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Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, NE 68583
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CTLs play a critical role in controlling viral infections, especially in the case of herpesvirus infections (7), where cell-to-cell spread occurs earlier than the hematogenous spread (8). The majority of CTLs are of the CD8+ phenotype and recognize antigenic peptides presented by MHC class I molecules (9). Presentation of viral peptides by class I molecules is the result of a highly intricate multistep pathway of Ag processing and presentation. It involves the cotranslational translocation of class I heavy chain and ß2-microglobulin into the lumen of the endoplasmic reticulum (ER), degradation of viral proteins into short peptides by the cytosolic proteasome, transport of these peptides from the cytosol into the ER by the TAP, binding of the peptides by class I molecule, and egress of this stable trimolecular complex from the ER via the Golgi apparatus to be expressed on the cell surface (10). As much as the complexity of this pathway provides great efficiency in elimination of viral pathogens, it also provides numerous opportunities for the viral proteins to interfere with the individual steps of this pathway, as a means of thwarting the CTL response of the host. Thus, many viruses have evolved mechanisms to interfere with the cell surface expression of class I molecules as a means of evasion of CTL recognition (11). Because PrV primarily infects the epithelial cells of the mucosal surfaces and spreads by cell-to-cell contact, this attenuation of surface class I expression may provide an opportunity to escape CTL recognition until sufficient number of virus progeny is generated to invade the surrounding tissues including neurons. Although, PrV has been shown to induce attenuation of MHC class I expression on porcine cells (12), the mechanism(s) of class I down-regulation, or the viral proteins responsible for the effect, have not been identified. In this study, we demonstrate that inhibition of peptide transport activity of the TAP is one of the mechanisms by which PrV down-regulates class I expression, and that one or more early proteins of this virus may be responsible for this effect.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Clones of porcine kidney (PK-15) and Madin Darby bovine kidney (MDBK) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). The cells were expanded and maintained in RDG medium (5 g/L each of RPMI and DMEM, 4.5 g/L of glucose, and 2.85 g/L of NaHCO3) supplemented with 10% FBS, 2 mM L-glutamine, and 20 µg/ml gentamicin (all from Life Technologies, Grand Island, NY). Indiana Funkhauser (IND-F) strain of PrV was obtained from Dr. Andrew Cheung (National Animal Disease Center, U.S. Department of Agriculture, Ames, IA). The virus was propagated in PK-15 cells and was purified over a potassium tartrate gradient. PrV modified live virus (MLV) vaccine strains PRV/Marker Gold (SyntroVet, Lenexa, KS) and Suvaxyn Herdfend PrV gpI- (SOLVAY Animal Health, Mendota Heights, MN) were also propagated in PK-15 cells.
IND-F strain of PrV, at 4 x 107 PFU, was diluted in 1.5 ml of RDG medium in petri dishes and irradiated under a UV source (Fotodyne trans-illuminator, Fotodyne, New Bwelin, WI) from a distance of 6 inches for 6 min. The efficiency of UV inactivation was assessed by absence of cytopathology following adsorption of the irradiated virus onto permissive PK-15 cells.
Antibodies
The monomorphic anti-porcine class I mAb, PT85A (IgG2a; Ref. 13), was purchased from VMRD (Pullman, WA), and the polymorphic anti-porcine class I mAb, 74.11.10 (IgG2b; Ref. 14), was kindly provided by Dr. Joan Lunney (U.S. Department of Agriculture, Beltsville, MD). Both mAbs PT85A and 74-11-10 recognize peptide-bound class I molecules. The anti-porcine pan-tissue marker mAb, 1030h-1-19 (IgM) was purchased from PharMingen (San Diego, CA). The anti-PrV glycoprotein mAbs were kindly provided by Dr. F. Zuckermann (University of Illinois, Urbana-Champaign). The anti-bovine class I mAb, IL-A88 (IgG2a), was obtained from International Livestock Research Institute (Nairobi, Kenya). The anti-human class I mAb, W6/32 (IgG2a), which cross-reacts with bovine class I molecules, was obtained from the ATCC. The mAb W6/32 binds peptide-bound class I molecules only, whereas IL-A88 binds class I molecules irrespective of peptide binding. The anti-PrV convalescent pig serum was a gift from Dr. Fernando Osorio (University of Nebraska, Lincoln). mAb MM113 (IgG2a) specific for BHV-1 glycoprotein D (15), mAb 6G11 (IgM) specific for BHV-1 glycoprotein gB (16), and normal pig serum were used as control Abs. PE-conjugated goat Abs to mouse IgG were purchased from Biomeda (Foster City, CA). FITC-conjugated goat Abs to pig IgG (H+L) and FITC-conjugated goat anti-mouse IgA, IgG, and IgM were purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD).
