INTRODUCTION

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Natural immunity to viruses can be mediated by humoral components of the immune system, including natural antibody (Ab) and complement (11). The complement system is generally activated by Ab that has bound to its target antigen, but it can also be activated by many membrane structures independently of Ab. For example, neutralization of retroviruses and paramyxoviruses has been reported to be mediated by Ab-independent mechanisms via the classical and alternative complement pathways, respectively (42, 44, 45). Retroviruses directly activate human complement through direct interaction of C1q with the murine retrovirus p15e or with the human retrovirus gp41 transmembrane protein (5, 13). Human and nonhuman complement sources act differently; although both human and guinea pig C1q bind the Moloney leukemia virus p15e, the human C1s is required to initiate the complement cascade (4).

Viruses act differently in their sensitivities to human complement. Murine retroviruses passaged through mouse cells are reported to activate complement sufficiently for it to lyse the virions, releasing virion RNA and reverse transcriptase (34, 44, 45). Conversely, activation of complement by human retroviruses passaged through human cells is reported to not be sufficient to lyse the virions. However, the deposition of complement on the human retroviruses results in opsonization of the virions and enhanced infection of complement receptor (CR)-expressing cells (20, 38).

It is clear that Ab is not required for the activation of human complement by retroviruses, because complement activation has been shown with purified viruses or viral proteins and purified complement components (4, 5, 10). It is also unlikely that Ab is essential for the reduction in infectivity and the lysis of nonhuman retroviruses by complement, as there are numerous reports that human serum can inactivate and lyse nonhuman retroviruses passed through human cells (34, 45), through other Old World primate cells (16, 34, 45), or through nonprimate cells lacking reactivity with Ab in human serum (44). In addition, agammaglobulinemic human sera retain the capacity to lyse murine retroviruses (44, 45). Nevertheless, Ab can accelerate the dynamics of complement deposition on membranes, and recent work has indicated that the level of retrovirus inactivation by normal human serum (NHS) can be greatly augmented by natural Ab if the viruses are passed through appropriate cell lines that express the epitope to which the Ab is directed (28, 30, 36).

Human sera contain very high levels of natural Ab specific for a carbohydrate moiety present on the surface of cells from most mammals but not from Old World primates, such as humans (15). This moiety, Gal1-3Gal1-4GlcNAc-R (Gal1-3Gal), is a product of the -1-3-galactosyltransferase (GT) enzyme that adds a terminal galactose onto glycoproteins and glycolipids in a specific 1-3 linkage. Humans do not express a functional GT enzyme and instead make high levels of Ab against Gal1-3Gal as a presumed consequence of environmental exposure (14, 15, 32, 39). Recent reports have shown that murine retroviruses and human immunodeficiency virus (HIV) passaged through cells expressing GT assimilate Gal1-3Gal onto the virion gp70 and gp120 envelope proteins, respectively (28, 30, 36). The anti-Gal1-3Gal Ab (anti-Gal) in NHS binds to the virion and greatly augments the ability of complement to lyse the virion. Unquestionably, the incorporation of Gal1-3Gal onto virion surfaces by passaging retroviruses through cells expressing this carbohydrate epitope augments their reactivity with complement in NHS by binding natural Ab, and such a host cell modification may well result in a species-dependent barrier for their transmission.

Earlier work with other enveloped viruses, such as lymphocytic choriomeningitis virus (LCMV), Newcastle Disease virus (NDV), and Sindbis virus (SV), has identified host cell modification of viruses as being important for virion reactivity with NHS (6, 19, 41, 42). Given the new information available on the role of anti-Gal natural Ab in complement-dependent lysis of retroviruses, we have reevaluated the mechanisms of NHS inactivation of a variety of other viruses that acquire their envelopes by budding through the plasma membrane. Our results show remarkably different patterns of inactivation, depending on the virus and the cell in which it is grown.

