Cattaneo, Roberto

ABSTRACT Many viruses utilize cell adhesion molecules of the immunoglobulin superfamily as receptors. In particular, viruses of different classes exploit nectins. The large DNA viruses, herpes simplex and pseudorabies viruses, use ubiquitous nectins 1 and 2. The negative-strand RNA virus measles virus (MeV) uses tissue-specific nectin-4, and the positive-strand RNA virus poliovirus uses nectin-like 5 (necl-5), also known as poliovirus receptor. These viruses contact the BC, C′C″, and FG loops on the upper tip of their receptor's most membrane-distal domain. This location corresponds to the newly defined canonical adhesive interface of nectins, but how viruses utilize this interface has remained unclear. Here we show that the same key residues in the BC and FG loops of nectin-4 govern binding to the MeV attachment protein hemagglutinin (H) and cell entry, nectin-4 homodimerization, and heterodimerization with nectin-1. On the other hand, residues in the C′C″ loop necessary for homo- and heterotypic interactions are dispensable for MeV-induced fusion and cell entry. Remarkably, the C′C″ loop governs dissociation of the nectin-4 and H ectodomains. We provide formal proof that H can interfere with the formation of stable nectin-1/nectin-4 heterodimers. Finally, while developing an alternative model to study MeV spread, we observed that polarized primary pig airway epithelial sheets cannot be infected. We show that a single amino acid variant in the BC loop of pig nectin-4 fully accounts for restricted MeV entry. Thus, the three loops forming the adhesive interface of nectin-4 have different roles in supporting MeV H association and dissociation and MeV-induced fusion. IMPORTANCE Different viruses utilize nectins as receptors. Nectins are immunoglobulin superfamily glycoproteins that mediate cell-cell adhesion in vertebrate tissues. They interact through an adhesive interface located at the top of their membrane-distal domain. How viruses utilize the three loops forming this interface has remained unclear. We demonstrate that while nectin-nectin interactions require residues in all three loops, the association of nectin-4 with the measles virus hemagglutinin requires only the BC and FG loops. However, we discovered that residues in the C′C″ loop modulate the dissociation of nectin-4 from the viral hemagglutinin. Analogous mechanisms may support cell entry of other viruses that utilize nectins or other cell adhesion molecules of the immunoglobulin superfamily as receptors.

ABSTRACT The genome of nonsegmented negative-strand RNA viruses is tightly embedded within a nucleocapsid made of a nucleoprotein (N) homopolymer. To ensure processive RNA synthesis, the viral polymerase L in complex with its cofactor phosphoprotein (P) binds the nucleocapsid that constitutes the functional template. Measles virus P and N interact through two binding sites. While binding of the P amino terminus with the core of N (N CORE ) prevents illegitimate encapsidation of cellular RNA, the interaction between their C-terminal domains, P XD and N TAIL is required for viral RNA synthesis. To investigate the binding dynamics between the two latter domains, the P XD F497 residue that makes multiple hydrophobic intramolecular interactions was mutated. Using a quantitative mammalian protein complementation assay and recombinant viruses, we found that an increase in P XD -to-N TAIL binding strength is associated with a slower transcript accumulation rate and that abolishing the interaction renders the polymerase nonfunctional. The use of a newly developed system allowing conditional expression of wild-type or mutated P genes, revealed that the loss of the P XD -N TAIL interaction results in reduced transcription by preformed transcriptases, suggesting reduced engagement on the genomic template. These intracellular data indicate that the viral polymerase entry into and progression along its genomic template relies on a protein-protein interaction that serves as a tightly controlled dynamic anchor. IMPORTANCE Mononegavirales have a unique machinery to replicate RNA. Processivity of their polymerase is only achieved when the genome template is entirely embedded into a helical homopolymer of nucleoproteins that constitutes the nucleocapsid. The polymerase binds to the nucleocapsid template through the phosphoprotein. How the polymerase complex enters and travels along the nucleocapsid template to ensure uninterrupted synthesis of up to ∼6,700-nucleotide messenger RNAs from six to ten consecutive genes is unknown. Using a quantitative protein complementation assay and a biGene-biSilencing system allowing conditional expression of two P genes copies, the role of the P-to-N interaction in polymerase function was further characterized. We report here a dynamic protein anchoring mechanism that differs from all other known polymerases that rely only onto a sustained and direct binding to their nucleic acid template.

