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The Journal of Immunology, 1999, 162: 69-77.
Copyright © 1999 by The American Association of Immunologists

Class I MHC Molecule-Mediated Inhibition of Sindbis Virus Replication1

Young S. Hahn*,{dagger}, Angelo Guanzon*, Charles M. Rice§ and Chang S. Hahn2,*,{ddagger}

* Beirne Carter Center for Immunology Research and Departments of {dagger} Pathology and {ddagger} Microbiology, University of Virginia Health Sciences Center, Charlottesville, VA 22908; and § Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The threshold for systemic viral infection relies on the amplification of virus at a primary infection site. We have identified that class I MHC molecules can trigger the inhibition of replication of Sindbis virus in a haplotype- and allele-specific manner. Class I MHC molecules of H-2d haplotypes exhibit a strong inhibitory effect whereas H-2k haplotypes show minimal inhibition of Sindbis viral replication. By a single gene transfection of H-2d class I MHC molecules, into cells that express class I MHC molecules of H-2k haplotype and are susceptible to viral replication, these cells became resistant to viral replication. The inhibition of viral replication by class I MHC molecules occurs neither during the stage of virus entry/endocytosis nor during virus maturation. Rather, viral-specific RNA replication, as well as viral gene expression, are inhibited in cells expressing inhibitory class I MHC molecules. This class I MHC molecule-mediated inhibition requires newly synthesized host gene products, implying the activation of an intracellular signaling mechanism that is triggered by specific class I MHC molecules.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Class I MHC molecules are well known for their role in presenting the antigenic moiety to CD8+ T cells (1, 2). However, several lines of evidence suggest that class I MHC molecules can play other roles in the biology of cellular metabolism. Class I MHC molecules can be associated with several cellular receptors at the cell surface (3, 4, 5). Studies involving several viruses, including mammary tumor virus (6), vaccinia virus (7), and lymphocytic choriomeningitis virus (8), suggest that there may be a genetic control of the susceptibility to viral infection that is mapped to the H-2 loci of the mouse. Also, the control of viral oncogenesis caused by several leukemia viruses has been mapped to the H-2 loci (9, 10, 11, 12, 13). In vitro studies with these viruses also show differences in virus propagation (14). The significance of these phenomena and their underlying mechanisms are largely unknown.

Sindbis virus (SIN)3 is the prototype virus of the Alphavirus genus and has been studied extensively because of the importance of this group as pathogens of human and domestic animals (for review see Refs. 15 and 16). SIN consists of a nucleocapsid that contains an RNA molecule of positive polarity and capsid proteins that are surrounded by a lipid bilayer containing two viral encoded glycoproteins called E2 and E1. Surface glycoprotein E2 is believed to interact with the virus receptor during attachment, and glycoprotein E1 may play an important role in low pH-dependent membrane fusion as well as hemagglutination. Upon infection, the viral genome acts as a messenger RNA to generate a replicase complex to initiate viral replication in the cytoplasm of the infected cells. During replication, 26S subgenomic RNA is synthesized from the internal promoter sequence of the viral genome and acts as a mRNA for structural proteins involved in viral maturation.

In this study, we have found that class I MHC molecules of a specific haplotype can modulate SIN replication such that virus production is reduced up to 100-fold, as compared with the cells with susceptible haplotype class I MHC molecules. We also identified that this inhibitory effect is not due to viral attachment/entry but rather effects on viral replication. And, finally, this class I MHC-mediated inhibition requires new host gene expression. This implies that class I MHC molecules may be involved in modulating the efficiency of virus replication by mechanisms distinct from the classical presentation of peptide Ags to CTL, and virus-MHC interactions may play an important role in keeping the delicate balance during early infection by determining susceptibility to infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and viruses

All cell lines unless specified below were originally obtained from American Type Culture Collection (ATCC, Manassas, VA) and were maintained according to the ATCC culture conditions. These include baby hamster kidney 21 cell clone 13 (BHK), L929 (murine fibroblast), L-M(TK-) (thymidine kinase mutant derived from L929), BW5147 (murine lymphoma), P815 (murine mastocytoma), EL4 (murine lymphoma), BALB/cCL.7 (murine fibroblast), J774A.1 (J774, murine monocyte-macrophage), A20 (murine B lymphoma), and S194/5 (murine myeloma). MC57 (murine fibroblast) was a generous gift from Dr. Rafi Ahmed (Emory University, Atlanta, GA). Stably transfected L cells, with individual genomic DNA of H-2d class I MHC molecules, Kd, Dd, and Ld (called L:Kd, L:Dd, and L:Ld, respectively) were generous gifts from Dr. Ted Hansen (Washington University School of Medicine, St. Louis, MO, for L:Ld), Dr. Carol Reiss (New York University, for L:Dd and L:Ld), and Dr. Lee Hood (University of Washington, Seattle, WA for L:Kd and L:Dd). These transfectant cell lines were maintained in DMEM supplemented with 2 mM glutamine, 10% FBS, and HAT (0.1 mM hypoxanthine, 0.4 µM aminopterin, and 16 µM thymidine). Cell surface expression of individual class I MHC molecules was examined using mAbs DO4 (Kd, Dd), 215 (Kd, Ld), 30-5-7 (Ld), 64-3-7 (Ld), SF1-1.1 (Kd), 11.4.1 (Kk), 15.5.5s (Dk), and L368 (ß2m). All mAbs except SF1-1.1 and L368 were generous gifts of Dr. Ted Hansen. L368 hybridoma was obtained from ATCC, and SF1-1.1 was purchased from PharMingen (San Diego, CA). Although many cell lines used in this study do not have clear passage histories, all experiments were performed within 10 cell passages for each cell line.