	MATERIALS AND METHODS

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Immunization of mice. GT null (GT-deficient) mice were mice with the 129/Sv (129) background derived from embryonic stem cells deleted by homologous recombination in the GT gene (37). These mice were licensed from the University of Michigan and were developed in a Howard Hughes Medical Institute Laboratory. 129 wild-type (WT) (GT-sufficient) mice used as controls were obtained from Jackson Laboratories. GT-deficient and GT-sufficient mice were immunized at 6 weeks of age with erythrocyte membranes isolated from fresh rabbit blood (Cocalico Biologicals, Inc., Reamstown, Pa.). Whole blood was centrifuged at 3,000 × g for 10 min, and the plasma was discarded. Cells were lysed with 0.1× phosphate-buffered saline (PBS), and membranes were pelleted at 40,000 × g for 30 min. The supernatant was removed, and membranes were resuspended in 0.1× PBS and centrifuged as before. After the procedure was carried out a third time, the membrane pellet was resuspended in 1× PBS and again centrifuged and resuspended in PBS to give 10¹⁰ erythrocyte membranes per ml. A total of 0.5 ml was injected intraperitoneally into each mouse. Four total injections were given at 4-week intervals. Anti-Gal Ab levels in the mouse sera were assayed by enzyme-linked immunosorbent assay (ELISA) using Gal1-3Gal-conjugated bovine serum albumin (BSA) in the plate coat.

Cells. L-929, a mouse fibroblast cell line derived from C3H mice, BHK-21, a Syrian hamster cell fibroblast line, N115, a neuroblastoma line from strain A mice, HeLa, a human epithelial cell line, and Vero, an African green monkey kidney cell line, are well-characterized cell lines cultivated on monolayers as previously described (8, 42). SK-N-MC (HTB 10), a human melanoma cell line (from the American Type Culture Collection, Rockville, Md.), was maintained as monolayers in RPMI medium (Mediatech, Inc., Herndon, Va.) supplemented with 10% fetal bovine serum (FBS), 100 penicillin (IU/ml), streptomycin (100 mg/ml), 2 mM glutamine, and 1 mM sodium pyruvate (R10 medium). GT-WT and GT-KO are mouse embryonic fibroblast lines cultured from GT-sufficient 129 WT mice and GT-deficient transgenic mice, respectively. They were cultivated as monolayers in Dulbecco modified Eagle medium (Gibco BRL, Grand Island, N.Y.) supplemented with penicillin, streptomycin, glutamine, pyruvate, and 10% FBS (D10 medium) as described above.

Generation of GT-modified SK-N-MC cells. The full-length porcine GT cDNA (39) was cloned into the pLXSN retroviral vector, and amphotropic retroviral particles were produced through the intermediate ecotropic packaging cell line GPE+86 (23). Briefly, GPE+86 cells were transfected with pLXSN containing the GT gene (pLGTSN) or pLXSN without insert, using the calcium phosphate method (2). Transfected cells were selected in D10 medium containing G418 (500 µg/ml, active weight). Transfectants were pooled, and a 24-h supernatant was harvested from cells at about 90% confluence. The ecotropic virus stock was used to transduce the amphotropic packaging cell line PA317 (26), which was also selected as a pool in G418. A 24-h supernatant was harvested from these cells at 90% confluence, and the supernatant was filtered through a 0.2-µm-pore-size filter and stored at 70°C. SK-N-MC cells were transduced with pLGTSN or pLXSN by adding 1 ml of amphotropic virus to cells in 9 ml of R10 medium containing Polybrene (8 µg/ml). Following an overnight incubation, virus was removed, and cells were selected as a pool in R10 medium containing G418 (500 µg/ml). The GT-modified cells were subcloned by limiting dilution and screened for surface expression of Gal1-3Gal by fluorescein isothiocyanate (FITC) staining (see below). One clone expressing high levels of the Gal1-3Gal epitope on its surface, designated SK-GT, was used in this study. The SK-N-MC cells transduced with pLXSN were selected as a pool and served as control cells (designated SK). Transduced cells were maintained in R10 medium containing G418 (500 µg/ml).

