Intracellular Transport of the Measles Virus Ribonucleoprotein Complex Is Mediated by Rab11A-Positive Recycling Endosomes and Drives Virus Release from the Apical Membrane of Polarized Epithelial Cells

ABSTRACTMumps virus (MuV) is an airborne virus that causes a systemic infection in patients.In vivo, the epithelium is a major replication site of MuV, and thus, the mode of MuV infection of epithelial cells is a subject of interest. Our data in the present study showed that MuV entered polarized epithelial cells via both the apical and basolateral surfaces, while progeny viruses were predominantly released from the apical surface. In polarized cells, intracellular transport of viral ribonucleoprotein (vRNP) complexes was dependent on Rab11-positive endosomes, and vRNP complexes were transported to the apical membrane. Expression of a dominant negative form of Rab11 (Rab11S25N) reduced the progeny virus release in polarized cells but not in nonpolarized cells. Although in this way these effects were correlated with cell polarity, Rab11S25N did not modulate the direction of virus release from the apical surface. Therefore, our data suggested that Rab11 is not a regulator of selective apical release of MuV, although it acts as an activator of virus release from polarized epithelial cells. In addition, our data and previous studies on Sendai virus, respiratory syncytial virus, and measles virus suggested that selective apical release from epithelial cells is used by many paramyxoviruses, even though they cause either a systemic infection or a local respiratory infection.IMPORTANCEMumps virus (MuV) is the etiological agent of mumps and causes a systemic infection. However, the precise mechanism by which MuV breaks through the epithelial barriers and achieves a systemic infection remains unclear. In the present study, we show that the entry of MuV is bipolar, while the release is predominantly from the apical surface in polarized epithelial cells. In addition, the release of progeny virus was facilitated by a Rab11-positive recycling endosome and microtubule network. Our data provide important insights into the mechanism of transmission and pathogenesis of MuV.

Download Full-text

Polarized Budding of Measles Virus Is Not Determined by Viral Surface Glycoproteins

Journal of Virology ◽

10.1128/jvi.72.6.5276-5278.1998 ◽

1998 ◽

Vol 72 (6) ◽

pp. 5276-5278 ◽

Cited By ~ 56

Author(s):

A. Maisner ◽

H.-D. Klenk ◽

G. Herrler

Keyword(s):

Epithelial Cells ◽

Measles Virus ◽

Apical Membrane ◽

Basolateral Membrane ◽

Envelope Glycoproteins ◽

Virus Release ◽

Membrane Domain ◽

Polarized Epithelial Cells ◽

Viral Surface ◽

Surface Glycoproteins

ABSTRACT For viruses that mature by a budding process, the envelope glycoproteins are considered the major determinants for the site of virus release from polarized epithelial cells. Viruses are usually released from that membrane domain where the viral surface glycoproteins are transported to. We here report that measles virus has developed a different maturation strategy. Measles virus was found to be released from the apical membrane domain of polarized epithelial cells, though the surface glycoproteins H and F were transported in a nonpolarized fashion and to the basolateral membrane domain, respectively.

Download Full-text

Faculty Opinions recommendation of Phase coexistence and connectivity in the apical membrane of polarized epithelial cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1030333.357550 ◽

2006 ◽

Author(s):

Akihiro Kusumi

Keyword(s):

Epithelial Cells ◽

Apical Membrane ◽

Phase Coexistence ◽

Polarized Epithelial Cells

Download Full-text

Sorting of Marburg Virus Surface Protein and Virus Release Take Place at Opposite Surfaces of Infected Polarized Epithelial Cells

Journal of Virology ◽

10.1128/jvi.75.3.1274-1283.2001 ◽

2001 ◽

Vol 75 (3) ◽

pp. 1274-1283 ◽

Cited By ~ 36

Author(s):

Christian Sänger ◽

Elke Mühlberger ◽

Elena Ryabchikova ◽

Larissa Kolesnikova ◽

Hans-Dieter Klenk ◽

...