SIN lab strain Toto 1101 and its double subgenomic derivatives capable of expressing CAT or C3 exoenzyme of Clostridium botulinum (called dsSIN:CAT and dsSIN:C3, respectively) were previously described (17, 18, 19). All viral stocks were generated from the full-length cDNA clones by in vitro transcription followed by transfection into BHK cells (17). All experiments involving virus manipulations were performed in a BL2 facility under the guidelines of the Centers for Disease Control (CDC) with the approval of the University of Virginia Institutional Biosafety Committee.

Virus infection

For infection of adherent cell lines, subconfluent monolayers of cells in either 6- or 12-well tissue culture plates were washed once with PBS, and the appropriate amount of virus was added in 0.5 ml PBS. For suspension cell lines, late logarithmic cultures (8 x 105 cells/ml) were collected and resuspended in PBS at a concentration of 4 to 5 x 106 cells/ml. Cells were incubated for 1 h at 37°C, viral inocula were removed, and appropriate media were added to produce a concentration of 5 x 105 cells/ml. Infected cells were collected for various assays as described below.

Virus binding and adsorption

Late logarithmic phase P815 cells were collected and resuspended in PBS at a concentration of 4 x 106 cells/ml. PBS (0.5 ml) or PBS containing P815 cells was placed in 12-well plates that were precoated with DMEM with 10% FBS to prevent nonspecific binding to plastic. L929 and L:Ld cells were placed in six-well plates at the concentration of 2 x 106 cells per well. Sindbis virus was added to each well at MOIs of 4 and 20 (1.2 x 107 and 6 x 108 pfu of viruses per well, respectively). Plates were incubated in a 37°C incubator with occasional rocking for 1 h. At the end of incubation, 2.5 ml of media were added to each well, and both supernatant and cell pellets were collected. Cell pellets were lysed by rapid freezing and thawing twice to release cell-associated viruses. Both supernatants and cell pellets were immediately titered in L929 cell to measure residual infectious virus titers.

Analyses of viral replication

The progeny virus generation was examined by two different methods: conventional plaque-forming assay of serial dilutions using chicken embryo fibroblast or BHK cells, or, alternatively, a lytic unit assay of two- to threefold serial dilutions in 96-well plates containing subconfluent L929 cells to ensure infectivity units in murine cell lines. All titrations were done in duplicate.

Virus-specific RNA synthesis was examined by purifying whole cellular RNA from the infected cells at several time points after infection. Total cellular RNAs were transferred to nylon membranes, and SIN-specific RNA was detected using a 32P-labeled cDNA strand RNA of a structural protein region of SIN as a probe.

Virus-specific gene expression was examined by both FACS analyses of cell surface expression of infected cells and immunoblot. FACS analyses of viral glycoprotein cell surface expression of infected cells were performed using rabbit polyclonal antisera against SIN glycoprotein E2 followed by FITC-conjugated goat anti-rabbit Ab at specified hours postinfection. L929 and P815 cells were infected with mock, or MOI of 5, 20, or 50 pfu per cell and incubated for 8 h. Cells were collected, and 2.5 x 105 cells were separated by SDS-PAGE and immunoblotted with rabbit polyclonal antisera against SIN glycoprotein E1. The binding of rabbit antisera was visualized by horseradish peroxidase-conjugated goat anti-rabbit Ab followed by development using an ECL (enhanced chemiluminescence) detection kit (Amersham, Arlington Heights, IL).

CAT reporter gene expression under the control of viral subgenomic promoter was examined by either enzymatic activity assay using 14C-labeled chloramphenicol or sandwich ELISA using rabbit polyclonal anti-CAT Ab (5'-3', Inc., Boulder, CO). All CAT assays were performed at least in duplicate. Total CAT polypeptide expression was divided by total infected cells to calculate the average CAT polypeptide expression per cell.

Generation of L cell transfectants expressing H-2d class I MHC molecules from cDNA

A eukaryotic expression vector based on the CMV promoter and a 3' mini exon of ß-globin (pBK/CMV, Stratagene, La Jolla, CA) was used to generate an expression plasmid capable of expressing either Kd or Ld molecules from cDNA clones. cDNAs of Kd and Ld were generous gifts of Drs. Lee Hood and Ted Hansen, respectively. cDNA of Kd or Ld molecules was placed immediately downstream of the CMV promoter. Resulting constructs were used to transfect L929 cells, and stable transfectants were selected by G418 selection (Life Technologies, Gaithersburg, MD; 0.5 mg/ml). Cell surface expression of individual class I MHC molecules was examined using allel- and haplotype-specific mAbs described above.

Inhibition of host gene expression upon viral infection

L929 and L:Ld cells were infected with dsSIN:CAT at an MOI of 20 pfu per cell. Upon infection, cells were treated with or without 800 ng/ml dactinomycin. Infected cells were collected at 3, 5, 7, and 15 h postinfection for L:Ld cells and 4, 7, and 10 h postinfection for L929 cells, and the accumulation of intracellular CAT polypeptides was determined by CAT sandwich ELISA. The supernatants were collected and examined for virus titers.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differential susceptibility of Sindbis virus replication in murine cell lines