Immunofluorescence and cell killing assays. Cells (5 × 10⁵) were reacted with polyclonal anti-Gal Ab (20 µg/ml) previously purified from human serum (30). FITC-conjugated goat anti-human immunoglobulin G (IgG) and FITC-conjugated goat anti-human IgM (Zymed Laboratories, South San Francisco, Calif.) were combined for use as secondary Ab (7.5 µg/ml [final concentration] for each). Fluorescence was measured in a FACSort (Becton Dickinson Immunocytometry Systems, San Jose, Calif.).

Viruses, propagation, and purification. The following viruses were used in these studies: herpes simplex virus type 1 (HSV-1), KOS1.1 strain (3); LCMV, Armstrong strain (8); NDV, AV strain (25); SV, originally from E. Pfefferkorn, Dartmouth Medical School (40); and vesicular stomatitis virus (VSV), Indiana strain (6). For purification, 75- or 150-mm² tissue culture flasks containing about 75% confluent monolayers of SK or SK-GT cells were infected with virus as described above and harvested at the peak of infection. On harvest, the culture fluid was twice cleared of cell debris by centrifugation at 2,000 rpm (900 × g) for 10 min. The supernatants were then layered onto discontinuous gradients of 25 and 75% Renografin-76 (Squibb Diagnostics, New Brunswick, N.J.) diluted in TES buffer (0.01 M Tris-HCl, 0.1 M NaCl, 0.001 M EDTA [pH 7.4]) as described previously (43). The samples were spun to equilibrium, and the 25%/75% interphase band was recovered, diluted in TES buffer plus 0.1% BSA, and pelleted in an SW41 Beckman centrifuge rotor at 35,000 rpm for 60 min in a Beckman ultracentrifuge. The pellet was resuspended in a standard sodium dodecyl sulfate-urea acrylamide gel preparation buffer and run on gels for Western analyses.

Western blots. Gradient-purified VSV and SV were disrupted, and their proteins were size fractionated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 12% gel. Viral samples were normalized for protein content by Coomassie blue staining of the gel. Proteins were transferred to nitrocellulose and exposed to either IB4 lectin or antiviral Ab in Western blots as described previously (28, 30). IB4 lectin selectively binds to Gal1-3Gal and is used like an Ab to detect expression of this sugar (30). Monoclonal Abs (MAbs) SV2 and SV127 to SV envelope proteins (used in combination) were a generous gift from Diane Griffin (Johns Hopkins University), and the anti-VSV MAb, which recognizes the VSV G envelope protein, was kindly provided by Kathy Hardgrave (Oklahoma Medical Research Foundation).

Serum inactivation studies. Virus was diluted in complete minimal essential medium (MEM) with 10% FBS to a concentration of 10⁵ PFU/ml, and 50 µl was mixed with 50 µl of MEM, freshly thawed NHS, or serum whose complement was inactivated by heating at 56°C for 30 min. Some experiments were miniaturized, with half of each volume used. The NHS was a human serum pool (Diamedix, Cambridge, Mass.) frozen at 70°C until use. The samples were incubated at 37°C for 45 min, 200 µl of MEM was added, and the reaction was stopped by putting the samples on ice. The virus was then titrated directly for plaque formation on Vero cell monolayers. In the disaccharide blocking studies, 10 µl of virus at a titer of 5 × 10⁵ PFU/ml was added to 40 µl of sera and 50 µl of disaccharide diluted in Hanks balanced salt solution (HBSS). The disaccharides were either Gal1-3Gal (Dextra Laboratories, Reading, England) or sucrose (Sigma Chemical Co., St. Louis, Mo.) at a concentration of 10 mg/ml before mixture (30). In some other experiments, mouse serum was substituted for NHS.

PEC. In some experiments serum-treated virus was added onto freshly isolated 129 mouse peritoneal exudate cells (PEC) previously adhered to 16-well plastic plates 2 to 4 h before infection. The PEC had been isolated by peritoneal lavage with RPMI 1640 and seeded at 10⁶ cells per well. Before infection, the medium was removed, and replaced with 1 ml of complete RPMI 1640 (R10 medium without the pyruvate), to which virus was added in 100 µl. After a 2- to 4-h adsorption period, the culture fluid containing unadsorbed virus was removed and replaced with 1 ml of RPMI medium. At 22 to 26 h postinfection, the culture fluid was harvested and used for viral titrations on Vero cell monolayers.

