Receptor-mediated cell entry of paramyxoviruses: Mechanisms, and consequences for tropism and pathogenesis.

Research in the last decade has uncovered many new paramyxoviruses, airborne agents that cause epidemic diseases in animals including humans. Most paramyxoviruses enter epithelial cells of the airway using sialic acid as a receptor and cause only mild disease. However, others cross the epithelial barrier and cause more severe disease. For some of these viruses, the host receptors have been identified, and the mechanisms of cell entry have been elucidated. The tetrameric attachment proteins of paramyxoviruses have vastly different binding affinities for their cognate receptors, which they contact through different binding surfaces. Nevertheless, all input signals are converted to the same output: conformational changes that trigger refolding of trimeric fusion proteins and membrane fusion. Experiments with selectively receptor-blinded viruses inoculated into their natural hosts have provided insights into tropism, identifying the cells and tissues that support growth and revealing the mechanisms of pathogenesis. These analyses also shed light on diabolically elegant mechanisms used by morbilliviruses, including the measles virus, to promote massive amplification within the host, followed by efficient aerosolization and rapid spread through host populations. In another paradigm of receptor-facilitated severe disease, henipaviruses, including Nipah and Hendra viruses, use different members of one protein family to cause zoonoses. Specific properties of different paramyxoviruses, like neurotoxicity and immunosuppression, are now understood in the light of receptor specificity. We propose that research on the specific receptors for several newly identified members of the Paramyxoviridae family that may not bind sialic acid is needed to anticipate their zoonotic potential and to generate effective vaccines and antiviral compounds.

Trans-endocytosis elicited by nectins transfers cytoplasmic cargo, including infectious material, between cells.

Here, we show that cells expressing the adherens junction protein nectin-1 capture nectin-4-containing membranes from the surface of adjacent cells in a trans-endocytosis process. We find that internalized nectin-1-nectin-4 complexes follow the endocytic pathway. The nectin-1 cytoplasmic tail controls transfer: its deletion prevents trans-endocytosis, while its exchange with the nectin-4 tail reverses transfer direction. Nectin-1-expressing cells acquire dye-labeled cytoplasmic proteins synchronously with nectin-4, a process most active during cell adhesion. Some cytoplasmic cargo remains functional after transfer, as demonstrated with encapsidated genomes of measles virus (MeV). This virus uses nectin-4, but not nectin-1, as a receptor. Epithelial cells expressing nectin-4, but not those expressing another MeV receptor in its place, can transfer infection to nectin-1-expressing primary neurons. Thus, this newly discovered process can move cytoplasmic cargo, including infectious material, from epithelial cells to neurons. We name the process nectin-elicited cytoplasm transfer (NECT). NECT-related trans-endocytosis processes may be exploited by pathogens to extend tropism. This article has an associated First Person interview with the first author of the paper.

Connections matter--how viruses use cell­cell adhesion components.

The epithelium is a highly organized type of animal tissue. Except for blood and lymph vessels, epithelial cells cover the body, line its cavities in single or stratified layers and support exchange between compartments. In addition, epithelia offer to the body a barrier to pathogen invasion. To transit through or to replicate in epithelia, viruses have to face several obstacles, starting from cilia and glycocalyx where they can be neutralized by secreted immunoglobulins. Tight junctions and adherens junctions also prevent viruses to cross the epithelial barrier. However, viruses have developed multiple strategies to blaze their path through the epithelium by utilizing components of cell­cell adhesion structures as receptors. In this Commentary, we discuss how viruses take advantage of the apical junction complex to spread. Whereas some viruses quickly disrupt epithelium integrity, others carefully preserve it and use cell adhesion proteins and their cytoskeletal connections to rapidly spread laterally. This is exemplified by the hidden transmission of enveloped viruses that use nectins as receptors. Finally, several viruses that replicate preferentially in cancer cells are currently used as experimental cancer therapeutics. Remarkably, these viruses use cell adhesion molecules as receptors, probably because--to reach tumors and metastases--ncolytic viruses must efficiently traverse or break epithelia.

The measles virus hemagglutinin stalk: structures and functions of the central fusion activation and membrane-proximal segments.