During infection of various murine cell lines with SIN, we found that an HR strain of SIN (called Toto1101) (17) exhibits a strong cell type-specific restriction of virus growth. We found that Toto1101 could productively replicate and generate progeny viruses in L929 cells, but not in the P815 cell line (mastocytoma cell lines from DBA mice) (Fig. 1GoA). To measure virus-specific gene expression in infected cells, we used recombinant double subgenomic virus capable of expressing CAT as a reporter gene under the control of a viral promoter (dsSIN:CAT) (18). As shown in Fig. 1GoB, CAT expression and progeny virus release were reduced over 200-fold in P815 cells compared with L929 cells when cells were infected with a multiplicity of infection (MOI) of 20. Virus-specific RNA synthesis showed similar results. These differences in SIN replication between L929 and P815 were amplified when cells were infected at an MOI of 0.2. At 24 h postinfection, CAT polypeptide expression in L929 and P815 cells was 4 x 106 per cell and 7 x 102 per cell, respectively. Progeny virus generation at 24 h postinfection in L929 and P815 cells was 600 and 0.07 infectious virus particles per cell, respectively. Extension of this susceptibility to SIN replication in several different inbred murine cell lines showed that there was a linkage between SIN replication and H-2 loci. H-2k cell lines such as L929 and BW5147 supported SIN replication well; on the other hand, H-2d cell lines such as P815, BALB/c CL7, J774, S194/5, and A20 showed limited SIN viral replication (data not shown). Interestingly, cell lines from H-2b mice such as MC57 and EL4 showed an intermediate level of viral replication (Fig. 1GoB and data not shown).



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  		FIGURE 1. Differential viral replication among L929, EL4, and P815. Both L929 and P815 cells were infected with a laboratory strain of SIN called Toto1101 and its double subgenomic recombinant capable of expressing CAT (dsSIN:CAT). Cells were infected with an MOI of 20 pfu per cell. A, Time course of the generation of progeny viruses from infected cells. The total number of viruses was divided by the number of infected cells to obtain the number of progeny viruses generated per infected cell. •, The mean value of P815 cells infected with Toto1101. {blacksquare}, The mean value of L929 infected with Toto1101. Illustrations are compiled data of 10 and 30 independent experiments for L929 and P815 cells, respectively. The virus titers were obtained by infecting L929 cells with limiting dilutions, and this titer was applied for all subsequent infection of different cell types. L929 experiments were terminated either 8 or 10 h postinfection due to the cells coming off from the plates due to the severe cytopathic effects. Although not shown in the figure, viral titers at 12 and 15 h postinfection in P815 cells were lower than 24 h postinfection. B, Number of CAT molecules expressed per cell as a measure of virus-specific gene expression (32). {diamond}, Represents mean value of P815 (H-2d) cells infected with dsSIN:CAT. •, The mean value of EL4 (H-2b) cells infected with dsSIN:CAT. {blacksquare}, The mean value of L929 (H-2k) infected with dsSIN:CAT. The SDs of CAT expression for infected cells are shown in thin bars. Data illustrate a compilation of 515, 130, and 50 data points for P815 cells, EL4 cells, and L929 cells, respectively.

 
Inhibition of Sindbis virus replication by class I MHC molecules of H-2d haplotype

Since a number of H-2d cell lines that we examined, including P815 and BALB/c CL7, do not express class II MHC molecules, the most notable difference in the H-2 loci is the class I MHC molecules. Thus, we examined whether the inhibition of SIN replication is linked to the class I MHC molecules of the H-2d haplotype by using stable transfectants of L cells expressing the individual genomic clones of H-2d class I MHC molecules Kd, Ld, and Dd. The L cells transfected with individual genomic DNA of H-2d class I MHC molecules (Kd, Ld, and Dd molecules), called L:Kd, L:Ld, and L:Dd, respectively, were examined for their ability to support SIN replication. L929 cells, as well as three transfected L cells (L:Kd, L:Ld, and L:Dd), were infected with dsSIN:CAT at an MOI of 20. SIN replication kinetics were measured by CAT reporter gene expression levels (Fig. 2GoA), SIN-specific RNA accumulation by dot blot with a SIN-specific radioactive RNA probe (Fig. 2GoB), and cumulative progeny virus generation after 24 h (Fig. 2GoC).



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  		FIGURE 2. The role of individual H-2d class I MHC molecules on SIN replication. L cell lines stably transfected with individual class I MHC molecules of H-2d genomic DNA (Dd, Kd, and Ld) were infected with dsSIN:CAT at an MOI of 20, and CAT expression, virus-specific RNA synthesis, and progeny virus generation were measured for SIN replication. A, Time course of CAT expression in L cell transfectants L:Dd, L:Kd, and L:Ld infected with dsSIN:CAT. Figure represents compiled data of eight independent experiments. B, Time course of RNA synthesis in L929 and L cell transfectants of L:Dd, L:Kd, and L:Ld at 4, 8, and 24 h postinfection. Total cellular RNA was measured by dot blot analysis using a radioactive SIN-specific RNA probe. Upon longer exposure, we can detect more prominent dots in L:Ld cells infected with Sindbis virus, and the intensity does not change during the duration of infection. These are representative data from five independent experiments. C, Cumulative progeny viruses of L cells and transfectants at 24 h postinfection from six independent experiments. D, Cell surface expression level of total class I MHC molecules examined by FACS analyses. Cell surface expression of the total class I MHC molecules, including endogenous Kk and Dk, as well as transfected class I MHC molecules of H-2d origin (Dd, Kd, or Ld), was examined using mAb L368 directed against human ß2m, which is capable of recognizing its murine counterpart. All three L cell transfectants (L:Dd, L:Kd, and L:Ld) exhibited similar levels of total class I MHC molecules.