Infection of cells
Cells (PK-15 or MDBK) grown to subconfluency in tissue culture flasks (25 cm2) or tissue culture dishes (100 x 20 mm) were infected at an appropriate multiplicity of infection (moi). Mock-infected cells were incubated with a similar volume of medium without the virus. Infection was allowed to proceed up to indicated time periods in a humidified incubator, containing 5% CO2 at 37°C.
Metabolic inhibition experiments
Cycloheximide (ChX) (Sigma, St. Louis, MO) was used at a concentration of 100 µg/ml to inhibit the viral protein synthesis in PrV-infected (10 moi) PK-15 cells, and the cells were subjected to flow cytometry by 6 h postinfection (h.p.i.) (17). To inhibit PrV late protein synthesis, cells were infected at 10 moi in the presence of phosphonoacetic acid (PAA) at 300 µg/ml (Sigma) (18) and the cells were subjected to flow cytometry by 6 h.p.i. ChX reversal experiment was performed to allow accumulation of large amounts of PrV IE180 protein in infected cells (19). Briefly, PK-15 cells were either mock infected or infected at 1 moi in the presence of ChX at 50 µg/ml, and infection was allowed to proceed for 5 h. Then the cells were extensively washed and incubated with growth medium containing actinomycin-D (Act-D) at 2.5 µg/ml (Sigma) to prevent further transcription of mRNA, while allowing the translation of already accumulated IE180 mRNA. At 20 h.p.i. the cells were subjected to flow cytometry.
Flow cytometry
PK-15 and MDBK cells, either mock-infected or infected with PrV at the indicated moi, were trypsinized and immediately suspended in FACS buffer (PBS containing 3% horse serum and 0.01% sodium azide) at selected times postinfection. Cells (2 x 105) were dispensed in duplicates into 96-well round-bottom plates in 50 µl volumes and were incubated with relevant mouse mAbs. After 45 min of incubation at 4°C, the cells were washed four times in FACS buffer and stained with FITC-conjugated goat anti-mouse IgA, IgG, and IgM (2.5 µg/ml) at 4°C for 30 min. After three subsequent washes in FACS buffer, the fluorescence exhibited by the cells was assessed using a Becton Dickinson FACScan flow cytometer (Mountain View, CA). For dual-labeling experiments, the cells were infected at 5 moi for 12 h. Mouse mAbs (1:1000) specific for porcine class I molecules were added, followed by porcine anti-PrV convalescent serum (1:500 dilution). PE-conjugated goat Abs to mouse IgG (5 µg/ml) and FITC-conjugated goat Abs to pig IgG (H+L) were added (2.5 µg/ml) in that order. All the incubations in the dual-labeling experiments were conducted for 30 min at 4°C. To exclude the dead cells, propidium-iodide (Calbiochem-Novabiochem, San Diego, CA) was added at a concentration of 1 µg/ml to all the samples before analysis (20).
In vitro transport assay
PK-15 or MDBK cells (6 x 106),
either mock-infected or infected with PrV, were trypsinized and washed
twice in propagation medium and twice in ice cold transport buffer
[130 mM KCl, 10 mM NaCl, 1 mM CaCl2, 2 mM EGTA,
2 mM MgCl2, and 5 mM HEPES (pH 7.3) with KOH].
