W. Paul Duprex

Hemagglutinin Protein of Wild-Type Measles Virus Activates Toll-Like Receptor 2 Signaling

ABSTRACT Pattern recognition via Toll-like receptors (TLR) by antigen-presenting cells is an important element of innate immunity. We report that wild-type measles virus but not vaccine strains activate cells via both human and murine TLR2, and this is a property of the hemagglutinin (H) protein. The ability to activate cells via TLR2 by wild-type MV H protein is abolished by mutation of a single amino acid, asparagine at position 481 to tyrosine, as is found in attenuated strains, which is important for interaction with CD46, the receptor for these strains. TLR2 activation by MV wild-type H protein stimulates induction of proinflammatory cytokines such as interleukin-6 (IL-6) in human monocytic cells and surface expression of CD150, the receptor for all MV strains. Confirming the specificity of this interaction, wild-type H protein did not induce IL-6 release in macrophages from TLR2−/− mice. Thus, the unique property of MV wild-type strains to activate TLR2-dependent signals might essentially contribute not only to immune activation but also to viral spread and pathogenicity by upregulating the MV receptor on monocytes.

Ligand-induced conformational changes allosterically activate Toll-like receptor 9

Microbial and synthetic DNA rich in CpG dinucleotides stimulates Toll-like receptor 9 (TLR9), whereas DNA lacking CpG either is inert or can inhibit TLR9 activation. The molecular mechanisms by which TLR9 becomes activated or is inhibited are not well understood. Here we show that TLR9 bound to stimulatory and inhibitory DNA; however, only stimulatory DNA led to substantial conformational changes in the TLR9 ectodomain. In the steady state, inactive TLR9 homodimers formed in an inactivated conformation. Binding of DNA containing CpG, but not of DNA lacking CpG, to TLR9 dimers resulted in allosteric changes in the TLR9 cytoplasmic signaling domains. In endosomes, conformational changes induced by DNA containing CpG resulted in close apposition of the cytoplasmic signaling domains, a change that is probably required for the recruitment of signaling adaptor molecules. Our results indicate that the formation of TLR9 dimers is not sufficient for its activation but instead that TLR9 activation is regulated by conformational changes induced by DNA containing CpG.

Predominant infection of CD150+ lymphocytes and dendritic cells during measles virus infection of macaques.

Measles virus (MV) is hypothesized to enter the host by infecting epithelial cells of the respiratory tract, followed by viremia mediated by infected monocytes. However, neither of these cell types express signaling lymphocyte activation molecule (CD150), which has been identified as the receptor for wild-type MV. We have infected rhesus and cynomolgus macaques with a recombinant MV strain expressing enhanced green fluorescent protein (EGFP); thus bringing together the optimal animal model for measles and a virus that can be detected with unprecedented sensitivity. Blood samples and broncho-alveolar lavages were collected every 3 d, and necropsies were performed upon euthanasia 9 or 15 d after infection. EGFP production by MV-infected cells was visualized macroscopically, in both living and sacrificed animals, and microscopically by confocal microscopy and FACS analysis. At the peak of viremia, EGFP fluorescence was detected in skin, respiratory and digestive tract, but most intensely in all lymphoid tissues. B- and T-lymphocytes expressing CD150 were the major target cells for MV infection. Highest percentages (up to 30%) of infected lymphocytes were detected in lymphoid tissues, and the virus preferentially targeted cells with a memory phenotype. Unexpectedly, circulating monocytes did not sustain productive MV infection. In peripheral tissues, large numbers of MV-infected CD11c+ MHC class-II+ myeloid dendritic cells were detected in conjunction with infected T-lymphocytes, suggesting transmission of MV between these cell types. Fluorescent imaging of MV infection in non-human primates demonstrated a crucial role for lymphocytes and dendritic cells in the pathogenesis of measles and measles-associated immunosuppression.

Early Target Cells of Measles Virus after Aerosol Infection of Non-Human Primates