 
All three L cell transfectants, L:Kd, L:Ld, and L:Dd, not only inhibited SIN replication, in terms of virus-specific RNA synthesis, CAT expression, and progeny virus generation when compared with L929 cells, but also exhibited various degrees of inhibition. L:Dd, L:Kd, and L:Ld showed an approximately 5-, 25-, and 200-fold decrease in both SIN-specific RNA and CAT expression, respectively, when compared with that of L929 cells (Fig. 2Go, A and B). The decrease in progeny virus generation was somewhat more dramatic, with 20-, 50-, and 500-fold decreases relative to L929 at 24 h postinfection in L:Dd, L:Kd, and L:Ld, respectively (Fig. 2GoC). Furthermore, unlike L929 cells infected at an MOI of 20 pfu per cell, in which virus-induced cytopathic effect was obvious by 6 to 8 h postinfection, L cell transfectants expressing the H-2d class I MHC molecule showed delayed cytopathic effect, if any. Notably, when L:Ld cells were infected with SIN, there was no detectable cytopathic effect even after several days; in addition, infected cells could be passaged with limited viral replication (data not shown). We also examined the Sindbis viral replication in an L cell line that is lacking thymidine kinase (L:tk-), from which all transfectants were generated. The viral replication in L-M(TK-) was comparable to that of L929 cells, when measured by its RNA synthesis, CAT expression, and progeny virus generation (data not shown).

These inhibitory phenomena were independent of the total cell surface expression level of class I MHC molecules since the overall class I MHC molecule levels in L929 and L:Dd, L:Kd, and L:Ld were comparable when examined by FACS using Ab L368 against ß2-microglobulin (ß2m), a common light chain subunit of class I MHC molecules (Fig. 2GoD). We also examined the expression level of endogenous class I MHC molecules of L929 cells and L cell transfectants, L:Ld and L:Dd, Kk and Dk, using Abs 11.4.1 and 15.5.5s, respectively. The level of cell surface expression in Dk molecules was virtually identical among three cell types we examined (data not shown). Also, expression levels of Kk were comparable among three cell types, except that L:Ld cells exhibited a slightly higher level of expression (data not shown). These data imply that the H-2d class I MHC molecules possess an inhibitory effect on SIN replication and, furthermore, that individual class I MHC molecules Kd, Ld, and Dd have different degrees of inhibitory effect. Again, similar to the P815 cells, infection of L:Dd, L:Kd, and L:Ld cells with low MOI (0.2 pfu/cell) yielded more dramatic inhibitory effects. Although L929 cells infected with low MOI display slower kinetics of viral replication, CAT expression reached 4 x 106 molecules per cell at 24 h postinfection, and 300 infectious progeny viruses were generated per cell during the same time period. On the other hand, L cell transfectants L:Dd, L:Kd, and L:Ld showed much slower replication kinetics of 9 x 104, 2 x 104, and 5 x 103 CAT polypeptide accumulation per cell and 0.4, 0.2, and 0.03 infectious progeny viruses per cell at 48 h postinfection, respectively.

Class I MHC expression level-dependent inhibition of Sindbis virus

To examine whether the level of cell surface expression of class I MHC can influence the inhibition of viral replication, we generated cDNA transfectants in L929 cells that were under the control of a CMV promoter and that exhibited decreased level of expression of the Ld molecules when compared with that of genomic transfectants. Individual clones of Ld cDNA transfectants were generated from four independent transfections. Over 10 independent clones were characterized for cell surface expression of Ld molecules, as well as for their inhibitory effect on SIN replication. Fig. 3Go shows representative data on the levels of cell surface Ld expression (Fig. 3GoA), the overall class I MHC expression level (Fig. 3GoB), and the time course of progeny virus generation (Fig. 3GoC). Compared with the genomic transfectant cell line L:Ld, Ld cDNA transfectant cell lines displayed 20- to 50-fold lower levels of Ld expression. When the effect of Ld expression on SIN replication was examined, all transfectant cell lines showed SIN replication approximately 20-fold higher than genomic Ld transfectants. Similar results were obtained for CAT expression (data not shown). We also examined the bulk cDNA of Ld transfectants upon selection for their ability to inhibit replication of Sindbis virus and essentially obtained the same results (data not shown).



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  		FIGURE 3. Dose-dependent inhibition of SIN replication in L cell transfected with class I Ld molecules. A, FACS profile of cell surface expression of Ld molecules in L cell transfectants. a, Vector alone transfection. b and c, Two independent stable transfectants of Ld cDNA under the control of the CMV promoter. d, Genomic DNA of the Ld transfectant. Cells were treated with mAb 30-3-7, which is Ld specific, followed by FITC-conjugated goat anti-mouse Ab. FACS profile of secondary Ab alone-treated cells, virtually indistinguishable from the vector transfected, was omitted for clarity. B, FACS profile of cell surface expression of total class I MHC molecules in L cell transfectants. a, Secondary Ab alone (control histogram). b, Vector alone transfection. c, A stable transfectant of Ld cDNA under the control of the CMV promoter. d, Genomic DNA of the Ld transfectant. Cells were treated with mAb L368, which is ß2m specific, followed by FITC-conjugated goat anti-mouse Ab. C, Time course of progeny virus generation in L929 and its transfectants infected with dsSINT:CAT at an MOI of 20 pfu per cell. {blacksquare}, Represents vector-transfected L929. •, Represents genomic Ld transfectant. Four open symbols ({triangleup}, {square}, {circ}, and {diamond}), Represent four independent Ld cDNA-transfectant clones generated from two independent transfections. Figure represents mean and SD of four independent experiments.

 
We also generated stable L929 cell transfectants expressing Kd molecules from cDNA under the control of a CMV promoter. Like cDNA transfectants of Ld in L929 cells, cell surface expression levels of Kd molecules were approximately 50-fold lower than those of L:Kd. When compared with vector-transfected cell lines, Kd cDNA-transfected L929 cell lines showed a fivefold decrease in CAT polypeptide expression when infected with dsSIN:CAT at an MOI of 20 pfu per cell, and progeny virus generation was decreased by ninefold. This suggests that the expression level of certain class I MHC molecules can alter the level of SIN replication inhibition. In all of the above experiments, the overall cell surface expression levels of total class I MHC molecules, including endogenous Kk and Dk molecules, examined by FACS analyses using Ab directed against ß2m were comparable to each other among vector transfectants, cDNA of Ld transfectants and genomic Ld transfectants (Fig. 3GoB).