ABSTRACT 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.

ABSTRACT The V proteins of paramyxoviruses control the innate immune response. In particular, the V protein of the genus Morbillivirus interferes with the signal transducer and activator of transcription 1 (STAT1), STAT2, and melanoma differentiation-associated protein 5 (mda5) signaling pathways. To characterize the contributions of these pathways to canine distemper virus (CDV) pathogenesis, we took advantage of the knowledge about the mechanisms of interaction between the measles virus V protein with these key regulators of innate immunity. We generated recombinant CDVs with V proteins unable to properly interact with STAT1, STAT2, or mda5. A virus with combined STAT2 and mda5 deficiencies was also generated, and available wild-type and V-protein-knockout viruses were used as controls. Ferrets infected with wild-type and STAT1-blind viruses developed severe leukopenia and loss of lymphocyte proliferation activity and succumbed to the disease within 14 days. In contrast, animals infected with viruses with STAT2 or mda5 defect or both STAT2 and mda5 defects developed a mild self-limiting disease similar to that associated with the V-knockout virus. This study demonstrates the importance of interference with STAT2 and mda5 signaling for CDV immune evasion and provides a starting point for the development of morbillivirus vectors with reduced immunosuppressive properties. IMPORTANCE The V proteins of paramyxoviruses interfere with the recognition of the virus by the immune system of the host. For morbilliviruses, the V protein is known to interact with the signal transducer and activator of transcription 1 (STAT1) and STAT2 and the melanoma differentiation-associated protein 5 (mda5), which are involved in interferon signaling. Here, we examined the contribution of each of these signaling pathways to the pathogenesis of the carnivore morbillivirus canine distemper virus. Using viruses selectively unable to interfere with the respective signaling pathway to infect ferrets, we found that inhibition of STAT2 and mda5 signaling was critical for lethal disease. Our findings provide new insights in the mechanisms of morbillivirus immune evasion and may lead to the development of new vaccines and oncolytic vectors.

ABSTRACT Measles virus (MV) lacking expression of C protein (C KO ) is a potent activator of the double-stranded RNA (dsRNA)-dependent protein kinase (PKR), whereas the isogenic parental virus expressing C protein is not. Here, we demonstrate that significant amounts of dsRNA accumulate during C KO mutant infection but not following parental virus infection. dsRNA accumulated during late stages of infection and localized with virus replication sites containing N and P proteins. PKR autophosphorylation and stress granule formation correlated with the timing of dsRNA appearance. Phospho-PKR localized to dsRNA-containing structures as revealed by immunofluorescence. Production of dsRNA was sensitive to cycloheximide but resistant to actinomycin D, suggesting that dsRNA is a viral product. Quantitative PCR (qPCR) analyses revealed reduced viral RNA synthesis and a steepened transcription gradient in C KO virus-infected cells compared to those in parental virus-infected cells. The observed alterations were further reflected in lower viral protein expression levels and reduced C KO virus infectious yield. RNA deep sequencing confirmed the viral RNA expression profile differences seen by qPCR between C KO mutant and parental viruses. After one subsequent passage of the C KO virus, defective interfering RNA (DI-RNA) with a duplex structure was obtained that was not seen with the parental virus. We conclude that in the absence of C protein, the amount of PKR activator RNA, including DI-RNA, is increased, thereby triggering innate immune responses leading to impaired MV growth.

ABSTRACT The pH-independent measles virus membrane fusion process begins when the attachment protein H binds to a receptor. Knowing that the central segment of the tetrameric H stalk transmits the signal to the fusion protein trimer, we investigated how. We document that exact conservation of most residues in the 92 through 99 segment is essential for function. In addition, hydrophobic and charged residues in the 104 through 125 segment, arranged with helical periodicity, are critical for F protein interactions and signal transmission.

