1991. 28 h postinfection. Comicroinjection of viral Flurazepam dihydrochloride RNA and fluorescent dextran in the presence of neutralizing computer virus antibody suggested that these protrusions mediated the spread of contamination from one cell to another prior to virus-induced cell lysis. Altogether, the CVB3-induced cellular protrusions could function as a hitherto-unknown nonlytic mechanism of cell-to-cell transmission exploited by enteroviruses. INTRODUCTION Enteroviruses induce fundamental changes in cell morphology. The molecular mechanisms of these changes and the ensuing cellular release of viral progeny are mostly unknown. It is generally assumed that enteroviruses, like many other nonenveloped viruses, require cell lysis for contamination spread. For example, the release of coxsackievirus B3 (CVB3) virions from infected cells depends on enhancement of cell membrane permeability caused by viral components (53). However, the lytic escape of enteroviruses can also be complemented by nonlytic computer virus release, as shown for poliovirus using an autophagosomal pathway (27, 49). Moreover, direct cell-to-cell spread has been suspected to occur with poliovirus in the central nervous system (40). Nonlytic transmission might be particularly important in prolonged contamination by enteroviruses and various other picornaviruses, such as Theiler’s murine encephalomyelitis computer virus, foot-and-mouth disease computer virus, and Nora computer virus, a picorna-like computer virus (17, 25, 41, 58). Such transmission could be envisioned to provide an important advantage for the computer virus by helping it to hide inside the Flurazepam dihydrochloride cell to avoid immune defense by neutralizing antibodies. CVB3, a nonenveloped RNA computer virus, is usually a member of the genus of the family resolution of 100 nm/voxel and of 350 to 500 nm/voxel. A 488-nm argon laser line was utilized for FITC-dextran excitation, and fluorescence was monitored with a 520- to 560-nm band-pass filter. TRITC-dextran was excited with a 543-nm HeNe laser and monitored with a long-pass filter of 560 Flurazepam dihydrochloride nm. For morphological characterization of uninfected cells, two cell populations individually transfected either with enhanced yellow fluorescent protein (EYFP) or enhanced cyan fluorescent protein (ECFP) were plated onto the same cell culture dish. The cells were imaged with a laser scanning confocal microsope (FV1000; Olympus) using the UPLSAPO 60 (NA = 1.2) water immersion objective. A 514-nm argon laser line was utilized for EYFP excitation, and fluorescence was monitored using a 530- to 630-nm band-pass filter. ECFP was excited by a 405-nm diode laser and was monitored with a band-pass filter of 460 to 500 nm. The pixel size was 210 nm in and and 460 nm in and 400 nm in = 240) expressed viral proteins, all with altered morphology, including protrusions. Open in a separate windows Fig. 1. Viral-RNA-induced time-dependent alterations of cells. Time-lapse imaging with a wide-field microscope was performed to examine the dynamics of morphological transformation of RNA-transfected cells. The images, taken at 23 to 26.5 h p.t., show the gradual emergence of membranous protrusions. Level bar, 10 m. Open in a separate windows Fig. 2. Time-related alteration of infected cells. Cells infected by computer virus inoculation, lipofection, or microinjection of viral RNA were incubated for 8, 12, 16, 20, 24, or 28 h prior to fixation, immunolabeling, and imaging. A general image (inset) and a close-up image of inoculated (A), lipofected (B), and microinjected (C) cells at each time point are shown. Capsid proteins were visualized with CVB3 Ab, followed by Alexa 488-conjugated anti-rabbit IgG (green), actin with TRITC-phalloidin (reddish), and nuclei with DAPI (blue). Level bars, 20 m. Open in a separate windows Fig. 3. Emergence of viral proteins Flurazepam dihydrochloride and infection-induced changes in cell viability. Circulation cytometry was performed at 8, 12, 16, 20, 24, and 28 h p.i. (A) Percentages of computer virus protein-expressing cells. The cells were fixed and labeled with CVB3 monoclonal antibody conjugated to Atto 488. Untreated cells (C?) and uninfected immunolabeled cells (C+) were used as controls. (B) The Antxr2 percentage of viable cells was analyzed with annexin V-Alexa 488/propidium iodide double staining. The viable cells were annexin V and PI unfavorable. (C) Percentages of apoptotic cells. Cells in early apoptosis were annexin V positive and PI unfavorable, Flurazepam dihydrochloride and cells in late apoptosis or necrosis were both annexin V and PI positive. STS- and CHX-treated cells were included as.