Class I MHC-mediated inhibition of Sindbis virus replication is targeted to viral replication, not viral attachment or entry

There are a number of possibilities to explain the inhibition of viral replication by class I MHC molecules of specific haplotype and allele. The most likely possibility is that, since class I MHC molecules are cell surface glycoproteins, they may interfere with viral infection during virus attachment or endocytosis. Alternatively, class I MHC molecules may be involved in inhibition of viral replication following endocytosis of viral particles. To discriminate these possibilities, we examined 1) the virus binding and uptake in both sensitive and resistant cells for viral replication, 2) the effect of increasing the dose of infectious particles, and 3) comparison of the effective infectivity of infectious particles between L929 and P815 cells.

We first examined viral adsorption to P815, L929, and L:Ld cells following incubation of appropriate cells with SIN for 1 h at an MOI of either 6 or 30 pfu per cell. Higher MOI was used to rule out the possibility of receptor saturation by virion particles. As shown in Table IGo, more than 95% of viruses were absorbed to the cell when incubated at an MOI of either 6 or 30 pfu per cell at 37°C for 1 h, suggesting that the observed class I MHC-mediated inhibition of viral replication does not occur at the level of viral binding. We chose this virus adsorption condition for the binding assay in an attempt to use conditions similar to those used for all other experiments described above, including virus-specific RNA synthesis, CAT polypeptides synthesis, and progeny virus generation. We also examined the infectivity of viruses that were associated with the P815 cells by measuring cell-associated infectious particles. As shown in Table IGo, more than 95% of virus particles associated with P815 cells became noninfectious within an hour at 37°C. Since receptor-mediated endocytosis of virion particles is suppressed at lower temperature, we examined the cell association of infectious particles at lower temperature. When this virus-binding experiment in P815 cells was repeated on ice instead of at 37°C, at least 80% of total input virus particles were recovered from the cell pellet as infectious particles (Table IGo). This implies that, during normal infection, attached viruses do not remain on the cell surface but progress through the viral replication cycle, including receptor-mediated endocytosis and the processing of viral particles that renders them noninfectious.


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  		Table I. Virus binding and adsorption in P815, L929 and L:L4 cells

 
To examine the level of virus-specific gene expression of the infected cells, we assayed the expression of viral glycoprotein E1 by immunoblot analysis. We compared viral glycoprotein E1 expression by Western blot analyses in P815 cells infected with Toto1101 at MOI of 5 and 20, which showed virtually identical, very low levels of expression of E1 glycoproteins when compared with those of L929 cells infected with the same MOI (Fig. 4Go). By the same token, CAT expression level was 3 to 6 x 104 molecules per cell when examined at 4, 8, and 24 h postinfection in P815 cells infected at MOI of 5, 16, or 50 pfu per cell. When the same experiment was performed in L929 cells, only an early time point postinfection (2 h postinfection) gave a significant difference that was proportional to the input MOI. At 4, 6, and 8 h postinfection, CAT expression values were within twofold of the values observed at an MOI of 20, shown in Fig. 1GoB.



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  		FIGURE 4. Western blot analyses of viral glycoprotein E1 expression in L929 and P815 cells. L929 and P815 cells were infected with appropriate MOI and incubated for 8 h, and the expression of viral glycoprotein E1 was detected by immunoblot.

 
Since a biochemical approach such as immunoblotting cannot discriminate between a majority of cells being infected and expressing a homogenous low level of viral proteins vs only a minority of cells being infected and expressing higher levels of viral protein, we examined cell surface expression of viral envelope glycoprotein E2 by FACS analysis (Fig. 5Go). We examined this in two different aspects. First, we examined the cell surface expression profile of viral envelope proteins at a high MOI and whether an increase in multiplicity of infection to very high MOI has an effect on the expression level of viral proteins. Second, we examined the specific infectivity of L929 and P815 cells using a low MOI of 1 pfu per cell.



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  		FIGURE 5. Efficiency of infection of P815 cells with a differential multiplicity of Sindbis virus. A, P815 cells were infected with MOI of 5, 50, and 500, as well as mock infection. At 17 h postinfection, cells were collected and treated with either secondary Ab (FITC-conjugated goat anti-rabbit Ab) alone (a), or with rabbit polyclonal Ab against SIN glycoprotein E2, followed by FITC-conjugated goat anti-rabbit Ab (b–e). Treated cells were fixed in PBS containing 1% formaldehyde and analyzed by FACScan for fluorescent intensity. At least 10,000 cells were analyzed for each sample. b, Mock-infected P815 cells. c, d, and e, P815 cells infected with Toto1101 at the multiplicity of 5, 50, and 500 pfu per cell, respectively. Similar results were obtained 8 and 24 h postinfection. The experiments were repeated three times and generated essentially the same results. B and C, P815 (B) and L929 (C) cells were infected with SIN at an MOI of 1 pfu per cell. Eight hours postinfection, cells were collected and examined for cell surface expression of SIN E2 glycoproteins by FACS as described above. a, Cells treated with secondary Ab alone. b, Mock-infected P815 and L929 cells treated with anti-E2 Ab. c, P815 and L929 cells infected with SIN at an MOI of 1 pfu per cell treated with anti-E2 Ab. As expected in a Poisson distribution curve, approximately two-thirds of the population exhibit expression of E2 glycoproteins at the plasma membrane of the P815 cells. Infected L929 cells exhibit a bimodal curve that indicates that approximately two-thirds of cells have a high level of glycoprotein E2 expression and that the rest of the population has low expression. The population that exhibits a lower level of E2 glycoprotein expression is due to infection of bystander L929 cells that were not infected initially but were infected subsequently by progeny viruses released from infected cells.