Then the cells were incubated at 37°C with 400 µl of transport
buffer containing streptolysin-O (Murex Diagnostics,
Dartford, U.K.) at a concentration of 1–2 U/ml, until 80%
permeablization was achieved, as judged by trypan blue (0.25%)
permeation into cells (21). Subsequently the cells were
washed with 10 ml of ice cold transport buffer to wash away the unbound
streptolysin-O. The cells were resuspended in 400 µl of
transport buffer, divided among four 1.5-ml ultra tubes, and treated
with 0.5 µl of ATP (200 mM) (Sigma) and/or apyrase (20 U/ml) (Sigma)
as indicated in Fig. 4
. Then the cells were incubated for 10 min at
37°C with 10 µl of 125I-labeled peptide
library (2 µM final concentration) composed of 2304 different
peptides each containing a tyrosine for radio-iodination and a
consensus glycosylation motif (NXT/S) (22, 23). The
peptide transport into the ER was terminated by the addition of 1 ml of
"stop buffer" (transport buffer containing 10 mM EDTA and 0.01%
NaN3) at 4°C. For the "0 min" samples, stop
buffer was added immediately after the addition of the radiolabeled
peptides. The samples were centrifuged at 15,000 x g
for 5 min and the supernatant fluid was removed. Then the cells were
incubated in ice cold lysis buffer (0.5% Nonidet P-40, 5 mM
MgCl2, and 50 mM Tris-HCl (pH 7.5)). Thirty
minutes later, the samples were centrifuged at 15,000 x
g for 10 min and the clarified lysate was incubated with 100
µl of Con A-Sepharose beads (Pharmacia Biotech, Piscataway, NJ) for
1 h at 4°C with gentle agitation. Finally, the Sepharose beads
were washed four times with ice cold lysis buffer and the bound
radioactivity was counted using an automated gamma-counter (ICN
Micromedic Systems, Huntsville, AL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PrV has been shown to down-regulate the expression of MHC class I
molecules on PK-15 cells (12). To confirm these findings,
PK-15 cells infected at 5 moi of IND-F strain of PrV were subjected to
flow cytometry at different h.p.i. Cell surface expression of the
porcine pan-tissue marker protein was measured as an internal control
for cellular protein synthesis in PrV-infected cells. There was a
progressive down-regulation of class I expression in PrV-infected PK-15
cells, whereas the expression of the pan tissue marker remained
unaffected (Fig. 1). Flow cytometric
analysis of class I expression on infected cells by this method,
however, does not ensure that a particular cell examined for class I
expression is in fact infected by the virus. Therefore, dual-labeling
analysis for simultaneous detection of class I molecules and virus
proteins was performed on PK-15 cells infected at 5 moi for 12 h.
Results of the dual-labeling analysis confirmed the results of the
previous experiment. At an moi of 5, almost 80% of the cells analyzed
were productively infected with PrV by 12 h.p.i (Fig. 2). The infected cells expressed lower
levels of class I molecules. A bovine cell line, MDBK, that is
permissive to PrV infection was also subjected to the same conditions,
but no detectable class I down-regulation was observed (data not
shown).
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To determine whether PrV MLV vaccine strains also are capable of
down-regulating class I molecules on infected cells, PK-15 cells were
infected at 5 moi with PRV/Marker gold or Suvaxyn vaccine strains. As
shown in Fig. 3, both the MLV vaccine
strains induced a pronounced down-regulation of expression of class I
molecules on PK-15 cell surface.
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To understand the mechanism(s) of class I down-regulation, we
tested the possibility of TAP inhibition by PrV. The peptide transport
activity of porcine TAP was measured by an in vitro transport assay.
The first set of experiments were conducted to ascertain that the
transport assay measured TAP-mediated transport of peptides. Previous
studies have shown that the peptide transport activity of TAP is
ATP-dependent (21). As expected, addition of exogenous ATP
increased the transport of peptides, as shown by the increased recovery
of glycosylated peptides (Fig. 4
A). Furthermore, addition of
apyrase, which results in the hydrolysis of ATP, profoundly impaired
the peptide transport activity (Fig. 4A), indicating that
the transport assay measured TAP-mediated transport of peptides. The
next set of experiments were conducted to determine the effect of PrV
on the transport of peptides. In PrV-infected PK-15 cells, inhibition
of peptide transport activity was observed as early as 2 h.p.i.,
which reached the maximum level by 6 h.p.i. (Fig. 4B).
