Department of Virology, SMI/Karolinska Institute, 171 82 Solna, Sweden 1
Department of Immunology, Microbiology, Pathology and Infectious Disease, Karolinska Institute, Box 12 773, 112 96 Stockholm, Sweden 2
Author for correspondence: Lennart Svensson.Fax +46 8 301635. e-mail Lensve{at}mbox.ki.se
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Abstract |
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Many fusogenic glycoproteins of viruses which fuse with the plasma membrane, e.g. cytomegalovirus (CMV) (Compton et al., 1992 ), human immunodeficiency virus (HIV) (Freed & Martin, 1995
; McClure et al., 1988
) and paramyxovirus (Lamb, 1993
), are rich in disulfide bonds and it is tempting to speculate on the role that disulfide bonds play during disassembly. Ryser et al. (1994)
have recently suggested that HIV and its target cell engage in a thioldisulfide interchange reaction and that reduction of critical disulfide bonds in viral glycoproteins may be the initial event that triggers the conformational changes required for HIV entry. In the present study we determined whether CMV contains free thiol groups that are essential for entry and infectivity.
CMV (strain AD 169) and herpes simplex virus-1 (HSV-1; strain F9004) were cultivated on human fibroblast cells until complete CPE. Infected cells were then harvested, cellular debris removed by centrifugation, the supernatant aliquoted and titres determined as described below.
To determine whether CMV or HSV-1 possess free thiol groups critical for infectivity, 1·2x104 p.f.u./10µl of virus was mock-treated or treated with various different concentrations of 5',5-dithiobis 2-nitrobenzoic acid (DTNB; Sigma) for 1 h at 37 °C or with 500 µM DTNB for various periods of time. DTNB is a membrane-impermeable thiol-blocking reagent which covalently modifies thiol groups (Buelt & Bernlohr, 1990 ; Li et al., 1994
; Smith et al., 1975
) and can be used to measure free thiol groups on proteins (Ploux et al., 1995
; Zhi et al., 1991
). After DTNB treatment virus was diluted 1/100 with serum- free Eagle's MEM and 100 µl volumes (equivalent to 1·2x103 p.f.u. were inoculated in duplicate in 96-well plates (Costar) containing confluent monolayers of human fibroblast cells. After 1 h incubation at 37 °C, the inoculum was removed and replaced with serum-free Eagle's MEM. At 8 h (HSV) or 24 h (CMV) post-infection, cells were fixed and the reduction in infectivity was determined by immunoperoxidase staining (Mirazimi & Svensson, 1998
; Mirazimi et al ., 1996
). Briefly, infected cells were fixed for 18 h with 2% paraformaldehyde in PBS, followed by treatment with 1% Triton X-100 for 10 min. Mouse anti-CMV early antigens [clone CCH2+DDG9 code no. M0854 (DAKO); CCH2 reacts with CMV delayed early DNA binding protein p52 and DDG9 reacts with a 76 kDa protein of Ad 169 antigen] and rabbit anti-HSV type 1 [code no. P0175 (DAKO)] were diluted in PBS containing 0·2% BSA and 0·1% Triton X-100 and incubated for 1·5 h at 37 °C on cells followed by three washes with PBS and a second incubation for 1·5 h at 37 °C with peroxidase-labelled goat anti-mouse IgG or goat anti-rabbit IgG (Bio-Rad). After three more washes the number of infected cells was determined (Mirazimi et al., 1996
).
The inhibitory effect of DTNB on CMV infectivity was dose-dependent and infectivity was completely abolished at a DTNB concentration of 250 µM (Fig. 1a). We have also obtained the same results with purified CMV(data not shown). Analysis revealed that the inhibitory effect was rapid and almost complete inactivation was obtained after 3045 min of treatment with 500 µM DTNB (Fig. 1b
).
