1 Departments of Microbiology & Immunology and Pediatrics, Medical College of Virginia Campus of Virginia Commonwealth University, 1101 E. Marshall Street, Richmond, Virginia 23298-01632, USA
2 Department of Parasitology, Hirosaki University School of Medicine, Hirosaki, Japan
Correspondence
Michael A. McVoy
mmcvoy{at}hsc.vcu.edu
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ABSTRACT |
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Several candidate vaccines utilizing live attenuated HCMV strains are in development (Balfour et al., 1984; Kemble et al., 1996
; Plotkin et al., 1991
, 1994
). These strains express a number of proteins designed to modify host immune responses, including four that down-regulate surface expression of MHC class I (Ahn et al., 1997
; Jones et al., 1995
, 1996
; Jones & Sun, 1997
; Wiertz et al., 1996
). As antigen presentation by class I is critical for induction of both cell- and antibody-mediated host immunity, down-regulation of class I by vaccine strains is counter-intuitive to induction of robust immune responses. Removal of the class I down-regulation genes should increase presentation of viral peptides on infected cell surfaces and may potentiate vaccine immunogenicity. However, replication in the host is also an important factor in live virus vaccines. Although reduced replication might improve vaccine safety, a class Idown-regulation-deficient virus might also be rendered so vulnerable to immune clearance that replication and persistence in the host would be inadequate to establish enduring immunity. Due to the difficulties of conducting human trials with recombinant HCMV viruses, the impact of class I down-regulation on HCMV vaccine efficacy will be difficult to determine.
Several animal cytomegaloviruses have been studied as models for HCMV disease. However, only guinea pig cytomegalovirus (GPCMV) provides a small animal model for congenital infection (Griffith & Aquino-de Jesus, 1991; Griffith et al., 1981
, 1990
; Liu & Biegalke, 2001
; Schleiss et al., 2000
). Congenital transmission of GPCMV can be induced experimentally following intraperitoneal, subcutaneous or intranasal inoculation of pregnant dams (Griffith et al., 1990
; Nankervis & Kumar, 1978
), and prior maternal immunity, induced either by natural infection or vaccination, can reduce transmission and protect against congenital disease (Bia et al., 1980
, 1982
; Fong et al., 1983
; Harrison et al., 1995
; Johnson & Connor, 1979
; Nankervis & Kumar, 1978
).
In order to develop GPCMV as a model to study the significance of class I down-regulation, we evaluated the ability of GPCMV to down-regulate class I. Guinea pig embryo fibroblast (GEF) cells were cultivated in culture medium consisting of Dulbecco's Modified Essential Medium, 10 % foetal bovine serum (FBS), 50 µg streptomycin ml-1 and 50 U penicillin (BioWhittaker) ml-1 as previously described (McVoy et al., 1997). Virus-infected cells were distinguished from uninfected cells by the use of a recombinant virus, GPCMV/EGFP, which expresses enhanced green fluorescent protein (EGFP) fused to puromycin N-acetyltransferase (Abbate et al., 2001
). Cells were mock-infected or infected with GPCMV/EGFP at different m.o.i. values, trypsinized at various time points and frozen at -70 °C in 90 % FBS/10 % DMSO. After thawing, cells were washed twice with staining buffer (PBS/10 % FBS) by centrifugation (500 g, 5 min, 4 °C) and incubated for 30 min on ice in 100 µl staining buffer containing monoclonal antibodies (mAbs) in ascites (0·5 µg total protein). Murine mAb HUSM-41, specific for guinea pig class I (Sato et al., 1997
), was used to detect class I, while mAb HSUM-49, specific for guinea pig class II (Sato et al., 1997
), served as an isotype-matched negative control. Cells were washed and then incubated for 30 min on ice in 100 µl staining buffer containing 0·1 µg biotin-conjugated rat mAb R8-140 (
-mouse Ig
) ml-1 (PharMingen). The cells were washed again, incubated 30 min on ice in 100 µl staining buffer containing 10 µg streptavidinR-phycoerythrin (Life Technologies) ml-1, washed and finally resuspended in PBS/1 % paraformaldehyde (Sigma). The cells were then analysed using a Beckman FACScan 2000 at wavelengths of 525 nm for EGFP and 575 nm for phycoerythrin.
As expected, the class II-specific mAb HUSM-49 failed to react with either infected or uninfected cells (Fig. 1A). Infected cells expressed EGFP whereas uninfected cells did not (Fig. 1A
). Virus-infected (EGFP+) cells underwent a decrease in class I surface expression and reached a maximal 4·5-fold reduction by 72 h post-infection (p.i.). The proportion of uninfected (EGFP-) cells decreased with increasing m.o.i. but the cells maintained normal levels of class I (Fig. 1B
). Similar results were obtained using a second class I-specific mAb, HUSM-20 (Sato et al., 1997
) (data not shown). Thus, GPCMV specifically down-regulated surface class I expression on infected cells but not on uninfected cells in the same culture.
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This result indicated that the viral proteins responsible for class I down-regulation must either be expressed with IE or early kinetics or be virion-associated and thereby carried into cells at the time of infection. The effects of virion-associated proteins can be distinguished from those requiring de novo expression by their lack of sensitivity to UV irradiation, which prevents viral gene expression by cross-linking the viral DNA (Jing et al., 2001). To determine if down-regulation is mediated by virion-associated proteins, replicate aliquots of viral inocula were exposed to increasing amounts of UV irradiation in a Stratalinker 1800 UV cross-linker (Stratagene) and then adjusted to 5 mM sodium pyruvate (Cellgro) (Fortunato et al., 2000
). GEF cells were incubated with irradiated inocula for 3 h, washed twice with culture media, incubated for 72 h in culture media containing 10 µg HPMPA ml-1 and then analysed for EGFP and surface class I expression. Prior to irradiation, the amount of virus in the inocula was such that approximately 50 % of the cells would be infected. The percentage of cells expressing EGFP decreased in a dose-dependent manner, confirming UV inhibition of de novo viral gene expression (Fig. 2
A). The percentage of class Ilow cells (i.e. those retaining the ability to down-regulate class I) also decreased in a manner that exactly paralleled the decrease in EGFP expression (Fig. 2A
), indicating that down-regulation required de novo gene expression and is therefore not mediated by virion-associated proteins. Thus, expression of one or more IE or early viral genes is responsible for class I down-regulation.
