Infection with an H2 recombinant herpes simplex virus vector results in expression of MHC class I antigens on the surfaces of human neuroblastoma cells in vitro and mouse sensory neurons in vivo

Allison Abendrothb,1, Anthony Simmons1, Stacey Efstathiouc,1 and Rosemarie A. Pereira1

Infectious Diseases Laboratories, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5000, Australia1

Author for correspondence: Tony Simmons. Fax +61 8 222 3543. e-mail tony.simmons{at}imvs.sa.gov.au


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
The majority of neurons in herpes simplex virus (HSV)-infected murine sensory ganglia are transiently induced to express MHC-I antigens at the cell surface, whereas only a minority are themselves productively infected. The aim of the current work was to determine whether MHC-I antigens can be expressed on the surfaces of infected neurons in addition to their uninfected neighbours. To address this aim a recombinant HSV type 1 strain, S-130, was used to deliver a mouse H2Kd gene, under control of the HCMV IE-1 promoter/enhancer, into human neuroblastoma cells in vitro and mouse primary sensory neurons in vivo. S-130 expressed H2Kd antigens on the surfaces of IMR-32 cells, a human neuroblastoma cell line that expresses very low levels of MHC-I constitutively. In K562 cells, which do not express MHC-I constitutively, H2Kd and {beta}2-microglobulin ({beta}2m) were shown to be co-expressed at the cell surface following S-130 infection. This observation was taken as evidence that class I heavy chain ({alpha}C) molecules encoded by the expression cassette in the HSV genome were transported to the cell surface as stable complexes with {beta}2m. Significantly, after introduction of S-130 into flank skin, H2Kd antigens were detected on the surfaces of primary sensory neurons in ganglia innervating the inoculation site. Our data show that HSV-infected murine primary sensory neurons and human neuroblastoma cells are capable of expressing cell-surface MHC-I molecules encoded by a transgene. From this, we infer that up-regulation of {alpha}C expression is, in principle, sufficient to overcome potential impediments to neuronal cell surface expression of MHC-I complexes.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
We showed previously that CD8+ cells play a pivotal role in terminating experimental herpes simplex virus (HSV) infection in the peripheral nervous systems of mice (Simmons & Tscharke, 1992 ). CD8+ cells recognize antigens displayed on the surface of target cells in the context of MHC class I molecules (Marrack & Kappler, 1987 ). Neurons, the main cell type infected by HSV in the peripheral nervous system, are classically regarded as MHC deficient, creating an apparent paradox. However, prior work from this laboratory showed that transcription (Pereira et al., 1994 ) and cell-surface expression (Pereira & Simmons, 1999 ) of MHC-I genes by neurons is up-regulated transiently in HSV-infected sensory nerve ganglia. MHC-I up-regulation was shown to be confined to ganglia infected with HSV (Pereira et al., 1994 ), but it has not been shown whether MHC-I molecules can be expressed on the surfaces of HSV-infected neurons in addition to their uninfected neighbours. The major impediment encountered in addressing this issue is that appearance of MHC-I on neurons in an HSV-infected ganglion is at a low density and coincident with clearance of infectious virus (Pereira & Simmons, 1999 ). Dual staining for HSV and MHC-I is therefore not a viable approach. In the current work, a novel strategy was used to address this question, namely delivery of a murine MHC-I heavy chain ({alpha}C) gene, H2Kd, directly into HSV-infected neurons using a recombinant HSV vector. The recombinant virus, designated HSV type 1 strain S-130, carries an H2Kd expression cassette regulated by the CMV immediate early promoter/enhancer. In vivo, neurons were targeted by inoculating S-130 into flank skin, thereby infecting primary sensory neurons by retrograde axonal transport of virus along spinal nerves.

Stability of MHC-I molecules at the cell surface is dependent on the formation of functional complexes comprising an {alpha}C linked non-covalently with a light chain, {beta}2-microglobulin ({beta}2m), and an antigenic peptide. The myelogenous leukaemia cell line, K562, does not express MHC-I owing to a failure to synthesize {alpha}Cs and, as a consequence, {beta}2m is not found at the cell surface (Lozzio & Lozzio, 1979 ). The current study exploited the knowledge that murine {alpha}Cs are able to associate in a stable manner with human {beta}2m (Van Agthoven et al., 1984 ). Detection of {beta}2m–{alpha}C complexes on the surface of S-130-infected K562 cells was taken as evidence that {alpha}Cs encoded by the expression cassette in the HSV genome were transported to the cell surface in association with {beta}2m.

We also demonstrate that H2Kd can be expressed by S-130-infected human neuroblastoma cells in vitro and murine primary sensory neurons in vivo. These data indicate that HSV-infected murine primary sensory neurons are capable of expressing the product of an MHC-I transgene, delivered by an HSV vector, at the cell surface. From this we infer that overexpressing or driving hard the transcription of {alpha}C genes is sufficient to overcome potential impediments to expression of stable MHC-I complexes on neuronal surfaces.


   Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Cells.
Vero (African green monkey kidney) cells (ATCC CCL 81) and IMR-32 (ATCC CCL 127; derived from a human abdominal neuroblastoma) cells were grown in HDMEM supplemented with 10% foetal calf serum (FCS). P815 (H2d mouse mastocytoma) cells (ATCC TIB 64) were grown in HDMEM with 10% heat-inactivated FCS. K562 (human myelogenous leukaemia) cells and HUT 78 (human T lymphoblastoid) cells were grown in RPMI supplemented with 10% FCS.