 
P815 cells were infected with Toto1101 at an MOI of 5, 50, and 500 and examined for cell surface expression of viral glycoprotein E2, using rabbit polyclonal Ab against E2 at 0, 8, 17, and 24 h postinfection. When P815 cells were incubated with different amounts of viral inocula for 1 h at 4°C or 37°C and immediately subjected to FACS analyses for E2 glycoprotein level, there were no differences in FACS histograms among mock-infected cells infected with MOIs of 5, 50, or 500 pfu per cell (data not shown). This implies that the potential background E2 staining due to the attachment of virus particles is below the detection limit.

Fig. 5GoA shows an overlapping FACS histogram of infected P815 cells with SIN at the MOI of 5, 50, and 500 at 17 h postinfection. The histograms show a single normal distribution curve, implying that a majority of cells were infected with viruses and expressed a homogenous low level of viral glycoprotein E2 in the plasma membrane. Furthermore, there was also no difference in glycoprotein E2 expression level in the plasma membrane even with a 100-fold increase of MOI (from an MOI of 5 to an MOI of 500). When these experiments were repeated at 8, 17, and 24 h postinfection with an MOI of 5, 50, and 500, the histograms of cell surface expression of E2 were basically superimposable. When histograms were compared with those of L929 cells infected with SIN, the level of fluorescence intensity in infected P815 cells was approximately 200 channels lower than that of L929 cells infected with the same virus at the MOI of 10 pfu per cell (data not shown, see below). These data imply that all the cells were infected with virus but that virus-specific gene expression is much lower in P815 cells, and a higher multiplicity of infection does not overcome this class I MHC molecule-mediated inhibitory effect.

The above data, Figs. 4Go and 5GoA, suggest that viral replication does not increase upon increasing input virus over 100-fold. Since we do not know the number of receptors in P815 cells, it is possible that even at an MOI of 5 pfu per cell, receptors of P815 cell may be saturated such that an increase of input viruses has no effect on the level of viral replication. This is important since we do not know the exact particle to infectious unit ratio, which can vary from 3 to 50 in our hands, depending on the virus preparation or cell types in which we determine the specific infectivity. To examine whether the specific infectivities of SIN in L929 and P815 cells are comparable, we infected L929 and P815 cells at an MOI of 1 pfu per cell. We anticipate that, if the specific infectivities of L929 and P815 cells are similar, approximately two-thirds of the cells in the population would be infected by SIN at an MOI of 1 pfu per cell, and one-third of the cells would not initially be infected with virus, according to a Poisson distribution. L929 and P815 cells were infected with SIN at an MOI of 1 pfu per cell, incubated for 8 h, and examined for cell surface expression of viral glycoprotein E2. As shown in Fig. 5Go, both P815 (Fig. 5GoB) and L929 (Fig. 5GoC) cells exhibit cell surface expression of E2 glycoproteins in approximately two-thirds of the population when measured by gating the higher fluorescent level population. The population of cells expressing viral envelope protein E2 is shown in Fig. 5GoB as a shoulder since the expression level is very low. However the expression level of viral glycoprotein E2 is dramatically different between P815 and L929 cells. The slight shift in peak that represents a population of presumably uninfected L929 cells is due to the subsequent infection by virus particles released from infected L929 cells. The cumulative virus release in L929 cells infected with SIN at an MOI of 1 pfu per cell was 4.5 ± 1.7 pfu per cell, assuming 67% of cells were infected, thus generating sufficient progeny virus to reinfect initially uninfected cells.

The above data imply that it is unlikely that the inhibition of viral replication is due to interference occurring at an early step of viral attachment/penetration into P815 cells. Rather, by some mechanism yet to be identified, viral replication is down-regulated after virus attachment and endocytosis so that infected cells have only limited viral replication. We extended this investigation to L cell transfectants to more clearly examine the effects of single gene product, class I MHC molecules of the H-2d haplotype. By taking advantage of the adherent nature of L cell transfectants, we examined the specific infectivity and replication of SIN in L cell transfectants using recombinant dsSIN capable of expressing the C3 exoenzyme of C. botulinum (dsSIN:C3) (19, 20, 21). C3 exoenzyme specifically inactivates the cellular small GTPase Rho by covalently modifying an Asn41 residue by transferring an ADP-ribose moiety of nicotinamide adenine dinucleotide (22). A prominent downstream event of Rho signaling is the control of actin microfilament stress fiber formation (23, 24). Upon microinjection of cells with C3 exoenzyme, cells lose their actin microfilament stress fibers, while focal adhesion to the extracellular matrix remains intact. As a consequence, cells appear like neurites with a central rounded cell body (25, 26, 27). Since only very small amounts of C3 exoenzyme are required to induce morphological change, this reagent provides us with a very sensitive measure of minimal virus gene expression.

Since C3 exoenzyme expression is placed under the control of viral subgenomic promoter, unless viral RNA replication and virus-specific gene expression occurs, C3 exoenzyme will not be expressed. When L:Ld cells were infected with dsSIN:C3 at an MOI of 5, within 2 h of infection, over 95% of cells showed the effect of C3 exoenzyme treatment (Fig. 6Go). In contrast, L:Ld cells infected with dsSINT:CAT showed no morphological change. This implies that almost all the cells were infected with dsSIN:C3, and, within 2 h postinfection, infected cells were expressing sufficient C3 exoenzyme under the control of the viral promoter. This phenomena was observed in similar efficiency with L929 cells L:Dd and L:Kd infected with dsSIN:C3 at an MOI of 5 pfu per cell (data not shown). This suggests that the viral attachment and endocytic steps of the replication cycle may not be affected by expression of certain specific class I MHC molecules such as Ld or Kd. However, viral RNA replication, viral gene expression, and progeny virus generation are inhibited dramatically, and these inhibitory effects are dependent upon which class I MHC molecules a given cell expresses. According to our data, cells expressing H-2d haplotype class I MHC molecules show the strongest inhibitory effects, followed by H-2b and H-2k. Even among H-2d haplotype class I MHC molecules, Ld molecules exhibit the strongest inhibitory effect on SIN replication, followed by Kd and Dd molecules.



