Vesicular stomatitis virus, the best-studied rhabdovi- rus, is composed of an RNA genome of negative sense, five viral proteins, and membrane lipids derived ...
Proc. Natl. Acad. Sci. USA Vol. 93, pp. 8268-8273, August 1996 Biochemistry
Migration of vesicular stomatitis virus glycoprotein to the nucleus of infected cells ANDREA T. DA POIAN, ANDRE M. 0. GOMES, RICARDO J. N. OLIVEIRA, AND JERSON L. SILVA* Departamento de Bioquimica Medica, Instituto de Ciencias Biomedicas, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, Brazil
Communicated by Lowell P. Hager, University of Illinois at Urbana-Champaign, Urbana, IL, April 29, 1996 (received for review January 18, 1996)
MATERIALS AND METHODS
A new means of direct visualization of the ABSTRACT early events of viral infection by selective fluorescence labeling of viral proteins coupled with digital imaging microscopy is reported. The early phases of viral infection have great importance for understanding viral replication and pathogenesis. Vesicular stomatitis virus, the best-studied rhabdovirus, is composed of an RNA genome of negative sense, five viral proteins, and membrane lipids derived from the host cell. The glycoprotein ofvesicular stomatitis virus was labeled with fluorescein isothiocyanate, and the labeled virus was incubated with baby hamster kidney cells. After initiation of infection, the fluorescence of the labeled glycoprotein was first seen inside the cells in endocytic vesicles. The fluorescence progressively migrated to the nucleus of infected cells. After 1 h of infection, the virus glycoprotein was concentrated in the nucleus and could be recovered intact in a preparation of purified nuclei. These results suggest that uncoating of the viral RNA occurs close to the nuclear membrane, which would precede transcription of the leader RNA that enters the nucleus to shut off cellular RNA synthesis and DNA replication.
Cell and Culture Medium. BHK21 cells were grown in monolayers at 37°C in a CO2 incubator using Dulbecco's Modified Eagle Medium (DMEM; Sigma) supplemented with 5% fetal bovine serum, 0.4% vitamins, and 1% nonessential amino acids and buffered with sodium bicarbonate. Virus Propagation and Purification. VSV type Indiana was used in all experiments. Virus was propagated for 16 h on a roller apparatus at 37°C. After propagation, the supematant was cleared of cellular debris in a Sorvall centrifuge (4000 x g for 10 min). The supernatant was spun in a Beckman Ti45 rotor at 30,000 rpm for 2.5 h. The pellet was resuspended in 3E buffer (0.12 M Tris/0.06 M sodium acetate/3.0 mM EDTA, pH 7.4), layered onto a continuous 5-40% sucrose gradient (in 3E buffer), and spun for 1 h at 36,000 rpm in a Beckman SW-41 rotor. The virus band was collected and pelleted in a Beckman type 40 rotor (35,000 rpm for lh) and resuspended in 10 mM Tris (pH 7.6). Viral stocks were kept at -700C. Infectivity Assays. Infectivity was evaluated by plaque assay as described by Brown and Cartwright (13). Confluent monolayers of BHK21 cells in 60-mm diameter dishes were infected with serial dilutions of VSV for 30 min at room temperature. After aspiration of the virus solution, 1% agarose in medium solution was added to each plate. The plates were left in a 37°C CO2 incubator for 24 h. After the agarose was peeled off the plates, the cells were stained with crystal violet and plaques were counted. Virus Labeling. The purified viral sample (0.5 mg of protein per ml) was incubated at 20°C with 57 ,uM FITC (F-143; Molecular Probes) in 100 mM phosphate buffer (pH 8.0) for 2 h, with constant stirring. The sample was then dialyzed against 50 mM Tris-HCl buffer (pH 7.5) and kept at 4°C, protected from the light. Isolation of Nuclei. Isolation of nuclei from infected and noninfected BHK21 cells in culture was carried out by modifying the method described by Nicotera et al. (14) for preparation of highly purified nuclei from rat liver. A monolayer of cells was incubated with trypsin for -2 min and washed to remove trypsin. Approximately 106 cells were resuspended in 3 ml of Tris buffer (50 mM Tris HCl, pH 7.5), supplemented with 0.25 M sucrose and 1 mM phenylmethylsulfonyl fluoride. The cells were homogenized with a glass/Teflon homogenizer. The nuclei were pelleted by centrifugation at 700 x g for 10 min. The pellets were homogenized in the supplemented buffer and centrifuged again at 700 x g for 10 min. The resulting pellet was resuspended in 1.5 ml of the same buffer by homogenization, and this suspension was added to a tube containing 3.0 ml of the Tris buffer supplemented with 2.3 M sucrose. The tube was gently mixed and a 1.5-ml cushion (Tris buffer containing 2.3 M sucrose) was carefully added to the bottom of each tube before centrifugation at 37,000 x g for 30 min. The resulting pellet was resuspended in a small volume of Tris buffer supplemented with 0.25 M sucrose. For observation
Vesicular stomatitis virus (VSV), the prototype of the Rhabdoviridae, interferes with various metabolic functions in the host cell (1). VSV is composed of an RNA genome of negative sense, five viral proteins, and membrane lipids derived from the host cell. The infectious component of VSV is the ribonucleoprotein core, where the RNA is tightly encased by the nucleocapsid protein, also associated with two minor proteins-L and NS. The VSV membrane contains two proteins: an integral glycoprotein (G protein) and a peripheral matrix protein that aligns in the inner surface of the virion membrane (2). Infection by VSV rapidly shuts off cellular RNA synthesis (3), as well as host-cell DNA replication (4) and protein synthesis (5, 6). It has been suggested that the ability of the virus to inhibit cellular RNA synthesis depends on the first viral transcript, the leader RNA (7-10). The leader RNA first appears in the cytoplasm of infected cells, whence it rapidly migrates to the nucleus (11). On the other hand, protein components of the virion may also play a role in shutting off synthesis of host cell macromolecules (12). Fluorescence digital imaging microscopy offers a direct means of following labeled viral proteins during the infection cycle. To study the early phase of viral infection, we have developed an approach to specifically label one of the protein components of the parental virus and to follow its fate. We show that VSV G protein labeled with fluorescein isothiocyanate (FITC) appears initially in endocytic vesicles and rapidly migrates to the nucleus of infected cells. On the other hand, when the virus has been inactivated by pressurization, labeled G protein is detected only at the cellular plasma membrane. In this paper, the great potential of selectively fluorescence labeling of viral proteins to follow the early events in virus infection is shown.
