The degradation of herpes simplex virus particles after uptake by phagocytes was studied, but, since lysis of the phagocyte also resulted in damage to the viral ...
Journal of General Virology (1990), 71, 1205-1209. Printed in Great Britain
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Degradation of herpes simplex virions by human polymorphonuclear leukocytes and monocytes Jos A. G. Van Strijp,* Lia A. M. Miltenburg, Marijke E. van der Tol, Kok P. M. Van Kessel, Ad C. Fluit and Jan Verhoef Eijkman-Winkler Laboratory o f Medical Microbiology, University o f Utrecht, Heidelberglaan 100, 3584 C X Utrecht, The Netherlands
The degradation of herpes simplex virus particles after uptake by phagocytes was studied, but, since lysis of the phagocyte also resulted in damage to the viral envelope, measurement of viral infectivity as a criterion of viral degradation after phagocytosis was not possible. Therefore we focused on later events in viral destruction, namely the degradation of macromolecules. We have demonstrated that polymorphonuclear leukocytes (PMN) and monocytes (MN) can rapidly degrade the membrane proteins of the phagocytosed herpesvirus virions. P M N and M N from a patient with
cllronic granulomatous disease showed a similar rate of degradation compared to P M N and M N from healthy donors, which excludes an important role for toxic oxygen species in viral protein degradation. Experiments using toxic oxygen species-generating systems supported this observation. In contrast to PMN, M N are also effective in the digestion of viral DNA. We conclude that P M N and M N are able to neutralize large amounts of phagocytosed HSV, so their role in antiviral defence has again been demonstrated.
As well as specific defence mechanisms, such as T cells, it has recently become clear that phagocytes also contribute to antiviral defence. For herpes simplex virus (HSV) the role of phagocytosis by polymorphonuclear leukocytes (PMN), at least in keratitis, seems to be established (Smith et al., 1986; Bingham et al., 1985). We (Van Strijp et al., 1989a,b) and others (Bingham et al., 1985; Fujisawa et aL, 1987; McCullough et al., 1988; Smith et al., 1986; West et al., 1987) have shown that viruses, including HSV, are efficiently phagocytosed by PMN and monocytes (MN). However, the importance of phagocytosis in antiviral defence can not be limited to the phenomenon of uptake alone. The ultimate question remains: can a phagocyte really destroy the virion in such a way that it is no longer infectious? The efficiency of this process will determine whether the phagocyte itself is at risk of becoming infected, or whether the phagocyte is a reservoir of live virus particles that are liberated and infect new targets elsewhere in the body, after the phagocyte has died. In the latter case the phagocyte could protect virus particles against other defence mechanisms and instead of being helpful to the host, the phagocyte may contribute to the capacity of the virus to invade target tissues. It could also be that the complex microbicidal activities of the phagocyte are virucidal in vivo and in vitro, in which case the phagocyte could be a key cell in the host defence against virus
infections. MN can be infected by viruses and the probability of being infected is greater when specific anti-virus antibodies are present. This antibody-enhanced infection has been described for several viruses, such as Sindbis virus (Chanas et al., 1982), dengue virus (Halstead, 1988), lactate dehydrogenase-elevating virus (Inada & Mims, 1985), rabies virus (King et al., 1984), West Nile virus (Peiris et al., 1981), human immunodeficiency virus (Takeda et al., 1988), BK virus (Traavik et al., 1988), yellow fever virus (Schlesinger & Brandriss, 1981) and fish rhabdoviruses (Clerx et al., 1978). The ability of phagocytes to digest their bacterial targets has been the subject of several investigations (Babior, 1984; Spitznagel, 1984). Because we are interested in the role of the phagocytic cell in the host defence against HSV, we studied the question of whether phagocytes could degrade HSV. The classic method of studying inactivation of virions is neutralization of infectivity, measured by titration of the virions in plaque assays or TCIDso experiments. Unfortunately, after the virion is taken up by a phagocyte it is impossible to develop a method for lysing the cell and to free the intact virions in order to assay them in virus titrations, because the viral membrane is also damaged by the procedure. Furthermore, virions are already partially neutralized by the opsonins needed for phagocytosis. Therefore, we directly measured the capacity of the phagocyte to