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To determine whether the plasma membrane possesses accessible free thiol groups which might participate in virus entry, human lung fibroblast monolayers were mock-treated or treated with 110 mM DTNB. After 1 h incubation at 37 °C, DTNB was washed out with Eagle's MEM and monolayers immediately infected with 1·2x103 p.f.u. of CMV; the number of infected cells was determined 24 h later. The results did not reveal any difference (<5% variation) between DTNB-treated and mock- treated cells, suggesting that if thiol groups had been accessible on the plasma membrane, they did not participate in the entry process of CMV. However, the observations do not rule out the possibility that binding of CMV to the plasma membrane causes conformational changes in cellular proteins such that thiol groups protrude and react with thiol groups on the virus.
The novel observation that CMV infectivity could be eliminated by blocking specific thiol groups on the virus surface led us to determine whether viral infectivity also could be restored if the DTNB-blocked thiol groups were reduced. To test this hypothesis, DTNB-inactivated virus with no remaining infectivity was mock-treated or treated with 1·25 or 2·5 mM of the disulfide-reducing agent dithiothreitol (DTT) for various times. Virus was subsequently diluted 1/100 in Eagle's MEM and inoculated onto cells, followed by determination of infectivity 24 h later. Most surprisingly, we found that a brief incubation with DTT restored viral infectivity by reducing disulfide bonds between the viral glycoprotein(s) and DTNB. As illustrated in Fig. 2(a), infectivity was already partly restored after 5 min incubation with DTT and increased to 65% after 15 min of treatment.
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The conclusion that the recovery of viral infectivity was due to reduction of covalent bond interactions between thiol groups on viral protein(s) and DTNB, and not to reduction of established inter-or intra- disulfide bonds of viral glycoproteins, is therefore supported by the results presented in Fig. 2(b).
In order to determine whether CMV retained its capacity to bind to target cells after inactivation by DTNB, a binding experiment was performed. Fibroblast monolayers (38x106 cells) were infected with CMV at an m.o.i. of 0·01. When 30% CPE was observed, the medium was replaced with medium free of methionine and cysteine except for 330 µCi [35 S]methioninecysteine (Trans-label; DuPont). Virus was collected 5 days later. Cell debris was removed by low-speed centrifugation and virus was pelleted by centrifugation at 25000 r.p.m. in a Beckman SW-28 rotor for 1 h through a 5 ml layer of 20% (w/v) d-sorbitol (Soderberg et al., 1993 ). The infectivity (p.f.u.) titre of the purified 35S-labelled virus was determined by peroxidase focus reduction test (Mirazimi et al., 1996
). Radioactivity was quantified by liquid scintillation counting, giving a specific activity of 6·7 c.p.m./p.f.u.
Different concentrations of purified 35S-labelled-CMV were mock-treated, DTNB-treated (500 µM) or heparan sulfate- treated (10 µg) and inoculated on fibroblast monolayers previously pretreated with PBSBSA (3%). After adsorption at 4 °C for 2 h, unbound virus was washed out and cells were rinsed three times before cell lysis and scintillation counting of bound radioactivity. The heparan sulfate treatment demonstrated the specificity of attachment of 35S-CMV to the cell-surface receptors, since it inhibits attachment of CMV to host cells (Compton et al., 1993 ; Kari & Gehrz, 1992
, 1993
). As expected, heparan sulfate treatment (Fig. 3
) inhibited attachment of CMV to the cell surface by 90% in our experiments (Fig. 3
). While infectivity of CMV was completely (<99·99%) eliminated by treatment with 0·5 mM DTNB, 70% of DTNB-treated virus still bound to the cells, suggesting that post-attachment events were inhibited by DTNB, but not the early attachment to cell receptor(s).
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As we have demonstrated in Fig. 1(a), the infectivity of HSV-1 was unaffected by treatment with DTNB. gB and gH of HSV-1 may have a conformation different from that of gB and gH of CMV, which could explain why HSV-1 does not expose free thiol groups critical for infectivity.
In summary, we have shown conclusively that CMV contains free thiol groups that play a critical role in viral infectivity and that after inhibition infectivity can be recovered following appropriate treatment. We believe that this new information can be useful to further explore molecular mechanisms behind entry and fusion events of CMV. Studies aimed at identifying viral and cell-surface proteins with free thiol groups are in progress.
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References |
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Received 8 January 1999;
accepted 16 July 1999.