Porcine Reproductive and Respiratory Syndrome Virus Utilizes Nanotubes for Intercellular Spread

Abstract

For many enveloped viruses, entry into a host cell is primarily through the binding of cellular receptors and subsequent endocytosis of the viral particle into the cells. The fusion of envelope with the endosomal membrane releases viral capsid into the cytosol of the infected cell (reviewed in reference 1). However, for some enveloped viruses, alternative pathways for cell-to-cell transmission have been described (reviewed in references 2 to 4). One emerging model proposes that some viruses can use long, filamentous intercellular connections (nanotubes) as a means to transport infectious viral materials to neighboring naive cells. Previously, intercellular nanotubes have been described as nanotubules, tunneling nanotubes, and bridging conduits (5-8; reviewed in reference 9). The fundamental feature of the intercellular nanotube is a long membrane-bound extension that connects two neighboring cells and can also link multiple cells together to form complex cellular networks (6). Nanotubes are 50 to 200 nm in diameter and can span several cell distances. These structures are primarily composed of filamentous actin (F-actin) and also contain myosin as a motor to drive the movement of organelles or other cargo into neighboring cells (6, 9). Intercellular nanotubes offer cellular communication over long distances, particularly for transporting relatively large cellular materials (10).In this study, we investigated whether porcine reproductive and respiratory syndrome virus (PRRSV) utilizes intercellular nanotubes as an alternative pathway to spread infection. PRRSV is an enveloped, positive-sense, single-stranded RNA virus. The viral genome is about 15 kb in length. The 5= two-thirds of the viral genome encodes two large replicase polyproteins, pp1a and pp1ab, which are proteolytically processed into at least 14 functional nonstructural proteins (nsp1 to nsp12, with nsp1 autocleaved into nsp1␣/nsp1␤ and nsp7 autocleaved into nsp7␣/ nsp7␤) (reviewed in reference 11). Recently, two novel proteins,

Influenza virus exploits tunneling nanotubes for cell-to-cell spread

“…In addition to these mechanism of viral spread, several papers describe that viral proteins including that of influenza virus can be transferred via TNTs7919. However, it is not clear whether such connections exist between infected and uninfected cells and whether viruses can exploit these existing connections to spread to neighboring cells.…”

Karyopherin Alpha 6 Is Required for Replication of Porcine Reproductive and Respiratory Syndrome Virus and Zika Virus

“…Whole-cell lysate in Laemmli sample buffer was subjected to SDS-PAGE and Western blotting as previously described (54,55). Antibodies against KPNA6 (Santa Cruz Biotechnology, Inc., Dallas, TX), KPNA1 (Santa Cruz), KPNA2 (Proscim, Inc., Poway, CA), PRRSV nsp2 protein (56,57), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz), flavivirus E protein (58), hemagglutinin (HA) tag (Thermo Fisher Scientific, Waltham, MA), cMyc tag (Thermo Fisher Scientific), ubiquitin (Santa Cruz), ␤-tubulin (Sigma-Aldrich), and FLAG tag (Sigma-Aldrich) were used in the blotting. Pig anti-PRRSV serum (59) was used to detect nsp2 of PRRSV strain VR-2332 since the monoclonal antibody against nsp2 (56, 57) did not work with this strain.…”

“…IFA. Immunofluorescence assay (IFA) was carried out as reported (63) with antibodies against HA tag (Thermo Fisher Scientific), cMyc tag (Thermo Fisher Scientific), KPNA6 (Santa Cruz Biotechnology), PRRSV nsp2 protein (56,57), and flavivirus E protein (58). The specific reactions were detected by the following conjugated secondary antibodies: goat anti-mouse IgG(H&L) Dylight 549, goat anti-mouse IgG(H&L) Dylight 488, and goat anti-rabbit IgG(H&L) Dylight 488 (Rockland Immunologicals).…”

Carbon Monoxide Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication by the Cyclic GMP/Protein Kinase G and NF-κB Signaling Pathway

“…The PRRSV-neutralizing assay was performed as previously described with the following modifications (60,61). A 2-fold dilution of the hyperimmune serum sample was prepared in a 96-well plate (100 l/well).…”