key: cord-0009152-qwngt8fw authors: Lucchiari, M.A.; Pereira, C.A.; Kuhn, L.; Lefkovits, I. title: The pattern of proteins synthesized in the liver is profoundly modified upon infection of susceptible mice with mouse hepatitis virus 3 date: 2007-09-22 journal: Res Virol DOI: 10.1016/s0923-2516(06)80111-1 sha: f210cb3da35e11e4e268372baad244fbdc140ab1 doc_id: 9152 cord_uid: qwngt8fw Susceptible BALB/c mice, after experimental infection with mouse hepatitis virus 3 (MHV3), revealed virus titres in the liver that increased gradually to a peak of 8 × 10(5) PFU/g of tissue after 3 days' infection, when the mice died of acute hepatitis. BALB/c mice were infected with MHV3, subsequently labelled in vivo with (35)S-methionine, and then the liver preparations from both infected and non-infected animals were subjected to two-dimensional gel electrophoresis. Comparisons of the patterns by computer image analysis revealed 17 gene products which increased, and 8 gene products which decreased, upon virus infection in their two-dimensional gel spot intensity. We conclude that during MHV3 infection of a susceptible strain of mice, a major modification in protein synthesis occurs. The pattern alterations were not related to the virus gene products but were mostly endogenous mouse proteins. Whether these proteins are a result of a defence attempt by the animal, or are dictated by the virus in order to prevent a protective response from happening, remains to be shown. Coronaviruses cause a wide variety of diseases in several hosts (McIntosh, 1985) . The murine hepatitis virus (MHV) group is widespread, occurring endemically in mouse colonies. MHV3 was isolated by Dick et al. (1956) and has been used as a model of viral infection in which resistance is dependent on the genetic background of the mouse strain (Arnheiter et al., 1982; Dupuy et al., 1984; Levy et al., 1981 ; Pereira, 1989, 1990; Virelizier and Gresser, 1978) . Adult BALB/c mice axe susceptible to MHV3 infection. After intraperitoneal (i.p.) inoculation with the virus, the animals develop acute hepatitis and die 3 to 5 days later. In the peritoneum, the virus replicates in the macrophages and reaches the liver via the blood stream where it replicates f'u'st in the Kupffer and endothelial cells of the liver sinusoid, and then Submitted March 25, 1992 , accepted July 10, 1992 (*) Corresponding author. continues to replicate in the hepatocytes (Arnheiter et al., 1982; Bang and Warwick, 1960; Pereira et al., 1984a) . A large number of local and systemic changes have been shown to be involved in the resistance of animals following infection (Eaton et al., 1982; Weinberg, 1984) . The changes in the concentration of plasma proteins are an important factor of the systemic response (Kushner et al., 1982) . Synthesis or degradation of several plasma protons are affected during infection and the change in components of the acute phase reactants is a prominent feature of the response. "Positive" and "negative reactants" are terms that have been proposed to describe the increase or decrease in the abundance of individual components during the acute phase (Kushner, 1981) . A wide variety of different stimuli can trigger an acute-phase reaction, and some of the inducers evoke a distinct response pattern (Allen et al., 1982) . A number of other changes in protein synthesis have been implicated, but have not been revealed until now, since no direct, sensitive methods were available for the detection of polypeptides present at concentrations considerably lower than those of acute-phase proteins. Two-dimensional gel electrophoresis, which allows simultaneous qualitative and quantitative analysis of numerous proteins in combination with in vivo labelling, turned out to be an ideal tool for the detection of changes (Pluschke et al., 1986) directly related to a course of infection. The results presented here show the changes observed in liver and serum proteins of BALB/c mice during infection with MHV3. plified on L929 cells to serve as the inoculum for future stocks (Martin et al., 1988) to limit spontaneous mutations. The stocks were titrated by plaque assay on L929 cells, as previously described (Pereira et ai., 1984b) . Aliquots containing 2x 105 plaqueforming units per millilitre (PFU/ml) were stored at -80°C for use in the experiments. BALB/c mice (4 to 8 weeks old) from the animal colony of the Max-Planck-Institut, Freiburg, Germany were used in all the experiments. MHV3 (10 a PFU) was inoculated intraperitoneally. The mice were observed during the following 3 