{blacksquare} Antibodies.
The following murine monoclonal antibodies (MAbs) were used: 34-1-2S (IgG2a; anti-H2Kd/b/Dd), from the hybridoma ATCC HB79); ID4.5 (IgG2b), directed against a salmonella antigen (O’Connor & Ashman, 1982 ); and 7B6 (IgG1), directed against human {beta}2m–{alpha}C complexes (Stoppini et al., 1997 ).

{blacksquare} Viruses.
Experiments were done with HSV-1 strains C3b or S-130. C3b (Lachmann et al., 1996 ) is a recombinant HSV which contains the E. coli lacZ gene, under the control of the immediate early (IE-1) promoter/enhancer of HCMV) inserted into a 168 bp HpaI deletion within the major LAT region of HSV-1 strain SC16 (Hill et al., 1975 ). S-130 is a recombinant HSV containing an MHC-I heavy chain (H2Kd) cDNA inserted within the major LAT region of HSV-1 strain C3b (Fig. 1). Virus working stocks were grown in Vero cells and titres determined by plaque assay using the suspension method of Russell (1962) .



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. Structure of HSV-1 strain S-130. The region spanning nucleotides 118500 to 123500 is expanded to show the major LAT region (black bar), which in turn is expanded to show the structure of the H2 expression cassette and its point of insertion into the viral genome. The position of the 381 bp subclone (pGEMAAC), used as a probe specific for transcripts from the cassette, is shown by the open box.

 
{blacksquare} Plasmids.
PSLAT-1 (Lachmann & Efstathiou, 1997 ) contains a 4·8 kb BamHI–Pst fragment from the LAT region of HSV-1 strain SC16 cloned into pBluescribe. pRC/CMV (Invitrogen) is a neomycin-resistant eukaryotic expression vector which contains a multiple cloning site flanked by the HCMV IE-1 promoter/enhancer and the bovine growth hormone (BgH) polyadenylation signal sequence. pLKd-R contains an H2Kd expression cassette comprising the HCMV IE-1 promoter/enhancer, a 1·1 kb H2Kd cDNA (Lalanne et al., 1983 ) and a BgH polyadenylation signal sequence. The H2Kd expression cassette was cloned into the HpaI site of pSLAT-1, resulting in HSV-1 LAT sequences flanking the H2Kd expression cassette. pGEMAAC (Fig. 1) was constructed by cloning a 381 bp BamHI fragment from pRC/CMV into pGEM-4Z (Promega).

{blacksquare} Construction of a recombinant HSV-1, S-130, encoding MHC class I heavy chain.
S-130 was constructed by co-transfection of sub-confluent Vero cell monolayers with pLKd-R DNA (3 µg) and purified C3b virion DNA (10 µg) by calcium phosphate co-precipitation as described by Lachmann & Efstathiou (1997) . The site of homologous recombination was designed to cause disruption of the lacZ locus in C3b such that recombinant viruses produced ‘white’ plaques in the presence of X-Gal. Non-{beta}-galactosidase producing recombinant virus was selected by plaque purification. Vero cells were infected with transfection progeny virus at a dilution resulting in approximately 20 plaques per 60 mm tissue culture dish (Nunc). After 2 days, white plaques were detected using an overlay of low melting temperature ME-agarose containing 300 µg/ml X-Gal (Boehringer Mannheim). Agar plates containing white plaques were picked with a Pasteur pipette and incubated at 4 °C for 1 h in HDMEM-MM. A further three rounds of plating and plaque picking were done in order to obtain pure white stocks.

{blacksquare} One-step virus growth curves.
Vero cell monolayers in 60 mm tissue culture dishes were infected at a multiplicity of 5 p.f.u. per cell in 1 ml of HDMEM-MM. After a 1 h adsorption period, the cells were washed with HDMEM-MM and incubated at 37 °C with HDMEM-MM. At various times after infection cells were harvested, lysed by three cycles of freezing and thawing and tested on Vero cells for infectious virus.

{blacksquare} Preparation of cultured cells for immunohistochemistry and rosetting.
Vero, K562 and IMR-32 cells grown in 25 cm2 flasks (Corning) were infected at 10 p.f.u. per cell. After virus adsorption for 1 h the inoculum was replaced with HDMEM-GM or RPMI-GM. Cells were harvested 5 h after infection, washed and resuspended in PBS (4x106 cells/ml) and smeared onto glutaraldehyde-activated aminopropyltriethoxysilane (APES)-coated slides. Smears were dried overnight at room temperature and fixed in methanol at 4 °C for 15 min. In rosetting experiments, cells were washed and resuspended in HDMEM containing 0·5% BSA (4x106 cells/ml).