In contrast, peptide transport activity in MDBK cells was not inhibited
even at 8 h.p.i. (data not shown) suggesting that the TAP
inhibition by PrV may be specific for porcine cells. To determine the
specificity of inhibition of peptide transport activity by PrV, a dose
titration was performed in the next set of experiments. A direct
correlation between the viral moi and the degree of TAP inhibition was
observed (Fig. 4C).
PrV early protein(s) are responsible for the down-regulation of class I molecules
To identify the viral protein(s) responsible for the
down-regulation of class I molecules, we first determined whether de
novo viral protein synthesis was necessary for class I down-regulation.
The cells were treated with UV-inactivated virus at 20 moi, and class I
expression was compared with that of the cells infected with live virus
at 2 moi. Although 2 moi of live PrV induced down-regulation of class I
molecules, even 20 moi of UV-inactivated PrV did not affect the
expression of class I molecules (Fig. 5).
To confirm these findings, cells were infected at 10 moi in the
presence of the protein synthesis inhibitor, ChX. As expected, no class
I down-regulation was observed on PK-15 cells infected in the presence
of ChX (data not shown). Taken together, the above findings confirmed
the requirement for newly synthesized viral proteins for the
down-regulation of class I molecules. The next set of experiments was
designed to infer the class of PrV proteins (immediate early, early, or
late) involved in the down-regulation. First, to ascertain the role of
viral proteins in class I down-regulation, the cells were infected in
the presence of PAA, an inhibitor of herpesvirus DNA synthesis. As
shown in Fig. 6, PAA was unable to
restore class I expression, ruling out the involvement of late proteins
in class I down-regulation. To determine whether PrV IE protein, IE180,
was responsible for this effect, a ChX reversal experiment was
conducted. Even in the presence of large quantities of IE180, class I
expression was not affected suggesting that one or more early proteins
are responsible for the down-regulation (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
Class I expression was reduced to 50% of that of mock-infected cells
by 8 h.p.i (Fig. 3). Continued expression of the porcine
pan-tissue marker protein on the infected cells indicated that the
down-regulatory effect of the virus was specific for class I molecules.
In addition, dual-labeling experiments, in which expression of both
class I molecules and viral proteins was measured simultaneously,
confirmed that the cells which exhibited attenuated class I expression
were in fact infected by the virus (Fig. 2). These findings, therefore,
confirm that PrV specifically down-regulates class I expression on
PK-15 cells.
Currently, MLV vaccines against PrV are being used extensively. If the
MLV vaccine strains are also capable of down-regulation of class I
molecules, immunization of pigs with MLV vaccines would result in
defective CTL priming, not only against PrV but also against other
viruses and intracellular pathogens. Both the vaccine strains that we
tested decreased the expression of class I molecules. In fact, the
extent of class I down-regulation by both the vaccine strains was
almost identical to that observed with the wild-type virus (Fig. 3).
Down-regulation of class I expression by MLV vaccines may explain, at
least partially, the failure of currently used MLV vaccines to induce
complete protection against this disease. In contrast to the findings
with the PK-15 cells, infection of the L14 cell line, an immortalized B
cell line originating from inbred miniature pigs has been reported to
result in an up-regulation of class I expression (24). The
reasons for this discrepancy are not clear. Because porcine B cells are
less permissive to PrV infection (25), the importance of
this finding in the pathogenesis of this virus is questionable.
Mellencamp et al. (12), who reported the down-regulation
of expression of class I molecules by PrV, did not elucidate the
mechanisms by which the virus causes the observed effect in the porcine
epithelial cells, the natural target cells of this virus. In this
study, we have demonstrated that PrV inhibits the peptide transport
activity of porcine TAP. The increase in the transport of peptides with
the addition of exogenous ATP, and the decrease in the transport of
peptides with the addition of apyrase (Fig. 4A), confirmed
that the transport assay that we utilized in this study measured the
TAP-mediated transport of peptides. TAP inhibition was detected as
early as 2 h.p.i. and reached a maximum by 6 h.p.i. (Fig. 4B). Direct correlation between the viral moi and the degree
of TAP inhibition indicated the specificity of inhibition of TAP by PrV
(Fig. 4C).