UNLABELLED: The measles virus (MeV) membrane fusion apparatus consists of a fusion protein trimer and an attachment protein tetramer. To trigger membrane fusion, the heads of the MeV attachment protein, hemagglutinin (H), bind cellular receptors while the 96-residue-long H stalk transmits the triggering signal. Structural and functional studies of the triggering mechanism of other paramyxoviruses suggest that receptor binding to their hemagglutinin-neuraminidase (HN) results in signal transmission through the central segments of their stalks. To gain insight into H-stalk structure and function, we individually replaced its residues with cysteine. We then assessed how stable the mutant proteins are, how efficiently they can be cross-linked by disulfide bonds, whether cross-linking results in loss of function, and, in this case, whether disulfide bond reduction restores function. While many residues in the central segment of the stalk and in the spacer segment above it can be efficiently cross-linked by engineered disulfide bonds, we report here that residues 59 to 79 cannot, suggesting that the 20 membrane-proximal residues are not engaged in a tetrameric structure. Rescue-of-function studies by disulfide bond reduction resulted in the redefinition and extension of the central fusion-activation segment as covering residues 84 to 117. In particular, we identified four residues located between positions 92 and 99, the function of which cannot be restored by disulfide bond reduction after cysteine mutagenesis. These mutant H proteins reached the cell surface as complex oligomers but could not trigger membrane fusion. We discuss these observations in the context of the stalk exposure model of membrane fusion triggering by paramyxoviruses. IMPORTANCE: Measles virus, while being targeted for eradication, still causes significant morbidity and mortality. Here, we seek to understand how it enters cells by membrane fusion. Two viral integral membrane glycoproteins (hemagglutinin tetramers and fusion protein trimers) mediate the concerted receptor recognition and membrane fusion processes. Since previous studies have suggested that the hemagglutinin stalk transmits the triggering signal to the fusion protein trimer, we completed an analysis of its structure and function by systematic Cys mutagenesis. We report that while certain residues of the central stalk segment confer specificity to the interaction with the fusion protein trimer, others are necessary to allow folding of the H-oligomer in a standard conformation conducive to fusion triggering, and still other residues sustain the conformational change that transmits the fusion-triggering signal.

Identification of putative interactions between swine and human influenza A virus nucleoprotein and human host proteins.

BACKGROUND: Influenza A viruses (IAVs) are important pathogens that affect the health of humans and many additional animal species. IAVs are enveloped, negative single-stranded RNA viruses whose genome encodes at least ten proteins. The IAV nucleoprotein (NP) is a structural protein that associates with the viral RNA and is essential for virus replication. Understanding how IAVs interact with host proteins is essential for elucidating all of the required processes for viral replication, restrictions in species host range, and potential targets for antiviral therapies. METHODS: In this study, the NP from a swine IAV was cloned into a yeast two-hybrid "bait" vector for expression of a yeast Gal4 binding domain (BD)-NP fusion protein. This "bait" was used to screen a Y2H human HeLa cell "prey" library which consisted of human proteins fused to the Gal4 protein's activation domain (AD). The interaction of "bait" and "prey" proteins resulted in activation of reporter genes. RESULTS: Seventeen positive bait-prey interactions were isolated in yeast. All of the "prey" isolated also interact in yeast with a NP "bait" cloned from a human IAV strain. Isolation and sequence analysis of the cDNAs encoding the human prey proteins revealed ten different human proteins. These host proteins are involved in various host cell processes and structures, including purine biosynthesis (PAICS), metabolism (ACOT13), proteasome (PA28B), DNA-binding (MSANTD3), cytoskeleton (CKAP5), potassium channel formation (KCTD9), zinc transporter function (SLC30A9), Na+/K+ ATPase function (ATP1B1), and RNA splicing (TRA2B). CONCLUSIONS: Ten human proteins were identified as interacting with IAV NP in a Y2H screen. Some of these human proteins were reported in previous screens aimed at elucidating host proteins relevant to specific viral life cycle processes such as replication. This study extends previous findings by suggesting a mechanism by which these host proteins associate with the IAV, i.e., physical interaction with NP. Furthermore, this study revealed novel host protein-NP interactions in yeast.