{blacksquare} Immunohistochemical detection of HSV and H2Kd antigens.
HSV and H2Kd antigens were detected in cell smears using a peroxidase–anti-peroxidase (PAP) method (Moriarty et al., 1973 ). The primary antibody for HSV antigen detection was rabbit anti-HSV-infected cells, diluted 1:50. Binding of primary antiserum was detected using swine antiserum against rabbit immunoglobulin (Ig), diluted 1:25, followed by rabbit PAP complex, diluted 1:200 (all antibodies from Dakopatts). The primary antibody for H2Kd antigens was 34-1-2S. Binding of primary antibody was detected using goat anti-mouse Ig, diluted 1:50 (Dakopatts), followed by mouse PAP complex, diluted 1:100 (Dakopatts). All antisera were diluted in Tris-buffered saline (TBS) containing 10% normal swine serum for HSV detection or 10% normal goat serum for H2Kd detection. All reactions were allowed to proceed for 30 min in a humidified atmosphere at 37 °C, with a 10 min wash in TBS between each step. Peroxidase activity was detected by immersing slides for 4 min in the dark in 0·5 mg/ml DAB–0·1% H2O2. Cells were lightly counterstained with rapid haematoxylin, dehydrated in ethanol and mounted with DePex (BDH).

{blacksquare} Rosetting assays.
Direct rosetting was done as described by Parish & McKenzie (1978) with the following modifications: sheep red blood cells (srbc) were coupled to MAb at 400 µg/ml with chromic chloride at 0·03125%. Indirect rosetting was done using protein A-coupled srbc as described by Sandrin et al. (1978) .

{blacksquare} Inoculation of mice.
C3H/HEJ (H2k) mice (>8 weeks of age) were obtained from the Specific Pathogen-Free facility, Animal Resource Center, Perth, Western Australia. Animals were inoculated with 1·0x105 p.f.u. in their left flanks, as previously described (Simmons & Nash, 1984 ). At various times after infection mice were killed by CO2 asphyxiation and consecutive dorsal root ganglia (T8–T13) ipsilateral to the inoculation site were removed, pooled and placed immediately in PBS at 4 °C.

{blacksquare} Preparation of cell suspensions from thoracic dorsal root ganglia.
Pooled ganglia were washed three times in PBS and enzymatically dissociated with collagenase–dispase (10 mg/ml; Boehringer Mannheim) for 1·5 h at 37 °C with occasional gentle tituration. For rosetting, ganglionic cells were washed in PBS and resuspended in HDMEM containing 0·5% BSA. Cells to be used for immunohistochemical staining were resuspended in PBS, smeared onto glutaraldehyde-activated APES-coated slides, dried at room temperature and fixed in methanol for 15 min at 4 °C.

{blacksquare} Northern hybridization.
H2Kd transcripts were detected in total RNA extracted from cells according to standard protocols (Sambrook et al., 1989 ). The probe (made from pGEMAAC) corresponded to sequences which could have been derived only from the H2Kd expression cassette, rather than endogenous MHC (Fig. 1). Filters were washed to a final stringency of Tm-10 °C (i.e. 10 °C below the 50% hybrid melting temperature) and bound probe was detected by autoradiography with X-AR film (Kodak).


   Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Construction and characterization of S-130, a recombinant HSV-1 encoding H2Kd
An expression cassette, pLKd-R, containing H2Kd cDNA driven by the HCMV major IE promoter/enhancer and flanked by HSV-1 major LAT sequences (Fig. 1) was transfected into Vero cells and shown by Northern blot analysis to synthesize full-length H2Kd RNA transcripts of the predicted size (1·8 kb; data not shown). The H2Kd expression cassette was inserted into both genomic copies of the LAT unit of the parental HSV-1 strain C3b by homologous recombination between pLKd-R LAT sequences and viral DNA from HSV-1 strain C3b (Fig. 1). S-130 was shown to contain the H2Kd expression cassette in both genomic copies of the LAT unit of parent virus C3b by Southern blot hybridization (data not shown). The LAT locus was chosen as the insertion site because disruption of this region does not inhibit virus replication in cultured cells, mouse peripheral tissues and neurons (Ho & Mocarski, 1989 ).

Single-step growth curves were done to determine whether the H2Kd recombinant virus S-130 had the same replication kinetics in vitro as its parent virus, C3b. Confluent Vero cell monolayers were infected (5 p.f.u. per cell) with S-130 or C3b. Infectious virus was quantified from cells harvested 2, 4, 8, 12, 16 and 24 h after infection (Fig. 2). Lag periods, growth rates and virus yields were the same for S-130 and C3b. Furthermore, the plaque morphologies of both viruses were identical. It was concluded that S-130 replicates in Vero cells in a manner indistinguishable from its parental virus, C3b.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2. Single-step growth curves for S-130 ({square}) and C3b ({blacktriangleup}). Vero cells were infected (5 p.f.u. per cell) and virus yield was quantified at the times shown.

 
To show that H2Kd was transcribed in infected cells, confluent Vero cell monolayers were infected (10 p.f.u. per cell) with S-130 and total RNA was examined by Northern blot analysis 2·5, 5 and 8 h later using a probe specific for the expression cassette (Fig. 1). A 1·8 kb RNA, corresponding to the expected size of full-length H2Kd transcripts from the expression cassette, was detected at all three time-points (Fig. 3). The intensity of the 1·8 kb band increased over the time studied.