	RESULTS

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Expression of Gal1-3Gal on cell lines. We reported previously that LCMV passaged in L-929 cells, but not in N115, HeLa, or BHK-21 cells, was effectively inactivated by NHS (42). To determine if this pattern of serum sensitivity correlated directly with Gal1-3Gal expression on the host cell surface, these cell lines, as well as the human melanoma cell line SK-N-MC modified to express GT, were reacted with purified anti-Gal and analyzed by flow cytometry. As expected, the unmodified human SK cells and human HeLa cells did not react with anti-Gal (Fig. 1). Similarly, the hamster cell line BHK-21, shown previously to be deficient in Gal1-3Gal expression (17), did not react with anti-Gal. In contrast, the mouse L-929 and modified human SK-GT cells reacted strongly with the anti-Gal. The mouse N115 cell line was weakly positive for Gal1-3Gal expression. These cell lines were also incubated at 37°C for 30 min with NHS, and cell viability was measured. There was a direct correlation between cell surface expression of Gal1-3Gal and cell killing in NHS (data not shown).

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Expression of Gal1-3Gal on cultured cells. Cells used in these experiments were stained with purified anti-Gal Ab and analyzed by flow cytometry as described in Materials and Methods. Peaks depict Gal1-3Gal expression on the surfaces of L-929, N115, HeLa, BHK-21, SK-N-MC (SK) cells or SK-N-MC cells transduced with recombinant GT (SK-GT), and cells from GT-deficient (GT-KO) or GT-sufficient (GT-WT) mice.

Inactivation of LCMV passaged through different cell lines. Passage of LCMV through L-929 cells resulted in a virus sensitive to inactivation by fresh but not heat-inactivated NHS (Table 1, experiment 1). Our previous studies had shown that this inactivation was mediated by an undefined natural Ab and by the classical complement pathway, which is heat sensitive (42). Passage of LCMV through HeLa and BHK-21 cells resulted in virus that resisted inactivation by NHS. Our earlier work also showed that passage of LCMV in N115 cells led to virus resistant to NHS, and it is noteworthy that N115 cells only dimly express Gal1-3Gal and are themselves resistant to lysis by NHS (Fig. 1). Passage of LCMV through SK-GT cells resulted in virus highly sensitive to inactivation, but passage of LCMV through unmodified SK cells led to resistant virus. These data suggest that incorporation of Gal1-3Gal onto the virion surface renders LCMV susceptible to the anti-Gal natural Ab in NHS.

Replication of LCMV in mice expressing Ab to Gal1-3Gal. To test the hypothesis that natural Ab-mediated complement inactivation of viruses passaged through cells expressing Gal1-3Gal may play an important role in natural resistance to virus infections in vivo, we examined the replication of LCMV in mice expressing Ab to this epitope. This experiment was complicated by the fact that mice endogenously express Gal1-3Gal and cannot be immunized against it because they are immunologically tolerant. Passive transfer of Ab into normal mice also is not feasible, as it would react with the recipient's tissues. To overcome these obstacles GT-sufficient and GT-deficient (GT-KO) 129 mice (37) were immunized with rabbit erythrocytes, which are a rich source of Gal1-3Gal (15). As assessed by binding to Gal1-3Gal-conjugated BSA by ELISA (data not shown), the GT-deficient mice developed high Ab titers to Gal1-3Gal, equaling or surpassing the anti-Gal titers in NHS. Both IgG and IgM anti-Gal Ab were present in the sera of these mice. The immunized GT-sufficient mice, because of immunological tolerance, did not make a detectable Ab response to the epitope. These mice were inoculated with LCMV passaged either in SK cells or in SK-GT cells and examined for viral titers in the spleens 3 days later. The mice received either a standard dose of 5 × 10⁴ PFU, which we use for assays on NK cell and cytotoxic T-cell function (8), or a lower dose of 5 × 10² PFU, in case any effects could more readily be seen at limiting amounts of virus. To ensure exposure of virus to high concentrations of serum components, virus was inoculated intravenously.