weeks and the mortality was recorded. In order to evaluate the virus growth kinetics in the liver during infection, selected mice were killed periodically, their livers removed, ground and resuspended in 2 ml of RPMI-1640 medium containing 5 % of foetal calf serum (FCS) and the virus titres determined by plaque assay on L929 cells (Pereira et ak, 1984b) . The in vivo experiments were performed at the Max-Planck Institut fiir Immunbiologie, Freiburg, Germany. The labelling procedure has been described in detail elsewhere (Pluschke and Lefkovits, 1984) . Briefly, the animals were inoculated intravenously (i.v.) with l03 PFU of MHV3, two days later they were injected i.v. with 1 mCi of 35S-methionine (Amersham International, UK), with a specific activity of 1,000-1,500 KCi/mol, in 100 ~l of isotonic saline for a period of 4 h. The mice were killed and the liver and serum removed. MHV3, originally obtained from Dr. J.L. Virelizier, Institut Pasteur, France, was cloned by limiting dilution. One plaque was selected and am- Liver slices (3-I0 rag) or serum samples (I0 ~I) were placed in Sarstedt tubes and instantaneously solubilized by mixing with 90 ~.I buffer containing 20 g/l of NP40 detergent (BDH, Z/irich, Switzerland), I0 g/l 2-mercaptoethanol (LKB, Bromma, Sweden) in 9 M urea (Schwartz-Mann, Orangeburg, NY, USA) and adjusted to pH 9.5. The labelled materials were subjected to 2D-SDS-PAGE by a previously described procedure (Lefkovits et al., 1985 ) based on the system originally developed by O'Farrell, 1975. In the first dimension, isolelectric focusing (IEF) from pH 3 to 9 was used. In the second dimension, SDS-PAGE with a l0 to 20 % polyacrylamide gradient was used. Both dimensions were run under reducing conditions on an "ISODALT" apparatus, which is described in detail elsewhere (Tollaksen et al., 1981) , as is the composition of the gel components and their preparation (Lefkovits et al., 1985) . Gels were immersed twice for 1 h each time in dimethyl sulphoxide (Merck), then incubated at room temperature for 3 h in a solution of 2,5-diphenyloxazole (130 g) dissolved in dimethyl sulphoxide (1 1). The gels were dried (3 h, 50°C, reduced pressure) and placed in contact with "XAR-5 Kodak" film at -70°C for the appropriate exposure interval. The film was developed in a "Kodak X-Omat Processor Model M7A". Details are given elsewhere (Kettman et al., 1986 ). Radiofluorographic images were scanned by a "Molecular Dynamics" laser scanner and the images submitted to "Kepler" image analysis system (LSB, Rockville, USA) for spot modelling and spot matching. The original radiofluorography image information is converted into so-called spot lists, in which each spot is defined by 5 bytes of information (2 bytes for the coordinate position x and y, and another 3 bytes for the spot volume [sx, sy, ampl]). Typically, a spot list containing say 600 spots will consist of 5x600=3,000 byte information. The original "pixel by pixer' information of the scanned image consisted of 1,700 x 1,700 = 3,000,000 bytes. Note that the spot list information is defined by three orders of magnitude less bytes than the scanned data. After completion of the image processing, one of the spot lists is chosen as a matrix to which all other spot lists will be compared. Such a list, to which the software program assigns a numbering system, is called master list or master pattern; all spot patterns will then be matched to the master pattern and individual spots will receive numbers congruent to the master spots. In this paper, the master list for liver samples (ML) and the master list for serum samples (MS) have independent numbering systems. For the evaluation of spot intensity, radiofluorographs with properly adjusted exposure intervals were used so that a quantitative comparison of spot intensity would be manageable. The relative values are valid throughout spot comparisons shown in this paper. Two types of histogram are displayed: firstly, comparison of absolute spot volumes for a given spot pair is shown, and secondly, comparison of spot pairs, in which the larger of the pair is adjusted to max y value, is shown. Such histograms are online features of the Kepler software system. All database information described in this paper is available from I. Lefkovits upon request. BALB/c mice infected with MHV3 were shown to be fully susceptible to virus infection (100 °70 mortality) and, as can be seen in figure 1 , the virus titres in the liver of infected mice increased gradually to a peak of 8 × 105 PFU/g of tissue after 3 