Keyword(s):

Epithelial Cells ◽

Apical Membrane ◽

Basolateral Membrane ◽

Surface Protein ◽

Hemorrhagic Fever ◽

Marburg Virus ◽

Virus Surface ◽

Mdckii Cells ◽

Vesicular Stomatitis ◽

Polarized Epithelial Cells

ABSTRACT Marburg virus, a filovirus, causes severe hemorrhagic fever with hitherto poorly understood molecular pathogenesis. We have investigated here the vectorial transport of the surface protein GP of Marburg virus in polarized epithelial cells. To this end, we established an MDCKII cell line that was able to express GP permanently (MDCK-GP). The functional integrity of GP expressed in these cells was analyzed using vesicular stomatitis virus pseudotypes. Further experiments revealed that GP is transported in MDCK-GP cells mainly to the apical membrane and is released exclusively into the culture medium facing the apical membrane. When MDCKII cells were infected with Marburg virus, the majority of GP was also transported to the apical membrane, suggesting that the protein contains an autonomous apical transport signal. Release of infectious progeny virions, however, took place exclusively at the basolateral membrane of the cells. Thus, vectorial budding of Marburg virus is presumably determined by factors other than the surface protein.

Download Full-text

Move your microvilli

The Journal of Cell Biology ◽

10.1083/jcb.201409059 ◽

2014 ◽

Vol 207 (1) ◽

pp. 9-11 ◽

Cited By ~ 1

Author(s):

Robert S. Fischer

Keyword(s):

Wound Healing ◽

Epithelial Cell ◽

Epithelial Cells ◽

Cell Polarity ◽

Apical Membrane ◽

Apical Surface ◽

Epithelial Cell Polarity ◽

Cell Biol ◽

Polarized Epithelial Cells

Polarized epithelial cells create tightly packed arrays of microvilli in their apical membrane, but the fate of these microvilli is relatively unknown when epithelial cell polarity is lost during wound healing. In this issue, Klingner et al. (2014. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201402037) show that, when epithelial cells become subconfluent, actomyosin contractions locally within the apical cortex cause their microvilli to become motile over the dorsal/apical surface. Their unexpected observations may have implications for epithelial responses in wound healing and disease.

Download Full-text

Arf6 regulates AP-1B–dependent sorting in polarized epithelial cells

The Journal of Cell Biology ◽

10.1083/jcb.201106010 ◽

2011 ◽

Vol 194 (6) ◽

pp. 873-887 ◽

Cited By ~ 27

Author(s):

Elina Shteyn ◽

Lucy Pigati ◽

Heike Fölsch

Keyword(s):

Epithelial Cells ◽

Dominant Negative ◽

Recycling Endosomes ◽

Membrane Recruitment ◽

Important Regulator ◽

Basolateral Plasma Membrane ◽

Polarized Epithelial Cells ◽

Clathrin Adaptor ◽

Basolateral Targeting

The epithelial cell–specific clathrin adaptor complex AP-1B facilitates the sorting of various transmembrane proteins from recycling endosomes (REs) to the basolateral plasma membrane. Despite AP-1B’s clear importance in polarized epithelial cells, we still do not fully understand how AP-1B orchestrates basolateral targeting. Here we identify the ADP-ribosylation factor 6 (Arf6) as an important regulator of AP-1B. We show that activated Arf6 pulled down AP-1B in vitro. Furthermore, interfering with Arf6 function through overexpression of dominant-active Arf6Q67L or dominant-negative Arf6D125N, as well as depletion of Arf6 with short hairpin RNA (shRNA), led to apical missorting of AP-1B–dependent cargos. In agreement with these data, we found that Arf6 colocalized with AP-1B and transferrin receptor (TfnR) in REs. In addition, we observed specific recruitment of AP-1B into Arf6-induced membrane ruffles in nonpolarized cells. We conclude that activated Arf6 directs membrane recruitment of AP-1B, thus regulating AP-1B’s functions in polarized epithelial cells.