Patterns of inactivation of other enveloped viruses. An evaluation of NHS inactivation of other enveloped viruses putatively modified by passage through cells either expressing or not expressing the Gal1-3Gal epitope was performed, and different patterns of inactivation were noted (Table 4). HSV passaged in either SK-GT or unmodified SK cells was totally inactivated by NHS, and this inactivation did not require complement, as heat-inactivated NHS also completely inactivated the virus (Table 4, experiment 1). This result could be explained by the fact that HSV is a ubiquitous human virus, and NHS contain high levels of anti-HSV neutralizing antibodies. The presence of anti-HSV Ab in a separately tested NHS preparation that gave the same pattern of inactivation was confirmed by exposing HSV-infected SK cells to NHS followed by an FITC-labeled anti-human Ig. We present these data with HSV to contrast them to studies with viruses that are not ubiquitous human pathogens. VSV, which has previously been shown to be inactivated by the classical complement pathway mediated by autologous natural Ab (6), was inactivated by NHS, regardless of the passage history of the virus (Table 4, experiments 1, 2, and 4). In contrast to HSV, heat inactivation of the serum considerably reduced the inactivation, suggesting a complement-mediated event. Similarly, NDV, which previously has been shown to be inactivated by the alternative complement pathway in the absence of Ab (41, 42), was inactivated over 100-fold by fresh NHS but not by heat-inactivated serum, again regardless of its passage history. Previous work with NDV has shown that host modification of the virus plays a role in its sensitivity to lysis by NHS (41), but in this case the virus was grown in similar cell lines either expressing Gal1-3Gal or not, and that defined variable did not alter the sensitivity of NDV to inactivation. NDV and VSV both had patterns of inactivation distinct from those of LCMV and the retroviruses, but SV demonstrated yet another pattern. SV passed through either SK-GT or SK cells resisted inactivation by NHS. Host modification of sialic acid determinants on SV has previously been implicated in its ability to activate human or gp C (19), but here it is clear that modulating only the cell expression of Gal1-3Gal did not affect the resistance of SV to neutralization by NHS.

View larger version (29K): [in this window] [in a new window]   FIG. 2.   Expression of Gal1-3Gal on virion proteins. Purified SV (A and B) and VSV (C and D) passaged in SK-GT (lanes 1) or SK (lanes 2) cells were analyzed by Western blotting for expression of viral envelope proteins by binding of anti-viral Ab (B and D) or for expression of Gal1-3Gal by binding of IB4 lectin (A and C) as described in Materials and Methods. Sizes are indicated in kilodaltons.

	DISCUSSION

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It is well established that enveloped viruses assimilate virtually unmodified cellular lipids into their virions and that their glycoproteins have glycosylation patterns similar to those of the glycoproteins of the cells in which they are passaged (29). It would therefore be expected that viruses passaged through cells containing a functional GT and expressing Gal1-3Gal should themselves express this carbohydrate epitope, and data shown here for VSV and SV (Fig. 2) and published elsewhere for HIV (28) and murine retroviruses (30, 36) support this conclusion. What is surprising is that the various enveloped viruses studied displayed different patterns of inactivation by NHS. LCMV was similar to the retroviruses in that Ab to Gal1-3Gal greatly augmented its inactivation by complement in NHS. In contrast, SV passed through GT-expressing human cells was resistant to inactivation by NHS, while NDV and VSV were inactivated by NHS, regardless of whether the Gal1-3Gal epitope was present.