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To determine if down-regulation is specific for class I and is not the consequence of a generalized virus-related inhibition of protein transport to the cell surface, we compared total surface protein profiles in both infected and uninfected cells. Cells were mock-infected or infected at an m.o.i. of 1 or 3 in the presence of 10 µg HPMPA ml-1 for 72 h. Cells were then trypsinized, washed three times with ice-cold PBS, counted, and incubated at room temperature for 30 min in PBS containing 20 µg Sulfo-NHS-LC-biotin (Pierce) to biotinylate surface-exposed proteins. A small aliquot was stained with trypan blue to confirm the integrity of the cell membranes. The remaining cells were lysed in gel loading buffer and subjected to SDS-PAGE (Sambrook et al., 1989). Separated proteins were transferred to a Nytran N membrane (Schleicher and Schuell). The membrane was blocked for 2 h at 4 °C in blocking buffer (Sambrook et al., 1989
) and incubated with streptavidin-conjugated horseradish peroxidase (Roche) according to the manufacturer's instructions. Biotinylated surface proteins were visualized by using Western Lightning chemiluminescence reagent plus (NEN) according to the manufacturer's instructions, followed by exposure to Hyperfilm MP X-ray film (Amersham). Numerous surface proteins were observed in extracts of uninfected cells that remained unchanged in infected cells (Fig. 3
A). One surface protein was clearly down-regulated by virus infection and had a molecular mass of approximately 46 kDa (Fig. 3A
). Although the identity of this protein is unknown, its size is consistent with the molecular masses of class I heavy chains from other species (Campbell & Slater, 1994
; Jones & Sun, 1997
) and therefore it is possible that this protein is the guinea pig class I heavy chain.
|
Many viruses and some bacteria have evolved the ability to inhibit class I antigen presentation on infected cells (for review, see Lorenzo et al., 2001). Down-regulation of class I during lytic replication has now been reported for all eight human herpesviruses and several animal herpesviruses (Ambagala et al., 2000
; Barnes & Grundy, 1992
; Campbell & Slater, 1994
; Cohen, 1998
; Hariharan et al., 1993
; Hirata et al., 2001
; Hudson et al., 2001
; Hunt et al., 2001
; Ishido et al., 2000
; Jones et al., 1995
; Nataraj et al., 1997
; Stevenson et al., 2000
; Tomazin et al., 1998
; Yamashita et al., 1993
; York et al., 1994
; Zeidler et al., 1997
). Thus, class I down-regulation is clearly an important, perhaps essential, component in the herpesviral arsenal of immune evasion mechanisms. Because HCMV is unique among the human herpesviruses in causing congenital disease, the roles of class I down-regulation in congenital transmission and pathogenesis to the foetus represent important questions. Furthermore, at least two vaccine projects are under way utilizing live attenuated HCMV viruses as candidate vaccines (Gonczol & Plotkin, 2001
). These strains retain all four class I down-regulation genes. How down-regulation of class I impacts on the ability of these vaccines to induce host immunity in vivo and protect against congenital disease is unknown.
Because GPCMV provides the only small animal model of congenital cytomegalovirus disease, we chose to investigate its ability to down-regulate class I. Our finding that down-regulation is mediated by viral genes that are expressed with IE or early kinetics should facilitate identification of the specific genes by eliminating a large number of late genes from initial consideration; however, our data do not rule out the possibility that, in addition to IE or early genes, late genes with down-regulation functions may also exist.
The various mechanisms by which herpesviruses down-regulate class I target virtually all stages of the class I expression pathway, including synthesis (Hirata et al., 2001), peptide transport (Ahn et al., 1997
; Ambagala et al., 2000
; Hill et al., 1995
; Hinkley et al., 1998
; Jugovic et al., 1998
; Zeidler et al., 1997
), transit to (Abendroth et al., 2001
; Ahn et al., 1996
; Campbell & Slater, 1994
; del Val et al., 1992
; Hudson et al., 2001
; Jones & Sun, 1997
; Jones et al., 1996
; Reusch et al., 1999
; Wiertz et al., 1996
) and stability on the cell surface (Ishido et al., 2000
). Although our data do not directly address mechanisms for class I down-regulation by GPCMV, our observation that the kinetics of class I loss in response to virus infection closely parallels the response to cycloheximide is consistent with a block to repopulation of surface class I; however, as multiple GPCMV genes may be involved, a detailed understanding of their mechanisms awaits identification of the viral genes and independent analyses of their effects.
Identification of the GPCMV class I down-regulation genes will permit construction of recombinant viruses with mutations in these genes, which can be used to investigate the importance of class I down-regulation in virus pathogenicity, congenital transmission and in utero disease. The impact of these genes can also be determined with regard to levels of induced maternal immunity and more importantly, prevention of congenital disease, when live GPCMVs are used as vaccines. Ultimately, the insights gained from the guinea pig model can be applied to HCMV disease and toward rational designs for improved HCMV live virus vaccines.
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ACKNOWLEDGEMENTS |
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Received 21 June 2002;
accepted 11 September 2002.
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