDNA able to express some 'empty' Kd that is heat-labile. Adding another Kd-motif peptide from influenza virus haemagglutinin (HAP) stabilized the surface expression of Kd and allowed the RMA-S-Kd cells to be lysed before or after heat shock. At 27 degrees C anti-Kd alloreactive CTL-lysed target cells in the presence and absence of HAP peptide. Alloreactive CTL appear to have a more stringent requirement for a high density of MHC class I on cell surfaces relative to peptide-specific MHC-restricted CTL. We conclude that while Kd-restricted CTL activity is strictly peptide-specific, anti-Kd-specific alloreactivity is MHC allele-specific, but peptide-nonspecific. This conclusion is at odds with the Standard Model of T-cell receptor (TCR) function, but consistent with the predictions of a Competing Model of TCR function.

Spontaneous mutation at position 114 in H-2Kd affects cytotoxic T cell responses to influenza virus infection.

Vaccinia virus (VV)-encoded MHC class I Kd molecules which differ by a single amino acid change from glutamine (Kdw, wild type) to histidine (Kdm, mutant) at position 114 located in the floor of the peptide binding groove were compared in terms of peptide binding and cytotoxic T (Tc) cell recognition. Most anti-viral Tc cells were not affected or only marginally affected. However, the Kdm molecule did not detectably present the immunodominant peptide (NPP147-155) of influenza virus nucleoprotein (NP), encoded by the full-length NP gene either in influenza A virus or recombinant VV. This defect could be overcome by using exogenous synthetic NPP147-155 or translation from a minigene encoding NPP147-155 in VV. Kdw presented NPP147-155 encoded by the full-length NP gene, but Kdw-NPP147-155 complexes were at least 100-fold less abundant than after translation from a minigene.

DNA-based and alphavirus-vectored immunisation with prM and E proteins elicits long-lived and protective immunity against the flavivirus, Murray Valley encephalitis virus.

The immunogenicity and protective efficacy of DNA-based vaccination with plasmids encoding the membrane proteins prM and E of the flavivirus Murray Valley encephalitis virus (MVE) were investigated. Gene gun-mediated intradermal delivery of DNA encoding the prM and E proteins elicited long-lived, virus-neutralising antibody responses in three inbred strains of mice and provided protection from challenge with a high titer inoculum of MVE. Intramuscular DNA vaccination by needle injection also induced MVE-specific antibodies that conferred resistance to challenge with live virus but failed to reduce virus infectivity in vitro. The two routes of DNA-based vaccination with prM and E encoding plasmids resulted in humoral immunty with distinct IgG subtypes. MVE-specific IgG1 antibodies were always prevalent after intradermal DNA vaccination via a gene gun but not detected when mice were immunised with DNA by the intramuscular route or infected with live virus. We also tested a Semliki Forest virus replicon as vector for a flavivirus prM and E protein-based subunit vaccine. Single-cycle infections in mice vaccinated with packaged recombinant replicon particles elicited durable, MVE-specific, and virus-neutralising antibody responses.

Signal peptidase cleavage at the flavivirus C-prM junction: dependence on the viral NS2B-3 protease for efficient processing requires determinants in C, the signal peptide, and prM.

Signal peptidase cleavage at the C-prM junction in the flavivirus structural polyprotein is inefficient in the absence of the cytoplasmic viral protease, which catalyzes cleavage at the COOH terminus of the C protein. The signal peptidase cleavage occurs efficiently in circumstances where the C protein is deleted or if the viral protease complex is present. In this study, we used cDNA of Murray Valley encephalitis virus (MVE) to examine features of the structural polyprotein which allow this regulation of a luminal cleavage by a cytoplasmic protease. We found that the inefficiency of signal peptidase cleavage in the absence of the viral protease is not attributable solely to features of the C protein. Inhibition of cleavage still occurred when charged residues in C were mutated to uncharged residues or when an unrelated protein sequence (that of ubiquitin) was substituted for C. Also, fusion of the C protein did not inhibit processing of an alternative adjacent signal sequence. The cleavage region of the flavivirus prM translocation signal is unusually hydrophobic, and we established that altering this characteristic by making three point mutations near the signal peptidase cleavage site in MVE prM dramatically increased the extent of cleavage without requiring removal of the C protein. In addition, we demonstrated that luminal sequences downstream from the signal peptidase cleavage site contributed to the inefficiency of cleavage.

Flavivirus-induced up-regulation of MHC class I antigens; implications for the induction of CD8+ T-cell-mediated autoimmunity.

Infection of a wide variety of cells of human, mouse and other species' origin by flaviviruses such as WNV, YF, Den, MVE, KUN and JE, increases the cell-surface expression of MHC class I. This MHC class I up-regulation is not due to increased MHC class I synthesis per se, but the result of increased peptide availability in the ER for MHC class I assembly. This is most likely due to the interaction of the viral polyprotein with the ER membrane during viral replication. Flavivirus infection can overcome peptide deficiency in TAP-deficient or non-permissive cell lines such as RMA-S and Syrian hamster cells, BHK and NIL-2. The consequence of this increased MHC class I expression manifests itself in reduced susceptibility to NK cells and augmented lysis by Tc cells. In mice, long-term flavivirus-immune Tc cell memory formation is impaired, following the appearance of strong anti-self Tc cell reactivity observed in in vitro cultures from splenocytes of flavivirus-primed animals. We hypothesize that flavivirus-induced MHC class I up-regulation leads to transient T-cell autoimmunity, followed by down-regulation of both autoimmunity and virus-specific Tc cell memory. Furthermore, we speculate that flavivirus infections of humans in the tropics may be responsible for the observed lower incidence of overt autoimmunity in these geographic regions than in temperate climates where flaviviruses are not endemic.

Protective immune responses to the E and NS1 proteins of Murray Valley encephalitis virus in hybrids of flavivirus-resistant mice.

The lack of an effective animal model has been a major obstacle in attempts to define the role of humoral and cellular immune responses in protection against flavivirus infection. We have used F1 hybrid mice (BALB/c x C3H/RV) that are heterozygous for the flavivirus resistance allele F1vr and show reduced virus replication in the brain after intracerebral inoculation. F1 hybrid mice challenged by intracerebral inoculation with Murray Valley encephalitis (MVE) virus developed encephalitis 2-3 days later than a genetically susceptible strain (BALB/c) but showed a similar mortality rate. This delay in the onset of disease provided more opportunity for virus clearance by primed immune responses. Using F1 hybrid mice we were able to demonstrate protective immunity induced by structural and non-structural proteins of MVE virus by immunization with pure NS1 protein or recombinant vaccinia viruses that expressed various regions of the MVE genome. These constructs included VV-STR (C-prM-E-NS1-NS2A), VV-delta C (prM-E-Ns1-NS2A) and VV-NS1 (NS1-NS2A). VV-delta C vaccinated mice were completely protected (100% survival)from challenge with 1000 infectious units of MVE virus, while mice inoculated with VV-STR, VV-NS1 or pure NS1 were partially protected (40%, 47% and 85% respectively). Analysis of prechallenge sera and in vivo depletion studies revealed that the solid protection induced by VV-delta C was mediated by neutralizing antibody to the E protein and did not require a CD8+ T cell response. The partial protection provided by VV-STR, VV-NS1 and pure NS1 occurred after induction of antibody to NS1. However, depletion of CD8+ cells prior to virus challenge ablated the protection provided by VV-NS1 indicating some requirement for class I restricted cytotoxic T cells.