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degrade viral protein and DNA. For the study of protein degradation we took advantage of the properties of Triton X-114 (Tx-ll4), a non-ionic detergent that can displace and replace much of the normal lipid environment around hydrophobic proteins, whereas hydrophilic proteins bind almost no detergent. This results in detergent micelles containing the integral membrane proteins and these micelles form aggregates at a temperature above 20 °C, resulting in a phase separation. After centrifugation they separate into a detergent-rich pellet containing hydrophobic proteins and a supernatant containing the soluble proteins and peptides (Bordier, 1981; Pryde, 1986). PMN, MN, cell lines, viruses and human sera were obtained as described (Van Strijp et al., 1989a, b). PMN were 98% pure and 97% viable, and MN were highly enriched (80 to 90% purity). In our earlier work (Van Strijp et al., 1989a, b) it was shown by flow cytometric analysis that viruses did not associate with the contaminating lymphocytes and that phagocytes react normally in, for instance, uptake of bacteria and metabolic burst. No difference was observed between phagocytes of donors that were seropositive or seronegative for HSV. Virus was radiolabelled using 20 rtCi [3H]thymidine per ml or methionine-free medium supplemented with 35 gCi [3SS]methionine per ml, and purified as described (Van Strijp et al., 1989a). Virus was stored at - 70 °C in small volumes at a standard concentration of 350 I.tg/ml. Purity was checked by electron microscopy and it was shown that over 95 % of the particles were enveloped viruses. To determine phagocytosis of the virus PMN or MN were mixed with purified fluorescein-labelled virus and human serum, incubated for various time periods and measured on a per cell basis in a FACStar flow cytometer as described (Van Strijp et al., 1989a). In the absence of opsonins no uptake by PMN of. HSV particles was observed, but spontaneous uptake of virions by MN reached a level of 25% after a 2 h incubation period. Addition of opsonins to this assay enhanced the uptake of virions for the phagocytes, but for MN the uptake process proceeded over a longer time (Van Strijp et al., 1989a). On the basis of the above mentioned measurements, for the following experiments, in which the degradation of HSV glycoproteins was studied, we used a 30rain phagocytosis time with radiolabelled virus. [3sS]Methionine was used to label the viral proteins. A total of 60 p.1 PMN or MN [3 × 107 cells/ml Hanks' balanced salt solution with 5 ~ foetal calf serum (HBSSf)] were mixed with 30 gl purified 35S-labelled virus and 30 gl human serum (diluted in HBSSf). The cells were incubated for 30 min in a shaking water-bath at 37 °C (150 r.p.m.) and the reaction was stopped by adding icecold phosphate-buffered saline. Cells were separated from free virus by centrifugation at 160 g and washed
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Time after phagocytosis (min) Fig. I. PMN and M N were assayed in their ability to degrade Tx-114associated membrane proteins into non-associated material. After phagocytosis for 30 rain at 37 °C cells were further incubated either at 37 °C ( ) or 4 °C ( ). Results are expressed as mean + standard error of three or more independent experiments with cells from different donors.
three times. The percentage uptake in 30 min, relative to the added amount of radioactivity, was 61 _+ 3% for PMN and 52 + 4% for MN, for the [3SS]methionine- as well as for the [3H]thymidine-labelled virions (about 8000 c.p.m, per 2 × 106 cells, depending on the virus batch). Thereafter the cells were washed, incubated further and then analysed for virus degradation. Cells were resuspended in 120 ~1 HBSSf and incubated further for various time periods at 37 °C in a shaking water-bath. After incubation the reaction mixtures were cooled on ice, solubilized in 1% Tx-114 in 500 gl and layered onto 500 gl 6% (w/v) sucrose. Tx-114 micelles were allowed to form aggregates at 37 °C and were then pelleted through the sucrose at 12000 g for 3 min. The Tx pellet and two 200 ~tl samples from the top supernatant were mixed with 2.5 ml scintillation cocktail and counted. Results were expressed as the solubilizable protein fraction: c.p.m, in supernatant fluid/(c.p.m, in pellet + supernatant fluid) × 100%. In an initial experiment using only purified [3SS]methionine-labelled virions it was determined that the virionassociated 35S label recovered in the detergent-rich phase remained the same after incubating the virion at 37 °C up to 2 h, demonstrating the validity of the choice for this system of using [3SS]methionine as a label (data not shown). The data in Fig. 1 (percentage of degradation after uptake) are expressed as the percentage of radioactivity that was associated with Tx-114 relative to the amount that was taken up by the leukocyte. The results indicated that degradation of virion proteins took place both in MN and granulocytes and was timedependent. In MN degradation was more effective than in granulocytes and reached values of over 70%, following an incubation period of 120 min after 30 min
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Table 1. Comparison of degradation rate of viral proteins by P M N from CGD patient and control cells
Uptake (~)* 0 mint 60 min 120 rain
Control PMN
CGD PMN
63.1 _+2.6 21-1 + 0-6 39.1 _+1.1 48.1 _+1.3
61-7 _+3-5 22.0 +_0.7 39.1 + 0.7 45-3 _+1-4
* Percentage uptake expressed as the PMN-associated radioactivity as a percentage of total added radioactivity. I"Time after phagocytosis.Tx-114non-associatedpercentage of total phagocytosed radioactivity.