ABSTRACT Wild-type measles virus (MV) strains use the signaling lymphocytic activation molecule (SLAM; CD150) and the adherens junction protein nectin-4 (poliovirus receptor-like 4 [PVRL4]) as receptors. Vaccine MV strains have adapted to use ubiquitous membrane cofactor protein (MCP; CD46) in addition. Recently solved cocrystal structures of the MV attachment protein (hemagglutinin [H]) with each receptor indicate that all three bind close to a hydrophobic groove located between blades 4 and 5 (β4-β5 groove) of the H protein β-propeller head. We used this structural information to focus our analysis of the functional footprints of the three receptors on vaccine MV H. We mutagenized this protein and tested the ability of individual mutants to support cell fusion through each receptor. The results highlighted a strong overlap between the functional footprints of nectin-4 and CD46 but not those of SLAM. A soluble form of nectin-4 abolished vaccine MV entry in nectin-4- and CD46-expressing cells but only reduced entry through SLAM. Analyses of the binding kinetics of an H mutant with the three receptors revealed that a single substitution in the β4-β5 groove drastically reduced nectin-4 and CD46 binding while minimally altering SLAM binding. We also generated recombinant viruses and analyzed their infections in cells expressing individual receptors. Introduction of a single substitution into the hydrophobic pocket affected entry through both nectin-4 and CD46 but not through SLAM. Thus, while nectin-4 and CD46 interact functionally with the H protein β4-β5 hydrophobic groove, SLAM merely covers it. This has implications for vaccine and antiviral strategies.

ABSTRACT After the contagion measles virus (MV) crosses the respiratory epithelium within myeloid cells that express the primary receptor signaling lymphocytic activation molecule (SLAM), it replicates briskly in SLAM-expressing cells in lymphatic organs. Later, the infection spreads to epithelia expressing nectin-4, an adherens junction protein expressed preferentially in the trachea, but how it gets there is not understood. To characterize the mechanisms of spread, we infected groups of 5 or 6 cynomolgus monkeys ( Macaca fascicularis ) with either a wild-type MV or its “N4-blind” derivative, which is unable to enter nectin-4-expressing cells because of the targeted mutation of two hemagglutinin residues. As expected, both viruses caused similar levels of immunosuppression, as monitored by reductions in white blood cell counts and lymphocyte proliferation activity. However, monkeys infected with the N4-blind MV cleared infection more rapidly. Wild-type virus-infected monkeys secreted virus, while marginal virus titers were detected in tracheal lavage fluid cells of N4-blind MV-infected hosts. Analyses of tracheal rings obtained at necropsy (day 12) documented widespread infection of individual cells or small cell clusters in the subepithelial lamina propria of monkeys infected with either virus. However, only wild-type MV spread to the epithelium, forming numerous infectious centers comprised of many contiguous columnar cells. Infected CD11c + myeloid (macrophage or dendritic) cells were frequently observed in the lamina propria below epithelial infectious centers. Thus, MV may use myeloid cells as vehicles not only immediately after contagion but also to infect epithelia of tissues expressing nectin-4, including the trachea.

ABSTRACT Hepatitis C virus (HCV) infection remains a serious public health problem worldwide. Treatments are limited, and no preventive vaccine is available. Toward developing an HCV vaccine, we engineered two recombinant measles viruses (MVs) expressing structural proteins from the prototypic HCV subtype 1a strain H77. One virus directs the synthesis of the HCV capsid (C) protein and envelope glycoproteins (E1 and E2), which fold properly and form a heterodimer. The other virus expresses the E1 and E2 glycoproteins separately, with each one fused to the cytoplasmic tail of the MV fusion protein. Although these hybrid glycoproteins were transported to the plasma membrane, they were not incorporated into MV particles. Immunization of MV-susceptible, genetically modified mice with either vector induced neutralizing antibodies to MV and HCV. A boost with soluble E2 protein enhanced titers of neutralizing antibody against the homologous HCV envelope. In animals primed with MV expressing properly folded HCV C-E1-E2, boosting also induced cross-neutralizating antibodies against two heterologous HCV strains. These results show that recombinant MVs retain the ability to induce MV-specific humoral immunity while also eliciting HCV neutralizing antibodies, and that anti-HCV immunity can be boosted with a single dose of purified E2 protein. The use of MV vectors could have advantages for pediatric HCV vaccination.

A candidate vaccine against Nipah and Hendra viruses, recently emerging zoonotic threats, looks promising.