View larger version (69K):
[in this window]
[in a new window]
 
Fig. 3. Detection of H2Kd RNA in S130-infected Vero cells by Northern hybridization. Cellular RNA (5 µg/lane) was transferred to nitrocellulose and hybridized with probe derived from pGEMAAC (Fig. 1). Filters were washed to a stringency of Tm-10 °C and exposed to Kodak X-AR film. Lane 1, uninfected cells; lane 2, cells 8 h after infection with C3b; lanes 3–5, cells at 2·5, 5 and 8 h after infection, respectively, with S-130. Arrowhead: expected size of full-length H2Kd transcripts. Migration of 28S and 18S RNAs is marked for reference. High molecular mass read-through transcripts are visible at the later time-points.

 
Detection of H2Kd on the surfaces of S-130-infected human neuroblastoma cells
H2Kd antigen expression was studied in IMR-32 cells, a human neuroblastoma cell line that expresses low levels of {alpha}C mRNA and cell-surface MHC-I (Marozzi et al., 1993 ). A rosetting assay was used to determine whether H2Kd antigens were expressed on the surfaces of S-130-infected cells. Rosetting was chosen because it is a sensitive and simple method for the detection of cell-surface molecules (Goding, 1976 ; Parish & McKenzie, 1978 ) which was shown recently to be suitable for characterizing H2 on neurons (Pereira & Simmons, 1999 ).

Confluent IMR-32 cell monolayers were infected (10 p.f.u. per cell) with S-130 or C3b and harvested 5 h after infection. Cell suspensions were reacted with 34-1-2S- (anti-H2Kd/b) coated srbc. P815 cells were included as a positive control for the presence of cell-surface H2Kd antigens (not illustrated). Uninfected or C3b-infected cells and ID4.5- (anti-salmonella) coated srbc were used to measure non-specific rosetting. S-130-infected IMR-32 cells formed rosettes with 34-1-2S-coated srbc, whereas background rosetting was minimal with S-130-infected cells incubated with ID4.5-coated srbc and C3b infected and uninfected cells incubated with 34-1-2S-coated srbc (Fig. 4). It was concluded that H2Kd is expressed on the surfaces of S-130-infected human neuroblastoma cells.



View larger version (89K):
[in this window]
[in a new window]
 
Fig. 4. Detection of H2Kd molecules on the surfaces of S-130-infected IMR-32 human neuroblastoma cells by rosetting. Panels show cells (x960) reacted with 34-1-2S- (anti-H2) coated srbc (A–C) and ID4.5- (anti-salmonella) coated srbc (D). Cells were infected with S-130 (A, D), C3b (B) or not infected (C).

 
Detection of H2Kd and {beta}2m on the surfaces of S-130-infected K562 cells
K562 cells are MHC-I deficient human myelogenous leukaemia cells (Lozzio & Lozzio, 1979 ) which are able to synthesize {beta}2m but cannot express it on their cell surfaces owing to an inability to make {alpha}Cs (Chen et al., 1986 ; Sutherland et al., 1985 ). Therefore, K562 cells were used to determine whether S-130-derived H2Kd was expressed in association with {beta}2m on the surfaces of S-130-infected cells. K562 cells were infected (10 p.f.u. per cell) with S-130 or C3b and 5 h after infection cell surface {beta}2m or H2Kd antigen expression was examined by indirect rosetting, using anti-human {beta}2m complexed with mouse {alpha}C (7B6) or anti-H2Kd/b/Dd (34-1-2S) MAbs. In these studies HUT 78 (a human lymphoblastoid cell line) and P815 cells were included as positive controls for cell-surface human {beta}2m and H2Kd antigen respectively. In addition, uninfected K562 cells and MAb ID4.5 were used to determine the extent of non-specific rosetting. {beta}2m–{alpha}C complexes and H2Kd were detected (with 7B6 and 34-1-2S, respectively) on the surfaces of a similar proportion of cells (46 and 50·3% respectively) in S-130-infected cells. In C3b-infected cultures, {beta}2m and H2Kd were not detected, confirming that {beta}2m is not expressed on K562 surfaces in the absence of {alpha}Cs. These results indicate that H2Kd is expressed on the surfaces of S-130-infected cells as an {alpha}C–{beta}2m heterodimer.

Detection of H2Kd expression by S-130-infected mouse ganglionic neurons
To show that S-130-infected ganglionic neurons synthesize H2Kd, immunohistochemistry was used to study groups of ten C3H (H2k) mice infected either with S-130 or C3b. Cell smears prepared from dissociated ganglionic cells 5 days after infection with S-130 stained strongly with 34-1-2S (anti-Kd) whereas no staining was seen in C3b-infected animals (Fig. 5). Staining for HSV antigens confirmed, however, that similar numbers of cells (15·9% vs. 16·0%) were infected in both groups of mice.



View larger version (53K):
[in this window]
[in a new window]
 
Fig. 5. Ganglionic neurons (x384) recovered from S-130- (A) or C3b- (B) infected C3H (H2k) mice stained immunohistochemically with MAb 34-1-2S (anti-H2Kd). Strong staining (black) was visible only in neurons from mice infected with S-130 (e.g. panel A).