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  		FIGURE 6. Efficiency of infection of Sindbis virus in L:Ld cells. L cell transfectants of genomic Ld (L:Ld) were infected with either dsSIN:CAT (a) or dsSIN:C3 (b) at the multiplicity of 5 pfu per cell. At 2 h postinfection, infected cells were examined for morphology change by inverted phase contrast microscopy. C3 exoenzyme inactivates the small GTPase Rho, which controls cellular stress fibers. As a consequence, cells that express C3 exoenzyme exhibit the morphology of cell rounding and neurite-like fibers. Virtually 100% of cells infected with dsSIN:C3, but not dsSIN:CAT, demonstrated cellular rounding, implying that almost all of the cells were infected and initiated replication, with expression of C3 exoenzyme under the control of subgenomic RNA.

 
Host gene expression is involved in class I MHC-mediated inhibition of Sindbis virus replication

The above data suggest that expression of specific class I MHC molecules can inhibit Sindbis virus replication. Furthermore, the inhibition of viral replication by H-2d class I MHC molecules is dominant over H-2k class I MHC molecules that may not interfere with viral replication. To investigate the potential involvement of host gene expression, we examined the role of host gene expression upon viral infection in the class I MHC-mediated inhibition of SIN replication. We infected L929 and L:Ld cells with dsSIN:CAT at an MOI of 20 pfu per cell followed by treatment with dactinomycin to inhibit host RNA transcription. Replication and transcription of SIN are not inhibited by dactinomycin. When infected L929 cells were examined for the kinetics of CAT expression and progeny virus generation, there were almost no differences between dactinomycin-treated cells and untreated controls (see Fig. 7Go; data not shown). On the other hand, when infected L:Ld were examined, there was a 5- to 10-fold increase in CAT expression upon treatment with dactinomycin. The time course of expression of CAT polypeptides in L:Ld cells infected with dsSIN:CAT in the presence or absence of dactinomycin is shown in Fig. 7Go. The progeny viruses released at 24 h postinfection were 138 ± 115 and 7.16 ± 6.64 per infected cell for L:Ld cells infected with dsSIN:CAT at an MOI of 20 with or without dactinomycin treatment, respectively. This strongly suggests that virus-induced host gene expression is required for maximal class I MHC molecule-mediated inhibition of SIN replication.



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  		FIGURE 7. Effect of host gene expression in Sindbis replication in L:Ld cells. The effects of host gene expression on viral replication upon infection in L:Ld cells were examined. •, Represents infected L929 cells. {blacksquare}, Represents infected L:Ld cells without dactinomycin treatment. Corresponding open symbols, Represent infected cells with treatment with dactinomycin. Since infected L929 cells detach from the plate at approximately 12 h postinfection due to the cytopathic effects, we collected the last time points at 10 h postinfection. The result is compiled data from three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A critical early step that determines successful viral infection of a host relies on viral replication in primary infection sites. This is especially significant during natural infection, since the threshold of infection is determined by the balance between viral replication within the primary infection site and the innate host immune response, such as leukocyte recruitment and inflammation. The virus has to overcome this innate immune response to successfully establish infection. If viral replication can be modulated by host factors during the early stage of infection, such modulation may explain the differential susceptibility to viral infection of individuals with different genetic backgrounds.

In this paper, we showed that class I MHC molecules of murine origin may be of potential importance in this respect. As shown in the results, class I MHC molecules can inhibit the replication of SIN by a mechanism yet to be defined. Furthermore, this class I MHC-mediated inhibition of SIN replication is not only haplotype-specific but also allele-specific. Sindbis viral replication was dictated by which haplotype class I MHC molecules a cell possesses. Among the cell types from three major haplotypes of H-2-inbred strains of mice that are used in laboratories, H-2d haplotypes inhibit viral replication in a very robust manner, H-2b moderately inhibit viral replication, and H-2k were least likely to inhibit viral replication. Even within H-2d haplotype class I MHC molecules, Ld inhibited viral replication in the most significant manner, followed by Kd and Dd, when susceptible cells (L929, H-2k) were transfected with the genes of the individual class I MHC molecules of H-2d. This implies that there is a gradient of effect on viral replication generated by different class I MHC molecules. These observations may be relevant to in vivo viral pathogenesis because our preliminary observations suggest that, upon low dose challenge in CBA (H-2k) and BALB/c (H-2d) mice with SIN, there are significant differences in viremia and CTL responses against SIN (Y. S. Hahn and C. S. Hahn, unpublished data). However, since the genetic background of these two mice are different, these experiments need to be examined in H-2 congenic mice such as D10 congenic lines.

Unlike class II MHC molecules, which are expressed only in the professional APCs or are inducible only in the presence of certain cytokines, virtually every nucleated cell in the body expresses class I MHC molecules. It is possible that this observed differential inhibitory effect on SIN replication by certain class I MHC molecules can create an all or none threshold difference in progression to systemic infection during natural infections that rely on small numbers of infectious particles. It is also of interest that, upon infection with RNA viruses, infected cells secrete type I IFN; this can up-regulate class I MHC molecules of adjacent cells, thus potentially amplifying the class I MHC molecule-mediated inhibition of viral replication (28).