days' infection, when the mice died of acute hepatitis. In figure 2 , the 2D-SDS-PAGE spot pattern of liver preparations from non-infected (A) and from MHV3-infected mice (B) is shown, as well as the master pattern (ML), which is based on the non-infected liver preparation spot pattern. The 2D-SDS-PAGE of the non-infected liver preparation revealed 276 spots. Upon matching, 187 spots were found to be present in both patterns. The master pattern, when created, contained 225 spots of the non-infected liver preparation, and then 32 spots from the infected liver preparation were added. Although all 89 spots could have been included in the master pattern, our conservative approach was to add only those spots which were reliably modelled. ,,= The ratio increase was calculated by volume comparison (arbitrary units). Histograms of spot intensities. n panel I, spot pairs are shown scaled separately for each spot pair. In panel II, a common scale for all spots is applied. --4 The ratio decrease was calculated by volume comparison (arbitrary units). decrease in abundance by a factor of 2 or more are indicated. Furthermore, there is a set of spots which appears only in the pattern from the infected liver preparation, many of which are known to be serum constituents. In figure 4 , the spot patterns of serum of noninfected (A) and MHV3-infected (B) mice are shown. There are a large number of spots which are profoundly altered upon MHV3 infection. At present, no complete computer evaluation permits reliable modelling of closely spaced glycosylated spots. Thus, at present, it is worth stating that several spots from the liver samples of MHV3-infected animals may be serum spots. In a conventional 2D gel approach (i.e. without labelling of the entire mouse), serum spots do not interfere since they are not labelled and hence are not revealed on the radiofluorography. The aim of the work presented here was to identify changes in liver protein expression upon infection of susceptible mice with MHV3. A powerful and also convenient procedure for identifying such changes is the labelling of the entire mouse with 35S-methionine during the course of infection. The experiment requires a relatively high dose of 35S-methionine which, as described earlier (Pluschke and Lefkovits, 1984) , consists of injecting 1 mCi of 35S-methionine i.v. into a mouse. Such treatment provides a pulse of radioisotope for the whole organism, and all the relevant biosynthetic processes make use of the provided radioisotope. The consequence is that not only do the cells become efficiently labelled but also the body fluid will contain proteins which were labelled intracellularly and then secreted into the extracellular space. Obviously the labelled polypeptides will be considerably diluted when secreted into the body fluids and will be detectable only above certain threshold concentrations. One hour after i.v. administration of 35S-methionine, sufficient labelling was achieved so that a 2D gel exposed for one month revealed several hundreds of spots on a radiofluorograph. Nevertheless, in vivo labelling imposed certain limitations upon the analysis; we did not detect 1,000-1,500 spots, as is the case for in vitro labelling but, as mentioned above, only about 300 spots. The label introduced into the animal is diluted in the body fluid (estimated volume 5 ml) and in the cytoplasmic content upon uptake into the cell, and the specific activity of the label is profoundly decreased by the rich amino acid pool of the animal. Thus, only those proteins which are synthesized at the highest rate will incorporate enough label to yield detectable spots. Nevertheless, most of these 300 spots could not be detected by any other means than by this methodology. Fine changes can be revealed only by in vitro labelling. Infection with MHV3 induced profound alterations in the spot pattern of proteins synthesized in the liver of BALB/c mice. We found that 17 major proteins increased their rate of synthesis by a factor of 2 to 20 (tables I and II). The abovementioned figures refer to an alteration in the rate of synthesis and/or post-translational modification rather than the overall abundance. Some spots from the liver sample are probably serum spots, as is the case for spot 110 which shows a 14-fold increase upon infection. It is advantageous to also consider the serum spots, since they may be in an as yet unknown fashion correlated to the events monitored in the liver. Using the quantitative 2D-SDS-PAGE approach, we were able to obtain a new insight into the events after