Connections matter − how viruses use cell–cell adhesion components

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The discovery that measles virus (MV) uses the adherens junction protein nectin-4 as its epithelial receptor provides a new vantage point from which to characterize its rapid spread in the airway epithelium. We show here that in well-differentiated primary cultures of airway epithelial cells from human donors (HAE), MV infectious centers form rapidly and become larger than those of other respiratory pathogens: human respiratory syncytial virus, parainfluenza virus 5, and Sendai virus. While visible syncytia do not form after MV infection of HAE, the cytoplasm of an infected cell suddenly flows into an adjacent cell, as visualized through wild-type MV-expressed cytoplasmic green fluorescent protein (GFP). High-resolution video microscopy documents that GFP flows through openings that form on the lateral surfaces between columnar epithelial cells. To assess the relevance of the protein afadin, which connects nectin-4 to the actin cytoskeleton, we knocked down its mRNA. This resulted in more-limited infectious-center formation. We also generated a nectin-4 mutant without the afadin-binding site in its cytoplasmic tail. This mutant was less effective than wild-type human nectin-4 at promoting MV infection in primary cultures of porcine airway epithelia. Thus, in airway epithelial cells, MV spread requires the nectin-4/afadin complex and is based on cytoplasm transfer between columnar cells. Since the viral membrane fusion apparatus may open the passages that allow cytoplasm transfer, we refer to them as intercellular membrane pores. Virus-induced intercellular pores may contribute to extremely efficient measles contagion by promoting the rapid spread of the virus through the upper respiratory epithelium. IMPORTANCE Measles virus (MV

Section: Discussionmentioning

confidence: 99%

The Nectin-4/Afadin Protein Complex and Intercellular Membrane Pores Contribute to Rapid Spread of Measles Virus in Primary Human Airway Epithelia

Singh

Hornick

Krishnamurthy

et al. 2015

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“…Remarkably, several viruses use nectins or other proteins of the apical junction complex as receptors for entry in polarized epithelial cells (31) even if most of these proteins are not readily accessible. MeV and the other negative-strand RNA viruses of the genus Morbillivirus use nectin-4 (8-11, 32, 33), large DNA viruses like herpes simplex viruses 1 and 2 use nectin-1 and -2 (34)(35)(36), and the small positivestrand RNA virus poliovirus uses the poliovirus receptor PVR (37), also known as nectin-like protein 5.…”

Section: Discussionmentioning

confidence: 99%

“…MeV and the other negative-strand RNA viruses of the genus Morbillivirus use nectin-4 (8-11, 32, 33), large DNA viruses like herpes simplex viruses 1 and 2 use nectin-1 and -2 (34)(35)(36), and the small positivestrand RNA virus poliovirus uses the poliovirus receptor PVR (37), also known as nectin-like protein 5. Moreover, other viruses use tight junction proteins, including occludins, claudins, and junctional adhesion molecules, as receptors (31). Exploiting adherens junction proteins is an effective way to overcome barriers within an organism in order to spread the infection.…”

Section: Discussionmentioning

confidence: 99%

Cell-to-Cell Contact and Nectin-4 Govern Spread of Measles Virus from Primary Human Myeloid Cells to Primary Human Airway Epithelial Cells

Singh

Mark

et al. 2016

Self Cite

Measles is a highly contagious, acute viral illness. Immune cells within the airways are likely first targets of infection, and these cells traffic measles virus (MeV) to lymph nodes for amplification and subsequent systemic dissemination. Infected immune cells are thought to return MeV to the airways; however, the mechanisms responsible for virus transfer to pulmonary epithelial cells are poorly understood. To investigate this process, we collected blood from human donors and generated primary myeloid cells, specifically, monocyte-derived macrophages ( IMPORTANCEMeasles virus spreads rapidly and efficiently in human airway epithelial cells. This rapid spread is based on cell-to-cell contact rather than on particle release and reentry. Here we posit that MeV transfer from infected immune cells to epithelial cells also occurs by cell-to-cell contact rather than through cell-free particles. In addition, we sought to determine which immune cells transfer MeV infectivity to the human airway epithelium. Our studies are based on two types of human primary cells: (i) myeloid cells generated from donated blood and (ii) well-differentiated airway epithelial cells derived from donor lungs. We show that different types of myeloid cells, i.e., monocyte-derived macrophages and dendritic cells, transfer infection to airway epithelial cells. Furthermore, cell-to-cell contact is an important component of successful MeV transfer. Our studies elucidate a mechanism by which the most contagious human respiratory virus is delivered to the airway epithelium. M easles virus (MeV) is extremely contagious and infects its human host via the respiratory route. For many years, MeV was thought to enter through the apical surface of airway epithelial cells (1), a misconception based on studies performed with polarized immortalized cell lines (2, 3). Using well-differentiated primary cultures of airway epithelial cells from human donors (HAE), we demonstrated that MeV has a clear preference for basolateral entry (4). HAE differentiate into a pseudostratified columnar epithelium sheet comprised of ciliated, nonciliated, basal, and goblet cells. This model system is highly representative of the in vivo airways (5). MeV infection of HAE results in the formation of infectious centers that, unlike syncytia, retain intact plasma membranes (4, 6, 7). MeV-mediated infectious-center formation in HAE differs from that of most paramyxoviruses (7) and may result from the unique receptor specificity of the Morbillivirus genus (8-11). Indeed, we have shown that infectious-center formation in HAE results from direct cell-to-cell spread and is facilitated by the nectin-4/afadin complex (7). These studies highlight the importance of using an appropriate model system to study MeV entry and spread.How MeV eventually reaches the airway epithelium during a natural infection is less clear. Initially, the infection may be ferried through the epithelium by myeloid cells that sample the airway lumen and express the primary MeV receptor signaling lymphocyte a...