Factors involved in Ab and complement inactivation of viruses include the amount of Ab bound to the virion, the proximity of Ab binding to the lipid bilayer (1), whether complement regulatory factors are incorporated into the virion (27), and the nature of virion lipid and carbohydrate structures (which could be influenced by the type of cellular membrane through which the virion buds and by such factors as the degree of sialation) (19, 21, 29). The fact that NDV and VSV were inactivated by NHS regardless of their modification with Gal(1-3)Gal can be explained by previous work showing that NDV can directly activate complement in the absence of Ab (41, 42) and that VSV binds to a distinct human natural IgM Ab that remains undefined but, based on the present study, is probably not specific for Gal1-3Gal (6). An explanation for the resistance of SV to NHS, even after it has incorporated Gal1-3Gal into its virion, is more problematic. Previous work has shown that SV could be lysed by complement in the presence of antiviral Ab (35). Other work showed that purified SV virions could activate the complement system in the absence of Ab, that the levels of activation depended on the cells through which SV was passaged, and that the activation was significantly enhanced by treatment of the virions with neuraminidase to cleave off sialic acid determinants (19). In this present study, neuraminidase treatment of SV passed through SK-GT cells did not render the virions significantly more sensitive to NHS (Table 4, experiment 5), suggesting that high levels of sialation were not responsible for its resistance to lysis. Although SV virions acquire host cell plasma membrane lipids and glycosylation patterns, the ratio of cholesterol to phospholipids is much higher than in cell membranes, and the SV membranes are more densely packed, less fluid, and have greater curvature than cellular membranes (21, 22, 29). It is possible that these properties contribute to the resistance of the virus to inactivation by NHS. Additionally, some viruses, such as HIV, acquire species-restricted complement regulatory factors from the cells through which they are passed (27). We detected significant expression of the complement regulatory factors CD55 and CD59 on SK-N-MC cells by immunofluorescence (data not shown). This seemed like an unlikely mechanism to explain the resistance of SV to inactivation by NHS, because the SK-GT cells themselves were quite sensitive to lysis (Fig. 2). Nevertheless, we examined the sensitivity of SV passaged through mouse GT-WT cells to NHS and found that it was sensitive to inactivation. Whether this means that SV acquired species-restricted anti-complement factors from human SK-GT cells but not from mouse GT-KO cells remains unresolved, but it demonstrates further the complexity of host modification factors determining whether or not a given virus is sensitive to inactivation by NHS.

The availability of GT-deficient mice, which can be immunized to mount Ab responses to Gal1-3Gal, led us to test our hypothesis that species-dependent differences in natural Abs to cellular membranes may play important roles in natural immunity against enveloped viruses (42). However, even though sera from the immunized mice had some moderate antiviral activity against LCMV passaged through GT-expressing cells, immune mice did not restrict the replication of that virus in vivo. Work on Ab-independent complement activation by human retroviruses has shown that the activation of complement through this mechanism is insufficient to trigger virolysis and consequently serves as an opsonin for viral infection of cells expressing CR (38). We therefore questioned whether the inactivation of LCMV vis à vis its growth in vero cells would be equivalent to its inactivation vis à vis its growth in macrophages, which express Fc receptors and CR and are the natural targets for LCMV in vivo (24). Table 3 shows that treatment with the same mouse sera that inhibited virus growth on Vero cells did not inhibit the viral infectivity on PEC. Complement levels in laboratory mice are much lower than in wild mice, guinea pigs, rabbits, or humans, and infectious virus-Ab-complement complexes can be isolated from the sera of LCMV persistently infected mice (46). Thus, the GT-deficient mouse model, due to its low complement activity, may not be an appropriate model to test the role of anti-Gal in immunity to viruses crossing species barriers, despite mounting anti-Gal Ab titers similar to that in humans. This model does, however, illustrate the probable importance of a highly efficient complement source, as the anti-Gal Ab that was present could apparently bind to the LCMV virions but was ineffective in the weak complement milieu of the mouse. The importance of complement in the control of viral infections has further been illustrated in studies in animals depleted of complement components (18) and is predicted by the observations that many viruses encode CR or complement regulatory proteins and that some viruses incorporate cellular complement regulatory proteins into their membranes (9, 27, 31).