Fig. 2. Autoradiogram of SDS-PAGE of total cell extracts of 3sSlabelled viral proteins after phagocytosis by PMN. (MN showed the same degradation pattern; data not shown.) Lane 1, normal HSV-1 protein pattern; lane 2, after 1 h phagocytosisby PMN; lane 3, after 2 h phagocytosis by PMN; lane 4, Mr markers.
phagocytosis. However, since the lines in this graph have the same slope, the difference between the two cell types must occur in the first 30 min. When P M N were allowed to take up virus particles at 37 °C for 30 min and were then further incubated at 4 °C, no additional degradation of viral proteins was observed after the initial breakdown demonstrated during the first 30 min. For analysis of the different viral proteins S D S - P A G E was performed. Samples with 3sS-labelled virus were taken after the phagocytosis assay, solubilized in SDS and then applied to a 7 ~ acrylamide gel, cross-linked with diallyltartardiamide (Cassai et al., 1975). The gel was fixed with methanol/acetic acid/water (40:10:50) and rinsed in enhancer (Amersham). The gel was then dried and autoradiography was carried out. Fig. 2 shows degradation of high MT proteins into lower Mr material. This is surely the simplest way to demonstrate the degradation
of proteins, but, since little radioactivity can be recovered in the bulk non-labelled cellular protein, these photographs needed very long exposure times and were difficult to quantify, which is the main reason why the Tx-114 method was developed. Radiolabelled virus (30 gl) was also incubated without cells under different conditions of low pH, proteinases or toxic oxygen species-generating systems and thereafter analysed with the same Tx-114 method. The condition of low pH, an isotonic sodium acetate buffer of p H 6 (as is observed in phagolysosomes), did not induce degradation of viral proteins on its own. Toxic oxygen species also played no major role in viral protein degradation, as toxic oxygen radical-generating systems, composed of either 1-25 mM-xanthine plus 0.1 units/ml xanthine oxidase or 15 mM-glucose plus 0"5 units/ml glucose oxidase, did not degrade m e m b r a n e proteins. N o significant amount of radioactivity was recovered in the Tx supernatant. Proteinases trypsin (0.5 mg/ml), elastase (70 units/ml) and Proteinase K (0.5 mg/ml) released, respectively, 11292 +_ 1232 c.p.m., 14968 _+ 2315 c.p.m. and 8328 _+ 1248 c.p.m, in the Tx supernatant of the 16342 +_ 1374 c.p.m, total added virion-associated radioactivity. The absence of a role for toxic oxygen species in viral protein degradation was further indicated by experiments carried out with P M N of a patient with chronic granulomatous disease (CGD). The results presented in Table 1 demonstrate that the phagocytic capacity of C G D P M N was the same as for control P M N and that even without the production of toxic oxygen species degradation of proteins occurred. To study the degradation of viral D N A , P M N or M N were incubated with [3H]thymidine-labelled virions in the presence of opsonins. Non-ingested virions were washed away and the cells were incubated further at 37 °C. Thereafter cells were resuspended in water, frozen and thawed, then an equal volume of 1 0 ~ T C A was added and incubated for 30 min at 4 °C. The precipitate
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Fig. 3. Degradation of viral D N A that was taken up by P M N (open bars) or MN (hatched bars). After 30 min the cells were washed free of non-incorporated virus and lysed directly (0), or further incubated for 120 min and then lysed (120). Results shown are TCA-soluble c.p.m, as a percentage of the total cell-associated c.p.m.