 
Detection of H2Kd on the surfaces of S-130-infected ganglionic neurons
To determine whether H2Kd antigens are synthesized and expressed on the surfaces of primary sensory neurons infected with S-130 in vivo, ganglionic cells (T8–T13) from flank infected mice were analysed by indirect rosetting, using 34-1-2S and protein A-coupled srbc. This procedure was shown previously to be more sensitive than antibody- and complement-mediated cytotoxic assays for detection of cell-surface MHC-I molecules (Sandrin et al., 1978 ). More recently, the method was found suitable for detecting low density cell-surface expression of MHC-I by neurons in HSV-infected ganglia (Pereira & Simmons, 1999 ). Two groups of ten adult C3H mice were infected with S-130 or C3b and cell suspensions from pooled spinal ganglia were prepared 5 days later. H2k mice were selected for this experiment in order to ensure that 34-1-2S detected MHC-I molecules derived from S-130, not host cells, which were recently shown to be induced to express MHC-I in HSV-infected ganglia (Pereira & Simmons, 1999 ). Cells dissociated from uninfected and C3b infected ganglia were included to measure non-specific rosetting. H2Kd specific rosettes (Fig. 6) were formed by 4·1% of neurons from S-130-infected mice. To determine the level of infection, ganglionic cell smears were stained with rabbit anti-HSV polyclonal serum. The proportion of ganglionic cells that were viral antigen positive (5%) was similar to the proportion that formed rosettes. It was concluded that the majority of mouse sensory ganglionic neurons infected with S-130 in vivo express H2Kd antigens on their surfaces.



View larger version (91K):
[in this window]
[in a new window]
 
Fig. 6. Detection of H2Kd molecules on the surfaces of ganglionic neurons (x750) by rosetting. (A)–(C), Cells reacted with 34-1-2S (anti-H2Kd). Neurons illustrated are from ganglia infected with S-130 (A) or C3b (B) and from uninfected mice (C). (D) Neuron from an S-130-infected ganglion reacted with MAb ID4.5 (anti-salmonella).

 
Virus clearance from S130-infected mice
Our previous data indicated that there is already MHC-I expression by neurons in HSV-infected ganglia (Pereira et al., 1994 ; Pereira & Simmons, 1999 ). The current data show that these observations may be extrapolated to HSV-infected neurons themselves, which constitute only a minority of the total number of neurons isolated from dissociated infected ganglia. This led us to hypothesize that up-regulating MHC-I using the S-130 construct would have no effect on clearance of infectious virus, which would have the implication that levels of MHC-I normally induced are sufficient for termination of infection by CD8+ cells. To address this hypothesis, groups of five mice were killed daily from 1 through 7 days after infection either with S-130 or C3b. Mean recovery of infectious virus was not significantly different at any of the times tested (not illustrated). Further, estimation of HSV DNA levels from groups of 25 mice killed 5 and 11 days after infection confirmed that virus clearance was not altered by enhanced MHC-I expression from the S-130 cassette (not shown).


   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Experiments presented in this report show that H2Kd can be synthesized and transported to the surfaces of HSV-infected human neuroblastoma cells in vitro and HSV-infected mouse neurons in vivo, following introduction of an H2Kd transgene encoded by S-130. In the absence of the transporter associated with antigen processing (TAP) and {beta}2m, {alpha}Cs are retained in the endoplasmic reticulum by calnexin, rather than being transported to the cell surface (Degen et al., 1992 ; Jackson et al., 1994 ; Rajagopalan et al., 1994 ) and degradation of {alpha}Cs is rapid unless they are complexed with {beta}2m and peptides. {alpha}C alone is, at best, expressed at very low levels and has not been reported for H2Kd (Allen et al., 1986 ; Bix & Raulet, 1992 ; Hansen et al., 1988 ; Neefjes et al., 1992 ; Ortiz-Navarrete & Hammerling, 1991 ). While neurons are normally deficient in expression of {beta}2m and the TAP, we showed previously that {beta}2m mRNA transcripts accumulate transiently in sensory neurons in ganglia of HSV-infected mice (Pereira et al., 1994 ). Subsequently, {beta}2m molecules were detected on the surfaces of a high proportion of neurons 13 days after infection (Pereira & Simmons, 1999 ). In addition, TAP has been shown to be switched on by IFN-{gamma} in neurons (Joly et al., 1991 ). It is therefore highly likely that detection of H2Kd in the cytoplasm and on the surfaces of S-130-infected cells indicates the formation of stable ternary complexes. In this report, cell-surface H2 expression was achieved by over-expression from a transgene, but it is not known to what extent over-expression and cytokine-mediated induction might overlap. The balance between the levels of expression of MHC-I genes and inhibitors of ternary complex formation, such as ICP47, governs whether functional MHC-I molecules appear at the cell surface. We draw attention to our previous demonstration that endogenous H2 genes can be induced to sufficiently high levels in uninfected neurons to result in appearance of cell-surface {alpha}C–{beta}2m heterodimers.