Measles virus (MV) is highly infectious, and has long been thought to enter the host by infecting epithelial cells of the respiratory tract. However, epithelial cells do not express signaling lymphocyte activation molecule (CD150), which is the high-affinity cellular receptor for wild-type MV strains. We have generated a new recombinant MV strain expressing enhanced green fluorescent protein (EGFP), based on a wild-type genotype B3 virus isolate from Khartoum, Sudan (KS). Cynomolgus macaques were infected with a high dose of rMVKSEGFP by aerosol inhalation to ensure that the virus could reach the full range of potential target cells throughout the entire respiratory tract. Animals were euthanized 2, 3, 4 or 5 days post-infection (d.p.i., nu200a=u200a3 per time point) and infected (EGFP+) cells were identified at all four time points, albeit at low levels 2 and 3 d.p.i. At these earliest time points, MV-infected cells were exclusively detected in the lungs by fluorescence microscopy, histopathology and/or virus isolation from broncho-alveolar lavage cells. On 2 d.p.i., EGFP+ cells were phenotypically typed as large mononuclear cells present in the alveolar lumen or lining the alveolar epithelium. One to two days later, larger clusters of MV-infected cells were detected in bronchus-associated lymphoid tissue (BALT) and in the tracheo-bronchial lymph nodes. From 4 d.p.i. onward, MV-infected cells were detected in peripheral blood and various lymphoid tissues. In spite of the possibility for the aerosolized virus to infect cells and lymphoid tissues of the upper respiratory tract, MV-infected cells were not detected in either the tonsils or the adenoids until after onset of viremia. These data strongly suggest that in our model MV entered the host at the alveolar level by infecting macrophages or dendritic cells, which traffic the virus to BALT or regional lymph nodes, resulting in local amplification and subsequent systemic dissemination by viremia.

Morbilliviruses and human disease

Morbilliviruses are a group of viruses that belong to the family Paramyxoviridae. The most instantly recognizable member is measles virus (MV) and individuals acutely infected with the virus exhibit a wide range of clinical symptoms ranging from a characteristic mild self‐limiting infection to death. Canine distemper virus (CDV) and rinderpest virus (RPV) cause a similar but distinctive pathology in dogs and cattle, respectively, and these, alongside experimental MV infection of primates, have been useful models for MV pathogenesis. Traditionally, viruses were identified because a distinctive disease was observed in man or animals; an infectious agent was subsequently isolated, cultured, and this could be used to recapitulate the disease in an experimentally infected host. Thus, satisfying Kochs postulates has been the norm. More recently, particularly due to the advent of exceedingly sensitive molecular biological assays, many researchers have looked for infectious agents in disease conditions for which a viral aetiology has not been previously established. For these cases, the modified Kochs postulates of Bradford Hill have been developed as criteria to link a virus to a specific disease. Only in a few cases have these conditions been fulfilled. Therefore, many viruses have over the years been definitely and tentatively linked to human diseases and in this respect the morbilliviruses are no different. In this review, human diseases associated with morbillivirus infection have been grouped into three broad categories: (1) those which are definitely caused by the infection; (2) those which may be exacerbated or facilitated by an infection; and (3) those which currently have limited, weak, unsubstantiated or no credible scientific evidence to support any link to a morbillivirus. Thus, an attempt has been made to clarify the published data and separate human diseases actually linked to morbilliviruses from those that are merely anecdotally associated. Copyright

Analysis of receptor (CD46, CD150) usage by measles virus

In order to investigate which measles virus (MV)-strains use CD46 and/or CD150 (signalling lymphocytic activation molecule, SLAM) as receptors, CHO cells expressing either recombinant CD46 or SLAM were infected with a panel of 28 MV-strains including vaccine strains, wild-type strains with various passage histories and recombinant viruses. We found that SLAM served as a common receptor conferring virus uptake and syncytium formation for all MV-strains tested. Predominantly vaccine and laboratory adapted strains, but also a minor fraction of the wild-type strains tested, could utilize both CD46 and SLAM. Using recombinant viruses, we demonstrate that the single amino acid exchange in the haemagglutinin (H) protein at position 481 Asn/Tyr (H481NY) determines whether the virus can utilize CD46. This amino acid alteration has no affect on the usage of SLAM as receptor, and as such demonstrates that the binding sites for SLAM and CD46 are distinct.

Dynamics of Viral RNA Synthesis during Measles Virus Infection

ABSTRACT We propose a reference model of the kinetics of a viral RNA-dependent RNA polymerase (vRdRp) activities and its regulation during infection of eucaryotic cells. After measles virus infects a cell, mRNAs from all genes immediately start to accumulate linearly over the first 5 to 6 h and then exponentially until ∼24 h. The change from a linear to an exponential accumulation correlates with de novo synthesis of vRdRp from the incoming template. Expression of the virus nucleoprotein (N) prior to infection shifts the balance in favor of replication. Conversely, inhibition of protein synthesis by cycloheximide favors the latter. The in vivo elongation speed of the viral polymerase is ∼3 nucleotides/s. A similar profile with fivefold-slower kinetics can be obtained using a recombinant virus expressing a structurally altered polymerase. Finally, virions contain only encapsidated genomic, antigenomic, and 5′-end abortive replication fragment RNAs.