“…The escape mutation R82G in domain A of E2 was generated under the neutralizing pressure of MAb 5F10 and was previously shown to promote the transmission of CHIKV isolates by direct cell-to-cell transfer of the virus (16). Although cell-to-cell transmission of viruses has been observed for many lipid-enveloped viruses (36)(37)(38), little is known about the exact mechanism. Nevertheless, it is known that cell-to-cell transmission is often accompanied by the increased concentration of molecules that can act as receptors (for instance, decay-accelerating factor) (39) for the free virus near cell junctions (39).…”

Section: Fitting the Fab Fragments Into The Cryo-em Densitiesmentioning

confidence: 99%

“…The former inhibits by covering the cellular receptor-binding site, and the latter inhibits by crowding around and restricting the movement of the B domain, but leaving the cellular receptor binding site vacant for use in cell-to-cell transmission. Although the requirements of cell-to-cell transmission are roughly known for quite a few membrane-enveloped viruses (36)(37)(38)40), this currently is the most detailed knowledge of the mechanism of this process.…”

Section: Fitting the Fab Fragments Into The Cryo-em Densitiesmentioning

confidence: 99%

Structural Studies of Chikungunya Virus-Like Particles Complexed with Human Antibodies: Neutralization and Cell-to-Cell Transmission

Porta

Prasad

Wang

et al. 2016

Chikungunya virus is a positive-stranded RNA alphavirus. Structures of chikungunya virus-like particles in complex with strongly neutralizing antibody Fab fragments (8B10 and 5F10) were determined using cryo-electron microscopy and X-ray crystallography. By fitting the crystallographically determined structures of these Fab fragments into the cryo-electron density maps, we show that Fab fragments of antibody 8B10 extend radially from the viral surface and block receptor binding on the E2 glycoprotein. In contrast, Fab fragments of antibody 5F10 bind the tip of the E2 B domain and lie tangentially on the viral surface. Fab 5F10 fixes the B domain rigidly to the surface of the virus, blocking exposure of the fusion loop on glycoprotein E1 and therefore preventing the virus from becoming fusogenic. Although Fab 5F10 can neutralize the wild-type virus, it can also bind to a mutant virus without inhibiting fusion or attachment. Although the mutant virus is no longer able to propagate by extracellular budding, it can, however, enter the next cell by traveling through junctional complexes without being intercepted by a neutralizing antibody to the wild-type virus, thus clarifying how cell-to-cell transmission can occur. IMPORTANCE Alphaviral infections are transmitted mainly by mosquitoes. Chikungunya virus (CHIKV), which belongs to the Alphavirus genus, has a wide distribution in the OldWorld that has expanded in recent years into the Americas. There are currently no vaccines or drugs against alphaviral infections. Therefore, a better understanding of CHIKV and its associated neutralizing antibodies will aid in the development of effective treatments. C hikungunya virus (CHIKV) is the causative agent of an emerging disease. The first outbreaks occurred in the 1950s in Tanzania (1). The virus later reemerged in late 2005 on the Reunion islands and, subsequently, also in several Southeast Asian countries (2). However, by 2013 large epizootics/epidemics of CHIKV had spread into the Caribbean and by 2014 into South America as well as the United States (3). CHIKV is a member of the Togaviridae family, Alphavirus genus. Some alphaviruses, such as Sindbis virus (SINV), generally cause only mild disease symptoms in humans (4). Although mortality rates from CHIKV infections are low, CHIKV and Venezuelan equine encephalitis virus (VEEV) can be lethal or permanently disabling (2, 3). Furthermore, there are currently no treatments or vaccines for alphaviral infections.The structures of a number of alphaviruses have been determined to resolutions better than 10 Å, including the structures of chikungunya virus-like particles (CHIK-VLPs), which have been determined to 5.3-Å resolution (5-7), and of VEEV, determined to 4.6-Å resolution (6). Alphaviruses have an external diameter of about 700 Å and are icosahedral with quasi-Tϭ4 symmetry. They have a nucleocapsid core that is completely surrounded by a lipid envelope, derived from a host membrane, into which is embedded an icosahedral array of glycoproteins (8). A single virus...