In this study, no evidence was found that S-130 infection prevented cell-surface H2Kd expression in IMR-32 cells or mouse neurons. Interestingly, an HSV IE protein, ICP47, can switch off TAP, preventing the formation of stable ternary complexes (Fruh et al., 1995 ; Hill et al., 1994 , 1995 ; York et al., 1994 ). This effect appears to be host- and cell type-specific, in that it has been demonstrated in fibroblast and epithelioid cells of human origin and not in mouse cells (Fruh et al., 1995 ; Hill et al., 1995 ). However, ICP47 does not appear to prevent antigen presentation in HSV-infected human B cells because they can be lysed by HSV-specific CD8+ CTLs (Koelle et al., 1993 ; Posavad & Rosenthal, 1992 ). Despite strong sequence and protein homology between human and murine TAPs (Monaco, 1992 ; Townsend & Trowsdale, 1993 ), introduction of murine TAP-1 and TAP-2 into ICP47-expressing human (HeLa) cells does not inhibit cell-surface MHC-I expression (Fruh et al., 1995 ). Nonetheless, Goldsmith et al. (1998) showed that ICP47 enhances HSV neurovirulence in mice by blocking the CD8+ T cell response. In the present study, cell-surface H2Kd antigens were detected on human IMR-32 and K562 cells infected with S-130. Hence it appears that HSV does not prevent cell surface {alpha}C expression in either of these human cell types, which are of neuroblastoma and myelogenous leukaemia origins, respectively. In the case of K562 cells, H2Kd was shown directly to be accompanied by expression of cell-surface {beta}2m, indicating that there is no fundamental problem with expression of {beta}2m in HSV-infected cells or its ability to complex with {alpha}Cs.

The cytokines responsible for up-regulation of MHC-I molecules, including {beta}2m and TAP, are not known. IFN-{gamma} is an obvious candidate because some cells of neuronal origin are known to respond to IFN-{gamma} by expressing MHC-I in vitro (Joly & Oldstone, 1992 ; Lampson & Fisher, 1984 ; Main et al., 1988 ). This hypothesis can be approached by determining whether components of the MHC-I antigen presentation pathway are up-regulated in neurons of IFN-{gamma} knockout mice. Another candidate is the neuronally derived IFN-{gamma} immunoreactive molecule described independently by two groups (Eneroth et al., 1991 ; Kiefer & Kreutzberg, 1990 ; Olsson et al., 1994 ). This molecule has not at the time of writing been characterized fully but its postulated role in stimulating neuronal MHC-I is consistent with our observation that the stimulus for MHC induction appears to derive from infected neurons (Pereira et al., 1994 ). We also note with interest that cultured neurons have recently been shown to synthesize conventional IFN-{gamma} (Neumann et al., 1997 ).

Our observations have two main implications. First, with the caveat that the {alpha}Cs described here were encoded by a transgene, the ability of HSV-infected cells to display H2Kd on their surfaces shows that MHC-I expression in HSV-infected murine ganglia need not be confined to uninfected cells. Second, the behaviour of IMR-32 neuroblastoma cells indicates, in principle, that over-expression of {alpha}C genes can overcome potential impediments to the appearance of MHC-I complexes at surfaces of cells of human neuronal origin. Cell-surface MHC-I expression is governed by the levels of transcription of components of MHC-I ternary complexes and inhibitors of complex formation, such as ICP47. In the current work, {alpha}Cs were over-expressed from a transgene encoded by a viral vector but the potential overlap between this degree of over-expression and maximal levels of cytokine-mediated induction is not known and has not been explored. However, we draw attention to the fact that we showed previously that HSV infection induces neurons in infected ganglia to express endogenously encoded MHC-I–{beta}2m heterodimers on neuronal surfaces (Pereira & Simmons, 1999 ), but in that report it was not possible to establish whether such a response could be achieved in infected cells. Expression of MHC-I molecules by neurons in HSV-infected ganglia (Pereira et al., 1994 ; Pereira & Simmons, 1999 ), including their presence on infected cells, may have profound implications for the mechanism by which ganglionic infection is terminated. Hence, the observations reported here represent a potentially important step towards elucidating the mechanisms responsible for control of HSV infection of neurons. We showed previously that clearance of infectious virus from the peripheral nervous systems of experimentally infected mice is dependent on CD8+ cells but not on death of infected neurons (Simmons & Tscharke, 1992 ). We have proposed that low density MHC-I expression by neurons favours a non-cytolytic cytokine-mediated response (Pereira & Simmons, 1999 ) and the molecular basis of the mechanism involved is a focus of future work.


   Acknowledgments
 
We thank David Johnson for critical review of the manuscript and Barry Slobedman for assistance and helpful discussion. This project was supported by National Health and Medical Research Council of Australia grant 96-0535. A.A. was the holder of a Royal Adelaide Hospital Postgraduate Research Scholarship.


   Footnotes
 
b Present address: Centre for Virus Research, Westmead, NSW 2145, Australia.

c Present address: Department of Pathology, University of Cambridge, UK.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Allen, H., Fraser, J., Flyer, D., Calvin, S. & Flavell, R. (1986). Beta 2-microglobulin is not required for cell surface expression of the murine class I histocompatibility antigen H-2Db or of a truncated H-2Db. Proceedings of the National Academy of Sciences, USA 83, 7447-7451.[Abstract]

Bix, M. & Raulet, D. (1992). Functionally conformed free class I heavy chains exist on the surface of beta 2 microglobulin negative cells. Journal of Experimental Medicine 176, 829-834.[Abstract]

Chen, E., Karr, R. W., Frost, J. P., Gonwa, T. A. & Ginder, G. D. (1986). Gamma interferon and 5-azacytidine cause transcriptional elevation of class I major histocompatibility complex gene expression in K562 leukemia cells in the absence of differentiation. Molecular and Cellular Biology 6, 1698-1705.[Medline]

Degen, E., Cohen-Doyle, M. F. & Williams, D. B. (1992). Efficient dissociation of the p88 chaperone from major histocompatibility complex class I molecules requires both beta 2- microglobulin and peptide.Journal of Experimental Medicine 175, 1653-1661.[Abstract]

Eneroth, A., Kristensson, K., Ljungdahl, A. & Olsson, T. (1991). Interferon-gamma-like immunoreactivity in developing rat spinal ganglia neurons in vivo and in vitro. Journal of Neurocytology 20, 225-231.[Medline]

Fruh, K., Ahn, K., Djaballah, H., Sempe, P., van Endert, P. M., Tampe, R., Peterson, P. A. & Yang, Y. (1995). A viral inhibitor of peptide transporters for antigen presentation. Nature 375, 415-418.[Medline]

Goding, J. W. (1976). Monoclonal Antibodies: Principles and Practice, 2nd edn, pp. 87–88. London: Academic Press.

