Transmission of Aerosolized Seasonal H1N1 Influenza A to Ferrets

Influenza virus is a major cause of morbidity and mortality worldwide, yet little quantitative understanding of transmission is available to guide evidence-based public health practice. Recent studies of influenza non-contact transmission between ferrets and guinea pigs have provided insights into the relative transmission efficiencies of pandemic and seasonal strains, but the infecting dose and subsequent contagion has not been quantified for most strains. In order to measure the aerosol infectious dose for 50% (aID50) of seronegative ferrets, seasonal influenza virus was nebulized into an exposure chamber with controlled airflow limiting inhalation to airborne particles less than 5 µm diameter. Airborne virus was collected by liquid impinger and Teflon filters during nebulization of varying doses of aerosolized virus. Since culturable virus was accurately captured on filters only up to 20 minutes, airborne viral RNA collected during 1-hour exposures was quantified by two assays, a high-throughput RT-PCR/mass spectrometry assay detecting 6 genome segments (Ibis T5000™ Biosensor system) and a standard real time RT-qPCR assay. Using the more sensitive T5000 assay, the aID50 for A/New Caledonia/20/99 (H1N1) was approximately 4 infectious virus particles under the exposure conditions used. Although seroconversion and sustained levels of viral RNA in upper airway secretions suggested established mucosal infection, viral cultures were almost always negative. Thus after inhalation, this seasonal H1N1 virus may replicate less efficiently than H3N2 virus after mucosal deposition and exhibit less contagion after aerosol exposure

Exhaled Aerosol Transmission of Pandemic and Seasonal H1N1 Influenza Viruses in the Ferret

Person-to-person transmission of influenza viruses occurs by contact (direct and fomites) and non-contact (droplet and small particle aerosol) routes, but the quantitative dynamics and relative contributions of these routes are incompletely understood. The transmissibility of influenza strains estimated from secondary attack rates in closed human populations is confounded by large variations in population susceptibilities. An experimental method to phenotype strains for transmissibility in an animal model could provide relative efficiencies of transmission. We developed an experimental method to detect exhaled viral aerosol transmission between unanesthetized infected and susceptible ferrets, measured aerosol particle size and number, and quantified the viral genomic RNA in the exhaled aerosol. During brief 3-hour exposures to exhaled viral aerosols in airflow-controlled chambers, three strains of pandemic 2009 H1N1 strains were frequently transmitted to susceptible ferrets. In contrast one seasonal H1N1 strain was not transmitted in spite of higher levels of viral RNA in the exhaled aerosol. Among three pandemic strains, the two strains causing weight loss and illness in the intranasally infected ‘donor’ ferrets were transmitted less efficiently from the donor than the strain causing no detectable illness, suggesting that the mucosal inflammatory response may attenuate viable exhaled virus. Although exhaled viral RNA remained constant, transmission efficiency diminished from day 1 to day 5 after donor infection. Thus, aerosol transmission between ferrets may be dependent on at least four characteristics of virus-host relationships including the level of exhaled virus, infectious particle size, mucosal inflammation, and viral replication efficiency in susceptible mucosa

Mosaic chromosomal alterations in peripheral blood leukocytes of children in sub-Saharan Africa

In high-income countries, mosaic chromosomal alterations in peripheral blood leukocytes are associated with an elevated risk of adverse health outcomes, including hematologic malignancies. We investigate mosaic chromosomal alterations in sub-Saharan Africa among 931 children with Burkitt lymphoma, an aggressive lymphoma commonly characterized by immunoglobulin-MYC chromosomal rearrangements, 3822 Burkitt lymphoma-free children, and 674 cancer-free men from Ghana. We find autosomal and X chromosome mosaic chromosomal alterations in 3.4% and 1.7% of Burkitt lymphoma-free children, and 8.4% and 3.7% of children with Burkitt lymphoma (P-values = 5.7×10-11 and 3.74×10-2, respectively). Autosomal mosaic chromosomal alterations are detected in 14.0% of Ghanaian men and increase with age. Mosaic chromosomal alterations in Burkitt lymphoma cases include gains on chromosomes 1q and 8, the latter spanning MYC, while mosaic chromosomal alterations in Burkitt lymphoma-free children include copy-neutral loss of heterozygosity on chromosomes 10, 14, and 16. Our results highlight mosaic chromosomal alterations in sub-Saharan African populations as a promising area of research

Downregulation of Major Histocompatibility Complex Class I by Human Ubiquitin Ligases Related to Viral Immune Evasion Proteins