Download Full-text

Human Respiratory Syncytial Virus Glycoproteins Are Not Required for Apical Targeting and Release from Polarized Epithelial Cells

Journal of Virology ◽

10.1128/jvi.00827-08 ◽

2008 ◽

Vol 82 (17) ◽

pp. 8664-8672 ◽

Cited By ~ 22

Author(s):

Melissa Batonick ◽

Antonius G. P. Oomens ◽

Gail W. Wertz

Keyword(s):

Respiratory Syncytial Virus ◽

Epithelial Cells ◽

Apical Membrane ◽

Human Respiratory Syncytial Virus ◽

Viral Gene ◽

Recombinant Viruses ◽

Intracellular Targeting ◽

Polarized Epithelial Cells ◽

Syncytial Virus ◽

Apical Sorting

ABSTRACT Human respiratory syncytial virus (HRSV) is released from the apical membrane of polarized epithelial cells. However, little is known about the processes of assembly and release of HRSV and which viral gene products are involved in the directional maturation of the virus. Based on previous studies showing that the fusion (F) glycoprotein contained an intrinsic apical sorting signal and that N- and O-linked glycans can act as apical targeting signals, we investigated whether the glycoproteins of HRSV were involved in its directional targeting and release. We generated recombinant viruses with each of the three glycoprotein genes deleted individually or in groups. Each deleted gene was replaced with a reporter gene to maintain wild-type levels of gene expression. The effects of deleting the glycoprotein genes on apical maturation and on targeting of individual proteins in polarized epithelial cells were examined by using biological, biochemical, and microscopic assays. The results of these studies showed that the HRSV glycoproteins are not required for apical maturation or release of the virus. Further, deletion of one or more of the glycoprotein genes did not affect the intracellular targeting of the remaining viral glycoproteins or the nucleocapsid protein to the apical membrane.

Download Full-text

Measles Virus Ribonucleoprotein Complexes Rapidly Spread across Well-Differentiated Primary Human Airway Epithelial Cells along F-Actin Rings

mBio ◽

10.1128/mbio.02434-19 ◽

2019 ◽

Vol 10 (6) ◽

Cited By ~ 1

Author(s):

Brajesh K. Singh ◽

Christian K. Pfaller ◽

Roberto Cattaneo ◽

Patrick L. Sinn

Keyword(s):

Epithelial Cells ◽

Airway Epithelium ◽

Measles Virus ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Particle Release ◽

Ribonucleoprotein Complex ◽

Human Airway ◽

Well Differentiated ◽

Actin Rings

ABSTRACT Measles virus (MeV) is a highly contagious human pathogen that continues to be a worldwide health burden. One of the challenges for the study of MeV spread is the identification of model systems that accurately reflect how MeV behaves in humans. For our studies, we use unpassaged, well-differentiated primary cultures of airway epithelial cells from human donor lungs to examine MeV infection and spread. Here, we show that the main components of the MeV ribonucleoprotein complex (RNP), the nucleocapsid and phosphoprotein, colocalize with the apical and circumapical F-actin networks. To better understand how MeV infections spread across the airway epithelium, we generated a recombinant virus incorporating chimeric fluorescent proteins in its RNP complex. By live cell imaging, we observed rapid movement of RNPs along the circumapical F-actin rings of newly infected cells. This strikingly rapid mechanism of horizontal trafficking across epithelia is consistent with the opening of pores between columnar cells by the viral membrane fusion apparatus. Our work provides mechanistic insights into how MeV rapidly spreads through airway epithelial cells, contributing to its extremely contagious nature. IMPORTANCE The ability of viral particles to directly spread cell to cell within the airways without particle release is considered to be highly advantageous to many respiratory viruses. Our previous studies in well-differentiated, primary human airway epithelial cells suggest that measles virus (MeV) spreads cell to cell by eliciting the formation of intercellular membrane pores. Based on a newly generated ribonucleoprotein complex (RNP) “tracker” virus, we document by live-cell microscopy that MeV RNPs move along F-actin rings before entering a new cell. Thus, rather than diffusing through the cytoplasm of a newly infected columnar cell, RNPs take advantage of the cytoskeletal infrastructure to rapidly spread laterally across the human airway epithelium. This results in rapid horizontal spread through the epithelium that does not require particle release.