Goldsmith, K., Chen, W., Johnson, D. C. & Hendricks, R. L. (1998). Infected cell protein (ICP)47 enhances herpes simplex virus neurovirulence by blocking the CD8+ T cell response.Journal of Experimental Medicine 187, 341-348.[Abstract/Free Full Text]

Hansen, T. H., Myers, N. B. & Lee, D. R. (1988). Studies of two antigenic forms of Ld with disparate beta 2-microglobulin (beta 2m) associations suggest that beta 2m facilitate the folding of the alpha 1 and alpha 2 domains during de novo synthesis. Journal of Immunology 140, 3522-3527.[Abstract/Free Full Text]

Hill, T. J., Field, H. J. & Blyth, W. A. (1975). Acute and recurrent infection with herpes simplex virus in the mouse: a model for studying latency and recurrent disease. Journal of General Virology 28, 341-353.[Abstract]

Hill, A. B., Barnett, B. C., McMichael, A. J. & McGeoch, D. J. (1994). HLA class I molecules are not transported to the cell surface in cells infected with herpes simplex virus types 1 and 2.Journal of Immunology 152, 2736-2741.[Abstract/Free Full Text]

Hill, A., Jugovic, P., York, I., Russ, G., Bennink, J., Yewdell, J., Ploegh, H. & Johnson, D. (1995). Herpes simplex virus turns off the TAP to evade host immunity. Nature 375, 411-415.[Medline]

Ho, D. Y. & Mocarski, E. S. (1989). Herpes simplex virus latent RNA (LAT) is not required for latent infection in the mouse.Proceedings of the National Academy of Sciences, USA 86, 7596-7600.[Abstract]

Jackson, M. R., Cohen-Doyle, M. F., Peterson, P. A. & Williams, D. B. (1994). Regulation of MHC class I transport by the molecular chaperone, calnexin (p88, IP90). Science 263, 384-387.[Medline]

Joly, E. & Oldstone, M. B. (1992). Neuronal cells are deficient in loading peptides onto MHC class I molecules. Neuron 8, 1185-1190.[Medline]

Joly, E., Mucke, L. & Oldstone, M. B. (1991). Viral persistence in neurons explained by lack of major histocompatibility class I expression. Science 253, 1283-1285.[Medline]

Kiefer, R. & Kreutzberg, G. W. (1990). Gamma interferon-like immunoreactivity in the rat nervous system. Neuroscience 37, 725-734.[Medline]

Koelle, D. M., Tigges, M. A., Burke, R. L., Symington, F. W., Riddell, S. R., Abbo, H. & Corey, L. (1993). Herpes simplex virus infection of human fibroblasts and keratinocytes inhibits recognition by cloned CD8+ cytotoxic T lymphocytes.Journal of Clinical Investigation 91, 961-968.[Medline]

Lachmann, R. H. & Efstathiou, S. (1997). Utilization of the herpes simplex virus type 1 latency-associated regulatory region to drive stable reporter gene expression in the nervous system. Journal of Virology 71, 3197-3207.[Abstract]

Lachmann, R. H., Brown, C. & Efstathiou, S. (1996). A murine RNA polymerase I promoter inserted into the herpes simplex virus type I genome is functional during lytic, but not latent infection. Journal of General Virology 77, 2575-2582.[Abstract]

Lalanne, J. L., Delarbre, C., Gachelin, G. & Kourilsky, P. (1983). A cDNA clone containing the entire coding sequence of a mouse H-2Kd histocompatibility antigen. Nucleic Acids Research 11, 1567-1577.[Medline]

Lampson, L. A. & Fisher, C. A. (1984). Weak HLA and beta 2-microglobulin expression of neuronal cell lines can be modulated by interferon. Proceedings of the National Academy of Sciences, USA 81, 6476-6480.[Abstract]

Lozzio, B. B. & Lozzio, C. B. (1979). Properties and usefulness of the original K-562 human myelogenous leukemia cell line. Leukemia Research 3, 363-370.[Medline]

Main, E. K., Monos, D. S. & Lampson, L. A. (1988). IFN-treated neuroblastoma cell lines remain resistant to T cell- mediated allo-killing, and susceptible to non-MHC-restricted cytotoxicity. Journal of Immunology 141, 2943-2950.[Abstract/Free Full Text]

Marozzi, A., Meneveri, R., Bunone, G., De Santis, C., Lopalco, L., Beretta, A., Agresti, A., Siccardi, A. G., Della Valle, G. & Ginelli, E. (1993). Expression of beta 2m-free HLA class I heavy chains in neuroblastoma cell lines. Scandinavian Journal of Immunology 37, 661-667.[Medline]