Poxviruses and gamma-2 herpesviruses share the K3 family of viral immune evasion proteins that inhibit the surface expression of glycoproteins such as major histocompatibility complex class I (MHC-I), B7.2, ICAM-1, and CD95(Fas). K3 family proteins contain an amino-terminal PHD/LAP or RING-CH domain followed by two transmembrane domains. To examine whether human homologues are functionally related to the viral immunoevasins, we studied seven membrane-associated RING-CH (MARCH) proteins. All MARCH proteins located to subcellular membranes, and several MARCH proteins reduced surface levels of known substrates of the viral K3 family. Two closely related proteins, MARCH-IV and MARCH-IX, reduced surface expression of MHC-I molecules. In the presence of MARCH-IV or MARCH-IX, MHC-I was ubiquitinated and rapidly internalized by endocytosis, whereas MHC-I molecules lacking lysines in their cytoplasmic tail were resistant to downregulation. The amino-terminal regions containing the RING-CH domain of several MARCH proteins examined catalyzed multiubiquitin formation in vitro, suggesting that MARCH proteins are ubiquitin ligases. The functional similarity of the MARCH family and the K3 family suggests that the viral immune evasion proteins were derived from MARCH proteins, a novel family of transmembrane ubiquitin ligases that seems to target glycoproteins for lysosomal destruction via ubiquitination of the cytoplasmic tail

The Poxviral RING Protein p28 Is a Ubiquitin Ligase That Targets Ubiquitin to Viral Replication Factories

We conducted an exhaustive search for three-dimensional structural homologs to the proteins of 20 key phylogenetically distinct nucleocytoplasmic DNA viruses (NCLDV). Structural matches covered 429 known protein domain superfamilies, with the most highly represented being ankyrin repeat, P-loop NTPase, F-box, protein kinase, and membrane occupation and recognition nexus (MORN) repeat. Domain superfamily diversity correlated with genome size, but a diversity of around 200 superfamilies appeared to correlate with an abrupt switch to paralogization. Extensive structural homology was found across the range of eukaryotic RNA polymerase II subunits and their associated basal transcription factors, with the coordinated gain and loss of clusters of subunits on a virus-by-virus basis. The total number of predicted endonucleases across the 20 NCLDV was nearly quadrupled from 36 to 132, covering much of the structural and functional diversity of endonucleases throughout the biosphere in DNA restriction, repair, and homing. Unexpected findings included capsid protein-transcription factor chimeras; endonuclease chimeras; enzymes for detoxification; antimicrobial peptides and toxin-antitoxin systems associated with symbiosis, immunity, and addiction; and novel proteins for membrane abscission and protein turnover.IMPORTANCE We extended the known annotation space for the NCLDV by 46%, revealing high-probability structural matches for fully 45% of the 9,671 query proteins and confirming up to 98% of existing annotations per virus. The most prevalent protein families included ankyrin repeat- and MORN repeat-containing proteins, many of which included an F-box, suggesting extensive host cell modulation among the NCLDV. Regression suggested a minimum requirement for around 36 protein structural superfamilies for a viable NCLDV, and beyond around 200 superfamilies, genome expansion by the acquisition of new functions was abruptly replaced by paralogization. We found homologs to herpesvirus surface glycoprotein gB in cytoplasmic viruses. This study provided the first prediction of an endonuclease in 10 of the 20 viruses examined; the first report in a virus of a phenolic acid decarboxylase, proteasomal subunit, or cysteine knot (defensin) protein; and the first report of a prokaryotic-type ribosomal protein in a eukaryotic virus

The Poxviral RING Protein p28 Is a Ubiquitin Ligase That Targets Ubiquitin to Viral Replication Factories

The poxviral RING protein p28 is a virulence factor whose molecular function is unknown. Many cellular RING-containing proteins act as ubiquitin ligases (RING-E3s) connecting selected substrate proteins to the ubiquitination machinery. Here we demonstrate that vaccinia virus p28 and its homologue in myxoma virus, M143R, can mediate the formation of polyubiquitin conjugates, while RING mutants of both p28 and M143R cannot. Furthermore, p28 is ubiquitinated in vivo and ubiquitin colocalizes with p28 to virus factories independently of an intact RING domain. These results implicate the ubiquitin system in poxviral virulence

Kaposi sarcoma herpesvirus K5 removes CD31/PECAM from endothelial cells

The transmembrane ubiquitin ligase K5/MIR2 of Kaposi sarcoma herpesvirus (KSHV) mediates internalization and lysosomal degradation of glycoproteins involved in antigen presentation and co-stimulation. In endothelial cells (ECs), K5 additionally reduced expression of CD31/platelet–endothelial cell adhesion molecule (PECAM), an adhesion molecule regulating cell-cell interactions of ECs, platelets, monocytes, and T cells. K5 also reduced EC migration, a CD31-dependent process. Unlike other K5 substrates, both newly synthesized and pre-existing CD31 molecules were targeted by K5. K5 was transported to the cell surface and ubiquitinated pre-existing CD31, resulting in endocytosis and lysosomal degradation. In the endoplasmic reticulum, newly synthesized CD31 was degraded by proteasomes, which required binding of phosphofurin acidic cluster sorting protein-2 (PACS-2) to acidic residues in the carboxyterminal tail of K5. Thus, CD31, a novel target of K5, is efficiently removed from ECs by a dual degradation mechanism that is regulated by the subcellular sorting of the ubiquitin ligase. K5-mediated degradation of CD31 is likely to affect EC function in KS tumors