Download Full-text

Glycoprotein targeting signals influence the distribution of measles virus envelope proteins and virus spread in lymphocytes

Journal of General Virology ◽

10.1099/vir.0.83407-0 ◽

2008 ◽

Vol 89 (3) ◽

pp. 687-696 ◽

Cited By ~ 11

Author(s):

Nicole Runkler ◽

Erik Dietzel ◽

Markus Moll ◽

Hans-Dieter Klenk ◽

Andrea Maisner

Keyword(s):

Epithelial Cells ◽

Measles Virus ◽

M Protein ◽

Target Cells ◽

Virus Spread ◽

Virus Propagation ◽

Virus Envelope ◽

Polarized Epithelial Cells

We previously demonstrated the presence of tyrosine-dependent motifs for specific sorting of two measles virus (MV) glycoproteins, H and F, to the basolateral surface in polarized epithelial cells. Targeted expression of the glycoproteins was found to be required for virus spread in epithelia via cell-to-cell fusion in vitro and in vivo. In the present study, recombinant MVs (rMVs) with substitutions of the critical tyrosines in the H and F cytoplasmic domains were used to determine whether the sorting signals also play a crucial role for MV replication and spread within lymphocytes, the main target cells of acute MV infection. Immunolocalization revealed that only standard glycoproteins are targeted specifically to the uropod of polarized lymphocytes and cluster on the surface of non-polarized lymphocytes. H and F proteins with tyrosine mutations did not accumulate in uropods, but were distributed homogeneously on the surface and did not colocalize markedly with the matrix (M) protein. Due to the defective interaction with the M protein, all mutant rMVs showed an enhanced fusion capacity, but only rMVs harbouring two mutated glycoproteins showed a marked decrease in virus release from infected lymphocytes. These results demonstrate clearly that the tyrosine-based targeting motifs in the MV glycoproteins are not only important in polarized epithelial cells, but are also active in lymphocytes, thus playing an important role in virus propagation in different key target cells during acute MV infection.

Download Full-text

Phosphatidylinositol 3,4,5-trisphosphate Localization in Recycling Endosomes Is Necessary for AP-1B–dependent Sorting in Polarized Epithelial Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e09-01-0036 ◽

2010 ◽

Vol 21 (1) ◽

pp. 95-105 ◽

Cited By ~ 40

Author(s):

Ian C. Fields ◽

Shelby M. King ◽

Elina Shteyn ◽

Richard S. Kang ◽

Heike Fölsch

Keyword(s):

Plasma Membrane ◽

Epithelial Cell ◽

Epithelial Cells ◽

Basolateral Membrane ◽

Apical Plasma Membrane ◽

Amino Acid Residues ◽

Recycling Endosomes ◽

C Terminus ◽

Polarized Epithelial Cells ◽

Clathrin Adaptor

Polarized epithelial cells coexpress two almost identical AP-1 clathrin adaptor complexes: the ubiquitously expressed AP-1A and the epithelial cell–specific AP-1B. The only difference between the two complexes is the incorporation of the respective medium subunits μ1A or μ1B, which are responsible for the different functions of AP-1A and AP-1B in TGN to endosome or endosome to basolateral membrane targeting, respectively. Here we demonstrate that the C-terminus of μ1B is important for AP-1B recruitment onto recycling endosomes. We define a patch of three amino acid residues in μ1B that are necessary for recruitment of AP-1B onto recycling endosomes containing phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3]. We found this lipid enriched in recycling endosomes of epithelial cells only when AP-1B is expressed. Interfering with PI(3,4,5)P3 formation leads to displacement of AP-1B from recycling endosomes and missorting of AP-1B–dependent cargo to the apical plasma membrane. In conclusion, PI(3,4,5)P3 formation in recycling endosomes is essential for AP-1B function.

Download Full-text