Abbreviations: VSV, vesicular stomatitis virus; G protein, glycoprotein; FITC, fluorescein isothiocyanate; EB, ethidium bromide. *To whom reprint requests should be addressed. e-mail: jerson@
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Biochemistry: Da Poian et al. in the microscope, nuclei suspension was immobilized in a 2% polyacrylamide gel polymerized over the coverslip. Fluorescence Microscopy. BHK21 cells were grown on coverslips overnight. Infection was carried out by adding the virus to a final concentration of 50 ,ug of protein per ml to the coverslips and incubating for different times at 37°C in the CO2 incubator. The multiplicity of infection was low (between one and three). After the coverslips were washed with culture medium, uninfected cells and cells infected with labeled and unlabeled virus samples were observed in a Zeiss Axioskop microscope with transmitted light and epifluorescence. For the fluorescence microscope measurements, illumination was provided by a short mercury arc lamp (Osram HBO 50 W; Osram, Berlin). Two sets of filters (Zeiss 487909 and 487914) were used, each one consisting of excitation and emission filters and a dichroic mirror. All images were collected with a Star I camera system (Photometrics, Tucson, AZ) consisting of a cooled camera head, a charge-coupled device (384 x 576 pixel), the camera controller, and a cooling liquid circulation unit (LC200) using processing software furnished by the manufacturer. Images were processed on an IBM/PC 486 DX2 computer equipped with a GPIB interface (NI-488.2; National Instruments, Austin, TX). The cells were observed both with transmitted light and with the filter set number 487909. A Corning filter (3-69) was also added to the emission pathway to guarantee blue excitation and a wavelength cuttoff at 510 nm. Isolated nuclei were observed with transmitted light, with the filter set number 487914 [for observation of the red fluorescence of ethidium bromide (EB)] and with the filter set number 487909 (for observation of the green fluorescence of FITC). High Pressure Equipment. The high-pressure bomb has been described (15) and was purchased from SLM Aminco (Urbana, IL). Size Exclusion Chromatography. High-performance liquid chromatography was performed using a prepacked SynChropak GPC-300 column [250 x 4.6 mm (i.d.)] (SynChropak, Linden, IN). The system was equilibrated in 50 mM Tris/0.2 M sodium acetate buffer, pH 7.0, containing 0.1% sodium dodecyl sulfate (SDS). A flow rate of 0.3 ml/min was used. Sample elution was monitored by fluorescence at 330 nm and 520 nm (excitation at 280 nm and 480 nm, respectively)
RESULTS AND DISCUSSION The early phases of viral infection have remained a relatively neglected field in spite of their obvious importance for understanding viral replication, cell tropism, and pathogenesis (16). Detailed mechanisms of infection remain unclear for most animal viruses probably because of experimental difficulties. The efficiency of productive entry is usually low, which means that it is difficult to obtain a good signal at a reasonable multiplicity of infection, since early events take place before virus amplification due to replication. The use of digital fluorescence microscopy has an enormous potentiality to follow the sequence of events between virus attachment and viral replication. To study the early phase of viral infection, we have developed an approach to specifically label one of the protein components of the virus and to follow its fate before virus replication. VSV was labeled with FITC, a fluorescent probe that reacts covalently with lysine side chains. Incorporation of FITC and localization of the probe among the different viral proteins was demonstrated by comparing the elution profile of viral proteins and FITC fluorescence in high-performance gel filtration chromatography in the presence of SDS. Labeled VSV was disassembled by incubation with 1% SDS, and its elution profile in a size exclusion column was monitored by tryptophan fluorescence (excitation at 280 nm and emission at 330 nm) and by FITC fluorescence (excitation at 480 nm and emission at 520 nm) (Fig. 1). A single peak of FITC fluorescence was detected, and it coincided with G protein elution, demonstrating that only G protein was labeled by treatment with the probe. Polyacrylamide gel
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