The PHD/LAP-Domain Protein M153R of Myxomavirus Is a Ubiquitin Ligase That Induces the Rapid Internalization and Lysosomal Destruction of CD4

The genomes of several poxviruses contain open reading frames with homology to the K3 and K5 genes of Kaposi's sarcoma-associated herpesvirus (KSHV) and the K3 gene of murine gammaherpesvirus 68, which target major histocompatibility complex class I (MHC-I) as well as costimulatory molecules for proteasomal or lysosomal degradation. The homologous gene product of myxomavirus (MV), M153R, was recently shown to reduce the cell surface expression of MHC-I. In addition, normal MHC-I surface expression was observed in cells infected with MV lacking M153R (J. L. Guerin, J. Gelfi, S. Boullier, M. Delverdier, F. A. Bellanger, S. Bertagnoli, I. Drexler, G. Sutter, and F. Messud-Petit, J. Virol. 76:2912-2923, 2002). Here, we show that M153R also downregulates the T-cell coreceptor CD4 and we study the molecular mechanism by which M153R achieves the downregulation of CD4 and MHC-I. Upon M153R expression, CD4 was rapidly internalized and degraded in lysosomes, whereas deletion of M153R from the genome of MV restored CD4 expression. The downregulation of both CD4 and MHC-I was dependent on the presence of lysine residues in their cytoplasmic tails. Increased ubiquitination of CD4 was observed upon coexpression with M153R in the presence of inhibitors of lysosomal acidification. Surface expression of CD4 was restored upon overexpression of Hrs, a ubiquitin interaction motif-containing protein that sorts ubiquitinated proteins into endosomes. Moreover, the purified PHD/LAP zinc finger of M153R catalyzed the formation of multiubiquitin adducts in vitro. Our data suggest that M153R acts as a membrane-bound ubiquitin ligase that conjugates ubiquitin to the cytoplasmic domain of substrate glycoproteins, with ubiquitin serving as a lysosomal targeting signal. Since a similar mechanism was recently proposed for KSHV K5, it seems that members of the unrelated families of gamma-2 herpesviruses and poxviruses share a common immune evasion mechanism that targets host cell immune receptors

Inhibitors of FAP-fluorogen interaction as a multiplex assay tool compound for receptor internalization assays

<p>A novel assay using fluorogen activating peptide (FAP) technology for G protein-coupled receptor (GPCR) activation and internalization was applied to the human β2AR. This technology avoids microscopy and antibody-based detection methods. A major goal for the project was to identify G-protein independent/β2AR ligands or β2AR biased ligands that induce β2AR internalization. Analysis of the most potent hits in the primary project revealed that they interfered with fluorogen activation by the FAP rather than interacting with the receptor itself. These molecules were pursued further because they had the potential to enable improved assay protocols to monitor receptor trafficking and receptor location in real time. A highly potent compound (ML342, CID 2953239) was declared as a Molecular Libraries Probe Center Network (MLPCN) probe molecule.</p

A Small Molecule Pan-Inhibitor of Ras-Superfamily GTPases with High Efficacy Towards Rab7

Low molecular weight guanine triphosphate hydrolases (GTPases) are GTP-binding enzymes that play pivotal roles in cell biology. Grouped into three subfamilies which are designated by function, Ras, Rho and Rab GTPases are involved in signal transduction, cytoskeleton modulation, and macromolecule cargo transport and degradation, respectively. Mutation and aberrant gene expression levels have been linked to human diseases including cancer, immunodeficiencies, and neurological disorders. A high through-put screen of the Molecular Libraries Small Molecule Repository (MLSMR) identified a compound, ML282, which potently inhibits GTPases from all three subfamilies. Importantly, this represents the discovery of the first reported compound to inhibit Rab GTPases, especially Rab7, the mutation of which is a causal factor in a heritable neuropathy called Charcot-Marie-Tooth Type 2B disease. ML282 inhibits Rab7 with an average EC50 = 53.2 ± 0.35 nM and with high response, as compared to other GTPases in the panel. The subsequent structure activity relationships (SAR) and secondary assays demonstrate its use as a molecular probe for both biochemical and cellular studies