Stronger together: Multi-genome transmission of measles virus.

Measles virus (MeV) is an immunosuppressive, extremely contagious RNA virus that remains a leading cause of death among children. MeV is dual-tropic: it replicates first in lymphatic tissue, causing immunosuppression, and then in epithelial cells of the upper airways, accounting for extremely efficient contagion. Efficient contagion is counter-intuitive because the enveloped MeV particles are large and relatively unstable. However, MeV particles can contain multiple genomes, which can code for proteins with different functional characteristics. These proteins can cooperate to promote virus spread in tissue culture, prompting the question of whether multi-genome MeV transmission may promote efficient MeV spread also in vivo. Consistent with this hypothesis, in well-differentiated primary human airway epithelia large genome populations spread rapidly through intercellular pores. In another line of research, it was shown that distinct lymphocytic-adapted and epithelial-adapted genome populations exist; cyclical adaptation studies indicate that suboptimal variants in one environment may constitute a low frequency reservoir for adaptation to the other environment. Altogether, these observations suggest that, in humans, MeV spread relies on en bloc genome transmission, and that genomic diversity is instrumental for rapid MeV dissemination within hosts.

Humidity as a non-pharmaceutical intervention for influenza A.

Influenza is a global problem infecting 5-10% of adults and 20-30% of children annually. Non-pharmaceutical interventions (NPIs) are attractive approaches to complement vaccination in the prevention and reduction of influenza. Strong cyclical reduction of absolute humidity has been associated with influenza outbreaks in temperate climates. This study tested the hypothesis that raising absolute humidity above seasonal lows would impact influenza virus survival and transmission in a key source of influenza virus distribution, a community school. Air samples and objects handled by students (e.g. blocks and markers) were collected from preschool classrooms. All samples were processed and PCR used to determine the presence of influenza virus and its amount. Additionally samples were tested for their ability to infect cells in cultures. We observed a significant reduction (p < 0.05) in the total number of influenza A virus positive samples (air and fomite) and viral genome copies upon humidification as compared to control rooms. This suggests the future potential of artificial humidification as a possible strategy to control influenza outbreaks in temperate climates. There were 2.3 times as many ILI cases in the control rooms compared to the humidified rooms, and whether there is a causal relationship, and its direction between the number of cases and levels of influenza virus in the rooms is not known. Additional research is required, but this is the first prospective study suggesting that exogenous humidification could serve as a scalable NPI for influenza or other viral outbreaks.

Sustained ELABELA Gene Therapy in High-salt Diet-induced Hypertensive Rats.

BACKGROUND: Elabela (ELA) is a recently identified apelin receptor agonist essential for cardiac development, but its biology and therapeutic potential are unclear. In humans, ELA transcripts are detected in embryonic stem cells, induced pluripotent stem cells, kidney, heart and blood vessels. ELA through the apelin (APJ) receptor promotes angiogenesis in vitro, relaxes murine aortic blood vessels and attenuates high blood pressure in vivo. The APJ receptor when bound to its original ligand, apelin, exerts peripheral vasodilatory and positive inotropic effects, conferring cardioprotection in vivo. METHODS: This study initially assessed endogenous ELA expression in normal and diseased rats and then characterized the effects of long-term ELA gene delivery by adeno-associated virus serotype 9 (AAV9) vectors on cardiorenal function in Dahl salt-sensitive rats (DS) on a high-salt diet over 3 months. RESULTS: Endogenous ELA was predominantly expressed in the kidneys, especially in the renal collecting duct cells and was not affected by disease. Rat ELA was overexpressed in the heart via AAV9 vector by a single intravenous injection. ELA-treated animals showed delayed onset of blood pressure elevation. Prior to high-salt diet, a reduction in the fractional sodium and chloride excretion was observed in rats given the AAV9-ELA vector. After three months on a high-salt diet, ELA preserved glomerular architecture, decreased renal fibrosis and suppressed expression of fibrosis-associated genes in the kidneys. CONCLUSION: ELA is constitutively expressed in renal collecting ducts in rats. Sustained AAV-ELA expression may offer a potential long-term therapy for hypertension and renal remodeling.