Quantitative Proteomic Analysis of A549 Cells Infected with Human Respiratory Syncytial Virus

Human respiratory syncytial virus (HRSV) is a major cause of pediatric lower respiratory tract disease to which there is no vaccine or efficacious chemotherapeutic strategy. Although RNA synthesis and virus assembly occur in the cytoplasm, HRSV is known to induce nuclear responses in the host cell as replication alters global gene expression. Quantitative proteomics was used to take an unbiased overview of the protein changes in transformed human alveolar basal epithelial cells infected with HRSV. Underpinning this was the use of stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS, which allowed the direct and simultaneous identification and quantification of both cellular and viral proteins. To reduce sample complexity and increase data return on potential protein localization, cells were fractionated into nuclear and cytoplasmic extracts. This resulted in the identification of 1,140 cellular proteins and six viral proteins. The proteomics data were analyzed using Ingenuity Pathways Analysis to identify defined canonical pathways and functional groupings. Selected data were validated using Western blot, direct and indirect immunofluorescence confocal microscopy, and functional assays. The study served to validate and expand upon known HRSV-host cell interactions, including those associated with the antiviral response and alterations in subnuclear structures such as the nucleolus and ND10 (promyelocytic leukemia bodies). In addition, novel changes were observed in mitochondrial proteins and functions, cell cycle regulatory molecules, nuclear pore complex proteins and nucleocytoplasmic trafficking proteins. These data shed light into how the cell is potentially altered to create conditions more favorable for infection. Additionally, the study highlights the application and advantage of stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS for the analysis of virus-host interactions.

Modulating the Function of the Measles Virus RNA-Dependent RNA Polymerase by Insertion of Green Fluorescent Protein into the Open Reading Frame

ABSTRACT Measles virus (MV) is the type species of the Morbillivirus genus and its RNA-dependent RNA polymerase complex is comprised of two viral polypeptides, the large (L) and the phospho- (P) proteins. Sequence alignments of morbillivirus L polymerases have demonstrated the existence of three well-conserved domains (D1, D2, and D3) which are linked by two variable hinges (H1 and H2). Epitope tags (c-Myc) were introduced into H1 and H2 to investigate the tolerance of the variable regions to insertions and to probe the flexibility of the proposed domain structures to spatial reorientation. Insertion into H1 abolished polymerase activity whereas introduction into H2 had no effect. The open reading frame of enhanced green fluorescent protein was also inserted into the H2 region of the MV L gene to extend these observations. This resulted in a recombinant protein that was both functional and autofluorescent, although the overall polymerase activity was reduced by over 40%. Two recombinant viruses which contained the chimeric L genes EdtagL(MMc-mycM) and EdtagL(MMEGFPM) were generated. Tagged L proteins were detectable, by indirect immunofluorescence in the case of EdtagL(MMc-mycM) and by autofluorescence in the case of EdtagL(MMEGFPM). We suggest that D3 enjoys a limited conformational independence from the other domains, indicating that the L polymerases of the Mononegavirales may function as multidomain proteins.

Measles Immune Suppression: Lessons from the Macaque Model

Measles remains a significant childhood disease, and is associated with a transient immune suppression. Paradoxically, measles virus (MV) infection also induces robust MV-specific immune responses. Current hypotheses for the mechanism underlying measles immune suppression focus on functional impairment of lymphocytes or antigen-presenting cells, caused by infection with or exposure to MV. We have generated stable recombinant MVs that express enhanced green fluorescent protein, and remain virulent in non-human primates. By performing a comprehensive study of virological, immunological, hematological and histopathological observations made in animals euthanized at different time points after MV infection, we developed a model explaining measles immune suppression which fits with the “measles paradox”. Here we show that MV preferentially infects CD45RA− memory T-lymphocytes and follicular B-lymphocytes, resulting in high infection levels in these populations. After the peak of viremia MV-infected lymphocytes were cleared within days, followed by immune activation and lymph node enlargement. During this period tuberculin-specific T-lymphocyte responses disappeared, whilst strong MV-specific T-lymphocyte responses emerged. Histopathological analysis of lymphoid tissues showed lymphocyte depletion in the B- and T-cell areas in the absence of apoptotic cells, paralleled by infiltration of T-lymphocytes into B-cell follicles and reappearance of proliferating cells. Our findings indicate an immune-mediated clearance of MV-infected CD45RA− memory T-lymphocytes and follicular B-lymphocytes, which causes temporary immunological amnesia. The rapid oligoclonal expansion of MV-specific lymphocytes and bystander cells masks this depletion, explaining the short duration of measles lymphopenia yet long duration of immune suppression.