Our results also demonstrate that inhibition of viral replication by class I MHC molecules is due not to the early entry step of viral infection but rather to host-mediated inhibition of viral replication subsequent to virus binding/penetration. There are several lines of evidence to support this. First, viral replication was not increased by increasing input virus by as much as a hundred-fold, from MOI of 5 to 500. If this class I MHC-mediated inhibition was rooted in efficiency in binding/entry events, one would expect that an increased viral inoculum should increase viral replication as measured by cell surface expression of viral envelope protein, which we did not find. Furthermore, when the adsorptions of virus inoculum in L929, L:Ld, and P815 cells were examined by titering both cell-bound virus and virus remaining in the inoculum, greater than 95% of viral inoculum was removed from the inoculum by cells when they were incubated for an hour at an MOI of either 6 or 30 pfu per cell. This argues against the possibility of limited receptors in H-2d cell types such as P815 or transfected cells such as L:Ld.

Second, upon high MOI infection of resistant cells, the majority of cells show low level viral envelope glycoprotein expression when examined by FACS analysis using rabbit polyclonal Abs against viral glycoprotein E2. Again, if the defect in viral replication is at the binding/entry level, we would expect a small subpopulation of cells that expresses normal envelope glycoprotein at the plasma membrane, with the remainder of cells expressing no detectable cell surface envelope glycoproteins. Furthermore, upon increasing the viral inoculum, the proportion of the cell population that expresses a high level of viral envelope glycoprotein would increase. Fig. 4GoA shows that, despite up to an MOI of 500, not only do all cells exhibit a homogenous low level of viral glycoprotein expression on the cell surface, but the expression level of envelope glycoprotein does not increase upon increase of viral inoculum by 100-fold.

Third, when both L929 and P815 cells were infected with an MOI of 1, both cell types demonstrated that approximately two-thirds of cells were originally infected with incoming virus as predicted by a Poisson distribution (Fig. 4Go, B and C). This implies that the specific infectivity of SIN for L929 and P815 cells was comparable. However, as shown in same figure, cell surface expression levels of viral glycoprotein differed dramatically between L929 and P815 cells. The shift in a peak representing a lower expression of viral glycoprotein E2 is likely due to subsequent infection of initially uninfected cells with viruses released from infected cells.

Fourth, to demonstrate that viral-specific gene expression is occurring in the majority of infected cells, we engineered recombinant double subgenomic Sindbis capable of expressing C. botulinum C3 exoenzyme that modifies and thus inactivates the small GTPase Rho in the cell, under the control of a second subgenomic promoter. The consequences of inactivation of Rho proteins are breakdown of cellular stress fibers that can be easily detected by morphological changes in fibroblast cell lines, which exhibit rounding up of the cell bodies and neurite-like extensions. As shown in Fig. 5Go, in comparison with cells infected with dsSIN:CAT, virtually 100% of dsSIN:C3-infected cells show morphologic changes that include rounding up of the cell body and neurite-like growth in as early as 2 h postinfection with an MOI of 5. This implies that inactivation of the small GTPase by C3 exoenzyme is occurring in the majority of cells in the field. The only way infected cells can express C3 exoenzyme is via viral RNA replication, since it is under the control of a second subgenomic promoter; this is a late promoter that requires not only viral nonstructural protein synthesis, but also replication of viral RNA to generate the negative sense RNA, which is a template for subgenomic RNA synthesis. From these experimental data, we have clearly demonstrated not only that all cells are infected with viruses, but also that active, although limited, viral replication is proceeding in most of the cells in both P815 cells and L cells transfected with Ld molecules.

Finally, when we examined the role of host gene expression on class I MHC-mediated inhibition of viral replication, host gene transcription was required for maximal inhibition of viral replication. This suggests that inhibition of viral replication by class I MHC molecules may be either directly or indirectly linked to the activation of specific host gene expression. There are several reports suggesting interactions between class I MHC molecules and cell surface receptors, including IL-2 receptor, insulin receptor, and insulin-like growth factor receptor (4, 29, 30, 31). It is possible that upon exposure to, or infection with, Sindbis virus, certain class I MHC molecules such as class I Ld, either by itself or in conjunction with other cell surface ligand receptors, may participate in activating gene expression that contributes to inhibition of viral replication. Our preliminary data suggest that class I MHC molecules and a SIN viral protein interacts in a specific manner and that the interaction is required for this class I MHC molecule-mediated inhibition of Sindbis virus (C.S.H., unpublished data).

Continuing studies on the mechanism of class I MHC molecule-mediated inhibition of viral replication will generate valuable information for our understanding of individual susceptibility patterns to viral infection as well as for the development of novel antiviral therapies.


   Acknowledgments
 
We appreciate Hannah Edelen, Kisha Martin, Chris Johnnides, Dien Luu, and Henry Cho, who participated in collecting data for Fig. 1Go. We also thank Dr. Jonathan Moorman for critical reading of this manuscript.


   Footnotes
 
1 This work was partially supported by Public Health Service Grants AI-33434 and AI-24134, the American Cancer Society Institutional Research Grant, and the Carter Foundation. Back

2 Address correspondence and reprint requests to Dr. Chang S. Hahn, Beirne Carter Center for Immunology Research, Box MR4–4012, University of Virginia Health Sciences Center, Charlottesville VA 22908. E-mail address: Back

3 Abbreviations used in this paper: SIN, Sindbis virus; ß2m, ß2-microglobulin; BHK, baby hamster kidney cell 21 clone 13; CAT, chloramphenicol acetyl transferase; CEF, chicken embryo fibroblast; MOI, multiplicity of infection; pfu, plaque-forming unit. Back

Received for publication March 20, 1998. Accepted for publication September 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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