formed was centrifuged for 10 min at 12000 g and washed with 5YooTCA. The pellet was suspended in Aqualuma, counted in a liquid scintillation counter and the TCA-precipitable radioactivity was measured. As expected, no breakdown of DNA was observed in PMN (Fig. 3). However, in MN after 30 min of incubation substantial DNA damage had already been observed. Two h later one-third of the phagocytosed DNA appeared to be broken into fragments that were no longer precipitable in 5 ~ T C A , and thus were smaller than about 20 bases (Cleaver & Boyer, 1972). Phagocytosis of viruses results, when IgG is present, in a metabolic burst of the phagocyte (Bingham et al., 1985; Van Strijp et al., 1989b; Weber & Peterhans, 1983). The question still remains as to whether the viruses are actually neutralized or even degraded by the phagocytes. There are several reasons to assume that degradation of virions occurs. After phagocytosis and phagosomelysosome fusion (Van Strijp et al., 1989a) it is likely that virus particles are inactivated and eventually degraded, due to the antiviral effects of lysosome constituents, such as defensins, cationic proteins (Rouse et al., 1980; Selsted & Harwig, 1987; Thorne et aL, 1984; Zerial et al., 1987; Daher et al., 1986) and the reactive oxygen species liberated by the PMN. It has been shown that toxic oxygen products are able to inactivate virus infectivity (Belding et al., 1970). Initially we monitored the loss of infectivity of herpes virions after phagocytosis by PMN and MN according to the method that we developed to follow the degradation of Escherichia coli (RozenbergArska et al., 1984). After phagocytosis the phagocyte was
lysed to release the virions, which were then quantified by titration on fibroblasts. The methods we used for disruption of the phagocyte (freezing and thawing, hypotonic shock, detergent lysis and sonication) destroyed the cells as well as the virions and resulted in nonreproducible titration curves of the released virions. Disruption using a Dounce homogenizer, a commonly used method for the lysis of virus-infected cells, unfortunately failed to work for these small amounts of phagocytes. To solve these technical problems we concentrated on a later stage of destruction of virions, namely the degradation of viral macromolecules, keeping in mind that when viral DNA or viral proteins are degraded the virus will no longer be infectious. Because one or more of the membrane proteins of HSV are responsible for the initiation of cell infection (Johnson et al., 1984) the degradation of membrane proteins can be viewed as a measure of loss of infectivity. In this study we showed that only MN were able to degrade the viral DNA to fragments that were no longer acid-precipitable. This indicated that the complete virion must have been fragmented beforehand, because enveloped virions are insensitive to DNase activity. In accordance With other findings for these cells, PMN were not capable of degrading viral DNA because they do not possess DNase (Lamers et al., 1981 ; Rozenberg-Arska et al., 1984). For the study of protein degradation we used Tx-114, which results in detergent micelles containing the integral membrane proteins that can be pelleted at temperatures above 20 °C (Bordier, 1981; Pryde, 1986). We showed a time-dependent decrease in the ability of radiolabelled proteins to integrate in Tx-ll4 micelles, mediated both by PMN and MN, and conclude that virions are broken down by these phagocytes and that MN perform more efficiently than PMN. But MN can become infected by several viruses and antibodyenhanced infection has been demonstrated in a number of cases. PMN escape infection, probably because of their differentiated state in which no DNA replication, RNA or protein synthesis can be observed (Bainton et al., 1971; Cline, 1966; Jack & Fearon, 1988). Although we were able to show that viruses are degraded in phagocytes, it is not possible to determine the contribution of oxygen radicals to damage. PMN from a patient with CGD showed the same rate of degradation and cell-free oxygen radical-generating systems were inactive, indicating a minor role for oxygen radicals in the degradation of proteins. Furthermore, from the 10000 virus particles able to be taken up by phagocytes (Van Strijp et al., 1989a) some might escape the destruction machinery of the phagocyte. We propose that PMN are the best candidates for the clearance of virions and although monocytes show faster degradation patterns, the total number of PMN present and the
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inability to be infected by viruses renders PMN more effective in rico. The authors wish to thank Edith Peters for handling the cell lines and virus stocks and Dr C. M. J. E. Vandenbroucke-Grauls for critically reading this manuscript.
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(Received 9 August 1989; Accepted 3 January 1990)