Previous studies in our laboratory have shown that bovine herpesvirus (BHV)-1 interferes with the TAP activity in MDBK cells (26). Although, MDBK cells (a bovine cell line) are fully permissive to PrV infection, no reduction in TAP activity was observed in infected cells, indicating that this inhibition of TAP by PrV may be species-specific. The inability of PrV to inhibit the TAP activity in MDBK cells also could explain the absence of down-regulation of expression of class I molecules on the surface of these cells.
HSV, human cytomegalovirus (HCMV), and BHV-1 also inhibit the TAP activity in infected cells (26, 27). Infected cell protein (ICP) 47, an IE protein of HSV, has been shown to bind to the peptide binding site of TAP1/2 heterodimer and inhibit its peptide transport activity (28, 29, 30, 31). ICP47 binds human TAP with a 100 fold more affinity than murine TAP (30, 31). Recent studies by Jugovic et al. (32) have shown that ICP47 inhibits porcine TAP more efficiently than the human TAP. However, we could not find any ICP47 homologue among the PrV protein sequences available to date. HCMV US6 gene product also interferes with the TAP activity, but by a different mechanism (33, 34). This ER-resident glycoprotein has been shown to associate with the TAP molecules from the ER luminal side and not interfere with peptide binding (35). Until the PrV-encoded putative protein responsible for the inhibition of peptide transport is precisely identified, the mechanism of TAP inhibition cannot be addressed.
In addition to the herpesviruses, other viruses have been shown to interfere with class I Ag presentation pathway. The highly oncogenic adenovirus 12 interferes with the transcription of most genes associated with Ag processing including TAP1 and TAP2 (36). Although we cannot exclude this possibility, this phenomenon may not discord our findings due to following reasons. Even though the precise half life of TAP is not known, it is believed to have a long half life (T. Elliot, personal communication). We observed the inhibition of peptide transport activity as early as 2 h.p.i. Therefore, even if PrV interferes with transcription and/or translation of TAP, it does not preclude our finding that PrV inhibits the transport of peptides from the cytosol into the lumen of the ER.
The inhibition of TAP by PrV may not be the only mechanism by which this virus down-regulates the cell surface expression of class I molecules. Mellencamp et al. (12) found reduced levels of class I molecules in PrV-infected mouse cells, which may be due to reduced synthesis or increased degradation of the newly synthesized class I molecules in the ER.
It is possible that PRV, like the cytomegaloviruses, may have evolved multiple mechanisms to perturb class I Ag presentation pathway. The immunoprecipitation experiments that we conducted with the PK-15 cells revealed reduced levels of class I molecules in PrV-infected cells (data not shown). However, the immunoprecipitation experiments were performed with an Ab that recognizes peptide-bound class I molecules only. Therefore, it is not possible to differentiate whether the synthesis or assembly (or both) are affected by the virus. Until an anti-porcine class I mAb that binds class I molecules irrespective of peptide binding becomes available, it is difficult to further elucidate these mechanisms. The failure of the anti-porcine pan-tissue marker mAb to function in immunoprecipitation experiments compounded this problem. The possibility of degradation of newly synthesized class I molecules also cannot be ruled out. Comparison of mRNA levels of virus-infected and mock-infected cells would shed light on this issue.
The second objective of this study was to identify the viral protein(s)
responsible for class I down-regulation. Like in other herpesviruses,
PrV gene expression occurs in a cascade fashion (37). The
IE or 
genes are expressed first, which induce the expression of
early or ß genes. The expression of early genes induces the onset of
the viral DNA replication which is followed by late or 
gene
expression. PrV encodes a single IE protein, IE180, which is a
regulatory protein that is essential for virus replication
(38). Early gene products are mostly involved in genome
replication, whereas the late gene products are mainly structural. Lack
of down-regulation of class I molecules in cells treated with
UV-inactivated virus or live virus in the presence of ChX, a protein
synthesis inhibitor, clearly indicated that virus replication was
essential for class I down-regulation. The involvement of PrV IE
protein, IE180, was ruled out by using the ChX reversal experiment. The
cells infected in the presence of ChX accumulates large amounts of
IE180 mRNA in the cytoplasm. Once ChX is removed and Act-D is added,
the already accumulated IE180 mRNA undergo translation and IE180
protein is synthesized in large amounts, whereas further mRNA synthesis
is inhibited. Failure to observe class I down-regulation in these cells
indicates that IE180 is highly unlikely to be responsible for the
effect. The suppression of late protein expression by PAA could not
restore the expression of class I to normal levels, indicating that the
late proteins are highly unlikely to be responsible for class I
down-regulation. Based on these data, we conclude that one or more
early PrV protein(s) may be responsible for the down-regulation of
class I expression on the cell surface. Additional evidence for our
conclusion is provided by the kinetic studies of PrV protein
expression. The early proteins are expressed as early as 1 h.p.i.
