We have studied "the role of hydrophobic interactions in the fusion activity of two lipid enveloped viruses, influenza and Sendai. Using the fluorescent probe ANS ...
Bioscience Reports, Vol. 14, No. 1, 1994
Role of Hydrophobic Interactions in the Fusion Activity of Influenza and Sendai Viruses Towards Model Membranes Jofio Ramalho-Santos, ~ Ricardo Negr~o, z and Maria da Conceiq~o P e d r o s o de Lima 2'3 Received November 20, 1993 We have studied "the role of hydrophobic interactions in the fusion activity of two lipid enveloped viruses, influenza and Sendai. Using the fluorescent probe ANS (1-aminonaphtalene-8-sulfonate) we have shown that low-pH-dependent influenza virus activation involves a marked increase in the viral envelope hydrophobicity. The effect of dehydrating agents on the fusion activity of both viruses towards model lipid membranes was studied using a fluorescence dequenching assay. Dehydrating agents such as dimethylsulfoxide and dimethylsulfone greatly enhanced the initial rate of the fusion process, the effect of dimethylsulfone doubling that of dimethylsulfoxide. The effect of poly(ethylene glycol) on the fusion process was found to be dependent on the polymer concentration and molecular weight. In general, similar observations were made for both viruses. These results stress the importance of dehydration and hydrophobic interactions in the fusion activity of influenza and Sendai viruses, and show that these factors may be generally involved in membrane fusion events mediated by many other lipid enveloped viruses. KEY WORDS: hydrophobicity; dehydration; influenza virus; Sendai virus; membrane fusion; fluores-
cence dequenching. Abbreviations: ANS- 1-aminonaphtalene-8-sulfonate; a.u.- arbitrary units; DMSO-dimethylsulfoxide;
DMSO2- dimethylsulfone; HA- influenza virus hemagglutinin; LUVs- large unilamellar vesicles, PCphosphatidylcholine; PE-phosphatidyl-ethanolamine; PEG 1500-poly(ethylene glycol), 1500 average molecular weight; PEG 3000- poly(ethylene glycol), 3000 average molecular weight; PEG 6000poly(ethylene glycol), 6000 average molecular; weight; PS-phosphatidylserine; R18octadecylrhodamine B chloride.
INTRODUCTION Lipid enveloped viruses infect cells by a membrane fusion event mediated by relatively well defined proteins included in the viral envelope [1-4]. These fusion proteins generally contain a certain number of consecutive hydrophobic residues 1Department of Zoology, Center for Cell Biology and Center for Neurosciences of Coimbra, University of Coimbra, 3049 Coimbra Codex, Portugal. 2 Department of Biochemistry, Center for Cell Biology and Center for Neurosciences of Coimbra, University of Coimbria, 3049 Coimbra Codex, Portugal. 3 To whom correspondence should be addressed. 15 0144-8463/94/0200-0015507.00/09 1994PlenumPublishingCorporation
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Ramalho-Santos, Negr~o and Pedroso de Lima
that are thought to be instrumental in membrane destabilization amd may therefore constitute the final trigger for fusion. In the case of influenza virus fusion is mediated by a well documented low-pH-dependent conformational change of the viral hemagglutinin (HA), which takes place in an intracellular acidic pre-lysosomal compartment following viral endocytosis [1-4]. The conformational change exposes a hydrophobic "fusion peptide" located in the amino terminal of the HA2 subunit of the hemagglutinin that is thought to somehow destabilize the contact point between the viral envelope and the target cellular membrane, thereby triggering fusion [5-8]. For Sendal virus, which is thought to penetrate its target cells by fusing with the cell plasma membrane at neutral pH, fusion is apparently mediated by a stretch of hydrophobic residues exposed on the surface of the viral fusion protein [3, 4]. In this paper we have studied the role of hydrophobic interactions in the fusion activity of influenza and Sendai viruses. Using ANS, a fluorescent probe normally employed to detect hydrophobic microenvironments in proteins [20, 21] and changes in membrane surface charge [23], we have shown that a marked increase in influenza virus envelope hydrophobicity correlates with the acquisition of viral fusion competence. This Observation prompted us to conduct studies on the fusion activity of influenza virus towards large unilamellar lipid vesicles composed of negatively charged and zwitterionic phospholipids as affected by dehydrating agents such as dimethylsulfoxide, dimethylsulfone and poly(ethylene glycol) of 1500 and 3000 average molecular weight. We have shown that dehydrating agents can greatly enhance the initial rate of the fusion process, although the effect of PEG is dependent on its concentration and molecular weight. We have also found that the fusion activity of Sendai virus is stimulated by dehydrating agents in a similar manner to that of influenza virus. Overall, our results demonstrate the importance of dehydration and hydrophobic interactions in the fusion activity of lipid enveloped viruses towards model membranes. MATERIALS AND METHODS DMSO and DMSO2 were from Sigma (St. Louis, MO), PEG 1500, PEG 3000 and EG 6000 were from B D H (Dorset, England), PS and PE were purchased from Avanti Polar Lipids Inc. (Birmingham, AL) and PC was from Avanti or Sigma. Virus and Liposome Preparations Influenza virus, A/PR/8/34 (HIN1) strain, was grown for 48 h at 37~ in the allantoic cavity of 11-day-old embryonated eggs, purified by discontinuous sucrose density gradient centrifugation and stored at -70~ in phosphate buffered saline
[ii]. Sendai virus (Hemagglutinating virus of Japan), Z strain, was grown for 72 h in the allantoic cavity of 10-day-old embryonated eggs, purified by differential centrifugation and stored at -70~ in phosphate buffered saline [19].