Marrack, P. & Kappler, J. (1987). The T cell receptor. Science 238, 1073-1079.[Medline]

Monaco, J. J. (1992). Genes in the MHC that may affect antigen processing. Current Opinion in Immunology 4, 70-73.[Medline]

Moriarty, G. C., Moriarty, C. M. & Sternberger, L. A. (1973). Ultrastructural immunocytochemistry with unlabeled antibodies and the peroxidase–antiperoxidase complex. A technique more sensitive than radioimmunoassay. Journal of Histochemistry and Cytochemistry 21, 825-833.[Medline]

Neefjes, J. J., Smit, L., Gehrmann, M. & Ploegh, H. L. (1992). The fate of the three subunits of major histocompatibility complex class I molecules. European Journal of Immunology 22, 1609-1614.[Medline]

Neumann, H., Schmidt, H., Wilharm, E., Behrens, L. & Wekerle, H. (1997). Interferon gamma gene expression in sensory neurons: evidence for autocrine gene regulation. Journal of Experimental Medicine 186, 2023-2031.[Abstract/Free Full Text]

O’Connor, C. G. & Ashman, L. K. (1982). Application of the nitrocellulose transfer technique and alkaline phosphatase conjugated anti-immunoglobulin for determination of the specificity of monoclonal antibodies to protein mixtures. Journal of Immunological Methods 54, 267-271.[Medline]

Olsson, T., Kelic, S., Edlund, C., Bakhiet, M., Hojeberg, B., van der Meide, P. H., Ljungdahl, A. & Kristensson, K. (1994). Neuronal interferon-gamma immunoreactive molecule: bioactivities and purification. European Journal of Immunology 24, 308-314.[Medline]

Ortiz-Navarrete, V. & Hammerling, G. J. (1991). Surface appearance and instability of empty H-2 class I molecules under physiological conditions. Proceedings of the National Academy of Sciences, USA 88, 3594-3597.[Abstract]

Parish, C. R. & McKenzie, I. F. (1978). A sensitive rosetting method for detecting subpopulations of lymphocytes which react with alloantisera. Journal of Immunological Methods 20, 173-183.[Medline]

Pereira, R. A. & Simmons, A. (1999). Cell surface expression of H2 antigens on primary sensory neurons in response to acute but not latent herpes simplex virus infection in vivo. Journal of Virology 73, 6484-6489.[Abstract/Free Full Text]

Pereira, R. A., Tscharke, D. C. & Simmons, A. (1994). Upregulation of class I major histocompatibility complex gene expression in primary sensory neurons, satellite cells, and Schwann cells of mice in response to acute but not latent herpes simplex virus infection in vivo. Journal of Experimental Medicine 180, 841-850.[Abstract]

Posavad, C. M. & Rosenthal, K. L. (1992). Herpes simplex virus-infected human fibroblasts are resistant to and inhibit cytotoxic T-lymphocyte activity.Journal of Virology 66, 6264-6272.[Abstract]

Rajagopalan, S., Xu, Y. & Brenner, M. B. (1994). Retention of unassembled components of integral membrane proteins by calnexin.Science 263, 387-390.[Medline]

Russell, W. C. (1962). A sensitive and precise assay for herpes virus. Nature 195, 1028-1029.[Medline]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Sandrin, M. S., Potter, T. A., Morgan, G. M. & McKenzie, I. F. (1978). Detection of mouse alloantibodies by rosetting with protein A-coated sheep red blood cells. Transplantation 26, 126-130.[Medline]

Simmons, A. & Nash, A. A. (1984). Zosteriform spread of herpes simplex virus as a model of recrudescence and its use to investigate the role of immune cells in prevention of recurrent disease. Journal of Virology 52, 816-821.[Medline]

Simmons, A. & Tscharke, D. C. (1992). Anti-CD8 impairs clearance of herpes simplex virus from the nervous system: implications for the fate of virally infected neurons. Journal of Experimental Medicine 175, 1337-1344.[Abstract]

Stoppini, M., Bellotti, V., Mangione, P., Merlini, G. & Ferri, G. (1997). Use of anti-(beta2 microglobulin) mAb to study formation of amyloid fibrils. European Journal of Biochemistry 249, 21-26.[Abstract]

Sutherland, J., Mannoni, P., Rosa, F., Huyat, D., Turner, A. R. & Fellous, M. (1985). Induction of the expression of HLA class I antigens on K562 by interferons and sodium butyrate. Human Immunology 12, 65-73.[Medline]

Townsend, A. & Trowsdale, J. (1993). The transporters associated with antigen presentation. Seminars in Cell Biology 4, 53-61.[Medline]

Van Agthoven, A. J., Lemonnier, F. A., Kkourilsky, F. M. & Jordan, B. R. (1984). Transformation of LMTK cells with purified HLA class I genes. V. A determinant on beta 2- microglobulin is controlled by heavy chain in a mouse–human hybrid complex: a biochemical analysis. Molecular Immunology 21, 175-179.[Medline]

York, I. A., Roop, C., Andrews, D. W., Riddell, S. R., Graham, F. L. & Johnson, D. C. (1994). A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes. Cell 77, 525-535.[Medline]

Received 8 March 2000; accepted 22 June 2000.