MHV3 infection, since our analysis was not restricted to following only one variable but rather to simultaneously monitoring all changes in the chosen sample. We identified and tagged 17 gene products which increased and 8 gene products which decreased in liver preparations upon MHV3 infection of BALB/c mice. In the near future, we shall attempt to identify some of these proteins by cDNA obtained from the gene catalogue of Lefkovits et al., 1990 , and we shall try to refine the above-described approach by analysing cell-free translation products from the mRNA obtained from infected and non-infected mice. This would lead us to the understanding of more refined regulatory events during the course of infection. We have attempted to complete this study by evaluating the changes in the protein pattern (upon virus inoculation) of resistant mice (A/J). Preliminary experiments show that certain spots detected in BALB/c mice are not present in A/J mice. Since the pattern from intact (noninoculated) A/J mice differs from the BALB/c pattern, rigorous evaluation will be possible only when there is an understanding of the genetic background differences (under study now). We interpret the protein modification as the result of a defence attempt by the animal, perhaps by the formation of stress proteins. We hope that blotting (or alternatively im-munoprecipitation and co-electrophoresis of the eluate) with defined antisera will shed some light on the nature of this response. Des souris BALB/c infect~es exp~rimentalement par le virus 3 de l'h6patite murine voient leur titre en virus croTtre progressivement jusqu'/t 8x l05 PFU/g de tissu 3 jours apr~s l'infection, moment of a les souris meurent d'h6patite aigu~. Apr~s l'infection par le virus, les souris ont ~t~ marquees tn vivo par la 35S-m6thionine et des preparations de foie des animaux infect6s et des animaux t6moins ont ~t~ soumises /~ une ~lectrophor~se bidimensionnelle. L'analyse compar~e des images ~ l'aide d'un ordinateur a r~v616 que 17 prot6ines ~taient d6tect6es en plus grande quantitY, alors que 8 autres ~taient moins abondantes chez les souris infect6es. Nous coneluons que, pendant l'infection par le virus, les animaux sensibles pr6sentent d'importantes perturbations des syntheses prot~iques. Ces alt&ations concernent les prot6ines produites par la souris et non des produits de synth~se du virus. I1 reste ~t d~terminer si ces alt~rations sont le r~sultat d'une d~fense de l'animal ou si elles sont dict6es par le virus afin d'enrayer une r6ponse de l'hfte. Mots-cl~s: Coronavirus, H6patite murine, Pro-t6ine; Synth~se, Foie, Marquage in vivo, D6fenses. Marker proteins in inflammation Adult mouse hepatocytes in primary monolayer culture express genetic resistance to mouse hepatitis virus type 3 Mouse macrophages as host cells for the mouse hepatitis virus and the genetic basis of their susceptibility Genetically determined resistance to mouse hepatitis virus type 3 is expressed in hematopoietic donor cells in radiation chimeras Haptoglobin: a natural bacteriostat Parameters of the labelling of mitogen-activated murine lymphocytes by 3SS-methionine for twodimensional gel electrophoresis. 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Genetically dependent resistance A major role of macrophage activation by interferon gamma during mouse hepatitis virus type 3 infection. --II Temperature sensitive mutants of mouse hepatitis virus type 3 (MHV3): isolation, biochemical and genetic characterization High resolution two-dimensional electrophoresis of proteins Interactions between mouse hepatitis viruses and primary cultures of Kupffer and endothelial liver cells from resistant and susceptible inbred mouse strains Induction of natural killer cells and interferon during mouse hepatitis virus infection of resistant and susceptible inbred mouse strains Two-dimensional gel pattern of proteins synthesized de novo in lymphoid organs of the mouse Complete two-dimensional gel electrophoresis pattern of de novo synthesized acute phase reactants Operation of the ISODALT system, report ANL-RIM-8 l-1 Role of interferon in the pathogenesis of viral diseases of mice as demonstrated by the use of anti-interferon serum. --V. Protective role in mouse hepatitis type 3 infection of susceptible and resistant strains of mice Iron withholding: a defense against infection and neoplasia M.A.L. was a recipient of the Deutscher Akademischer Austauschdients (DAAD) during this research. The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche and Co. Ltd., Basel, Switzerland.