Membrane Dehydrationand Viral Fusion Acitivity
17
LUVs of pure PS, PS/PC 1:1, PS/PC 1:4 or PS/PE 1:1 (molar ratio) were prepared in 150mM NaC1, 10 mMHepes, pH 7.4 as described [9]. The vesicles were sized through 0.1/xm polycarbonate filters and their concentration datermined by a phosphate assay.
Hydrophobicity Measurements For ANS experiments 30/xg of viral protein were added to 2 ml of a 23/zM ANS solution in appropriate buffer containing 150 mM NaC1 and 10 mM HEPES, pH7.4. In each case a ANS fluorescence emission scan was taken between 450-560 nm with the fixed excitation set at 350 nm using a SPEX Fluorolog fluorometer. The buffer pH was then lowered to 5.0 and (following a 15 min incubation) another ANS emission scan was recorded. Changes in the maximum fluorescence intensity of ANS (a.u.), as weli as in the emission maximum of ANS fluorescence (nm) were measured. Increases in hydrophobicity were assessed by a concomitant increase in ANS fluorescence and a simultaneous blue shift in the emission maximum of the fluorescent probe [20, 21]. It should be noted that ANS fluorescence in solution, as described before [20], does not change when the pH is lowered from 7.4 to 5.0 (not shown). Viral fusion activity. Both viral preparations were labeled with octadecylrhodamine B chloride (R18, Molecular Probes Inc., Eugene, OR) as described previously [10, 11, 22]. The final concentration of added probe corresponded to approximately 5 mole % of total viral lipid and that of ethanol was less than 1% (v/v). The mixture was incubated in the dark for 30 min at room temperature. R18-1abeled virus was separated from noninserted fluorophore by chromatography on Sephadex G-75 (Pharmacia, Uppsala, Sweden) using 150 mM NaC1, 10 mM Hepes, pH 7.4 as elution buffer. The protein concentration of the labeled virus was determined by the Lowry assay. Fusion, initiated by rapid injection of R18-1abeled virus into a cuvette containing LUVs in a final volume of 2 ml was monitored continuously using the fluorescence dequenching assay as described [10, 11, 22]. All experiments were carried out at 37~ either in 150 mM NaC1, 10 mM sodium acetate, pH 5.0 (with or without dehydrating agents) for influenza virus or in 150mMNaCI, 10 mM HEPES, pH 7.4 (with or without dehydrating agents) for Sendai virus. In the case of influenza virus 1/xg of viral protein and 100 nmoles of LUV lipid were used per experiment, while in the case of Sendai virus 10/xg of viral protein and 200 nmoles of LUV lipid were used per experiment. In each experiment the fluorescence scale was calibrated such that the initial fluorescence of R18 labeled virus and LUV suspension was set at 0% fluorescence. The value obtained by detergent lysis after each experiment with Triton X-100, at a final concentration of 1% (v/v), was set at 100% fluorescence. Fluorescence measurements were performed in a Perkin-Elmer LS-50 luminescence spectrometer or in a SPEX Fluorolog fluorometer with excitation at 560 nm and emission at 590nm. The sample chamber was equipped with a magnetic stirring device, and the temperature was controlled with a thermostated circulating water bath.
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Ramalho-Santos, Negrao and Pedroso de Lima
For both viruses the initial rate of fluorescence dequenching was calculated in the first instants following the onset of fusion. The extent of fluorescence dequenching was measured after 5 min in the case of influenza virus and after 15 min in the case of Sendai virus. In inactivation experiments influenza virus was preincubated at pH 5.0 and 37~ for 1 h before fusion activity was monitored. To test for unspecific probe transfer the virus was preincubated at pH 5.0 (37~ 1 h) in the presence of 5 % (v/v) glutaraldehyde prior to the measurements of viral fusion activity. For Sendai virus the possibility of fusion-independent probe exchange was also assessed by preincubating the virus in the presence of 5% (v/v) glutaraldehyde (1 h, 37~ pH 7.4). RESULTS Effect of pH on Influenza and Sendai Viruses-Mediated Changes in ANS Fluorescence As depicted in the Introduction we have studied the effect of influenza virus on the fluorescence of ANS, a probe used to detect hydrophobic microenvironments in proteins [20, 21]. Results displayed in Fig. 1 show that lowering the pH of the medium from 7.4 to 5.0 in the presence of influenza virus results in a drastic increase in ANS fluorescence and a simultaneous blue shift in the emission maximum of this fluorescent probe. Apparently this may reflect a decrease in polarity caused by the exposure of the hydrophobic fusion peptide of the viral HA. When the same experiment is repeated with inactivated influenza virus (virus that has been preincubated at 37~ and pH 5.0 for 1 h) the changes in ANS fluorescence upon lowering the pH are much less dramatic. This was to be
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