and reach the maximum levels by 2 h.p.i., which accounts for the
onset of down-regulation of cell surface class I expression by 4 h
(37). Furthermore, since TAP inhibition was seen as early
as 2 h.p.i., it is likely that an early protein(s) may be
responsible for the inhibition of TAP. PrV IE180 shows a high degree of
homology with ICP4 of HSV-1, IE140 of varicella-zoster virus, IE1 of
equine herpesvirus, and p180 of BHV-1. It contains a nuclear
localization signal and accumulates in the nuclei of the infected cells
(39). In contrast, both the proteins that are known to
interfere with the TAP activity are localized either in the cytosol
(HSV ICP47) or in the ER (HCMV US6 gene product), where they have easy
access to the TAP. Furthermore, PrV encodes only a single IE protein;
therefore, at least some of the functions of HSV IE proteins have to be
fulfilled by PrV early proteins (37). Therefore, it is
likely that PrV encodes an early protein(s) which has functional
similarity to either HSV-encoded ICP47 or HCMV-encoded US6 gene
product.
PrV encodes several early proteins. Identification of early protein(s) that inhibits the transport of peptides by porcine TAP (and possibly other steps in the Ag presentation pathway), necessitates the development of transfectants expressing the individual early proteins, or the development of mutants from which individual early proteins are deleted. There are not many PrV mutants lacking early genes available, and the few that are available have not helped to identify the gene(s) responsible for the down-regulation of the expression of class I molecules (L. W. Enquist, and T. Mettenleiter, personal communication). Alternatively, anti-porcine TAP Abs could be used to coprecipitate the TAP binding PrV early protein(s) along with the TAP. Again, anti-porcine TAP Abs are not available, and the currently available anti-human TAP and anti-murine TAP Abs do not cross-react with porcine TAP. Abs that recognize porcine class I molecules irrespective of peptide binding would help in further elucidation of the mechanisms of class I down-regulation by PrV. Production of anti-porcine TAP and anti-porcine class I Abs is underway in our laboratory.
Note.
During the review of this manuscript, Sparks-Thissen and Enquist (40) reported the results of their study of class I down-regulation by PrV in the murine system. They observed a differential regulation of Dk and Kk molecules on the surface of L929 cells. Their study also suggested an early PrV protein to be responsible for the down-regulation of class I molecules during the early phase of infection. They also showed that the down-regulation of class I molecules during the early phase of infection was not due to inhibition of synthesis or transport of the complex through the secretory system implying that PrV might interfere with the transport of peptides, which supports our finding in the porcine system.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. S. Srikumaran, Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, NE 68583-0905. E-mail address:
3 Abbreviations used in this paper: PrV, pseudorabies virus; Act-D, actinomycin-D; BHV-1, bovine herpesvirus 1; ChX, cycloheximide; ER, endoplasmic reticulum; HCMV, human cytomegalovirus; h.p.i., hours postinfection; ICP, infected cell protein; IND-F, Indiana Funkhauser; IE, immediate early; MDBK, Madin-Darby bovine kidney; MLV, modified live virus; moi, multiplicity of infection; PAA, phosphonoacetic acid; PK, porcine kidney.
Received for publication October 15, 1998. Accepted for publication October 14, 1999.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), PRV/Marker Gold (
), or Suvaxyn (
) vaccine virus strains at
an moi of 5, were subjected to flow cytometric analysis at indicated
h.p.i. with the anti-class I mAb PT85A. MHC class I expression by
infected cells is shown as a percentage of that of mock-infected cells.
The mAb MM113 was used as the isotype-matched control. Dead cells
were gated using propidium iodide, and 10,000 viable cells were
analyzed for each sample. The results represent the means of three
independent experiments.
