Comprehensive Rare Variant Analysis via Whole-Genome Sequencing to Determine the Molecular Pathology of Inherited Retinal Disease

Inherited retinal disease is a common cause of visual impairment and represents a highly heterogeneous group of conditions. Here, we present findings from a cohort of 722 individuals with inherited retinal disease, who have had whole-genome sequencing (n = 605), whole-exome sequencing (n = 72), or both (n = 45) performed, as part of the NIHR-BioResource Rare Diseases research study. We identified pathogenic variants (single-nucleotide variants, indels, or structural variants) for 404/722 (56%) individuals. Whole-genome sequencing gives unprecedented power to detect three categories of pathogenic variants in particular: structural variants, variants in GC-rich regions, which have significantly improved coverage compared to whole-exome sequencing, and variants in non-coding regulatory regions. In addition to previously reported pathogenic regulatory variants, we have identified a previously unreported pathogenic intronic variant in $\textit{CHM}$ in two males with choroideremia. We have also identified 19 genes not previously known to be associated with inherited retinal disease, which harbor biallelic predicted protein-truncating variants in unsolved cases. Whole-genome sequencing is an increasingly important comprehensive method with which to investigate the genetic causes of inherited retinal disease.This work was supported by The National Institute for Health Research England (NIHR) for the NIHR BioResource – Rare Diseases project (grant number RG65966). The Moorfields Eye Hospital cohort of patients and clinical and imaging data were ascertained and collected with the support of grants from the National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital, National Health Service Foundation Trust, and UCL Institute of Ophthalmology, Moorfields Eye Hospital Special Trustees, Moorfields Eye Charity, the Foundation Fighting Blindness (USA), and Retinitis Pigmentosa Fighting Blindness. M.M. is a recipient of an FFB Career Development Award. E.M. is supported by UCLH/UCL NIHR Biomedical Research Centre. F.L.R. and D.G. are supported by Cambridge NIHR Biomedical Research Centre

Insight into Influenza Manipulation of its Target Cell and the Countermeasures Taken by the Host to Limit Infection

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2012.Influenza A viruses remain a major public health concern, as is evident by the occurrence of annual epidemics and occasional pandemics. Insight into the interactions that occur between influenza and its host will help to uncover targets for novel therapies. One approach is to better understand the cellular signaling events that occur following infection. Our examination of this revealed that mice deficient in the MLK3 kinase disrupted MAPK signaling and resulted in high viral titers present in the lung at late time points following infection. This was a consequence of prolonged survival of infected cells, which in turn were able to produce virus for an extended time. These data implicate MLK3 as a pro-survival regulator during influenza infection. Additionally, it is well established that following infection with influenza a dramatic reduction of cellular protein synthesis ensues, a phenomenon referred to as shutoff. Although evidence in the literature has suggested a role for the viral polymerase complex in this process, how influenza does this remains unknown. Therefore, to investigate the contribution of the polymerase complex to shutoff, we utilized reporter gene assays to evaluate the role of polymerase subunits on host protein expression. In this study, we found that the PA subunit is a critical factor in the initiation of shutoff. Of interest, in the strains that we characterized, PA of avianorigin, such as the pandemic H1N1, were more efficient at inducing shutoff than human-origin PA, such as WSN. This phenotype was confirmed in comparison of protein synthesis following infection with a recombinant WSN virus containing Cal PA and wild-type WSN. Chimeric analysis revealed that the activity of PA responsible for shutoff was localized to the amino terminal domain of this protein, with the lysine at position 134 being critically involved in the phenotype. Although this is the catalytic lysine within the endonuclease active site, endonuclease activity was not required for shutoff. Together, these results implicate PA as a critical determinant in the inhibition of cellular protein synthesis following infection. Overall, these findings uncover novel influenza-host interactions and protein functions that should be considered in the design of future therapeutic agents

Specific residues in the 2009 H1N1 swine-origin influenza matrix protein influence virion morphology and efficiency of viral spread in vitro.

In April 2009, a novel influenza virus emerged as a result of genetic reassortment between two pre-existing swine strains. This highly contagious H1N1 recombinant (pH1N1) contains the same genomic background as North American triple reassortant (TR) viruses except for the NA and M segments which were acquired from the Eurasian swine lineage. Yet, despite their high degree of genetic similarity, we found the morphology of virions produced by the pH1N1 isolate, A/California/04/09 (ACal-04/09), to be predominantly spherical by immunufluorescence and electron microscopy analysis in human lung and swine kidney epithelial cells, whereas TR strains were observed to be mostly filamentous. In addition, nine clinical pH1N1 samples collected from nasal swab specimens showed similar spherical morphology as the ACal-04/09 strain. Sequence analysis between TR and pH1N1 viruses revealed four amino acid differences in the viral matrix protein (M1), a known determinant of influenza morphology, at positions 30, 142, 207, and 209. To test the role of these amino acids in virus morphology, we rescued mutant pH1N1 viruses in which each of the four M1 residues were replaced with the corresponding TR residue. pH1N1 containing substitutions at positions 30, 207 and 209 exhibited a switch to filamentous morphology, indicating a role for these residues in virion morphology. Substitutions at these residues resulted in lower viral titers, reduced growth kinetics, and small plaque phenotypes compared to wild-type, suggesting a correlation between influenza morphology and efficient cell-to-cell spread in vitro. Furthermore, we observed efficient virus-like particle production from cells expressing wild-type pH1N1 M1, but not M1 containing substitutions at positions 30, 207, and 209, or M1 from other strains. These data suggest a direct role for pH1N1 specific M1 residues in the production and release of spherical progeny, which may contribute to the rapid spread of the pandemic virus

Identification of the N-terminal domain of the influenza virus PA responsible for the suppression of host protein synthesis.

Cellular protein synthesis is suppressed during influenza virus infection, allowing for preferential production of viral proteins. To explore the impact of polymerase subunits on protein synthesis, we coexpressed enhanced green fluorescent protein (eGFP) or luciferase together with each polymerase component or NS1 of A/California/04/2009 (Cal) and found that PA has a significant impact on the expression of eGFP and luciferase. Comparison of the suppressive activity on coexpressed proteins between various strains revealed that avian virus or avian-origin PAs have much stronger activity than human-origin PAs, such as the one from A/WSN/33 (WSN). Protein synthesis data suggested that reduced expression of coexpressed proteins is not due to PA's reported proteolytic activity. A recombinant WSN containing Cal PA showed enhanced host protein synthesis shutoff and induction of apoptosis. Further characterization of the PA fragment indicated that the N-terminal domain (PANt), which includes the endonuclease active site, is sufficient to suppress cotransfected gene expression. By characterizing various chimeric PANts, we found that multiple regions of PA, mainly the helix α4 and the flexible loop of amino acids 51 to 74, affect the activity. The suppressive effect of PANt cDNA was mainly due to PA-X, which was expressed by ribosomal frameshifting. In both Cal and WSN viruses, PA-X showed a stronger effect than the corresponding PANt, suggesting that the unique C-terminal sequences of PA-X also play a role in suppressing cotransfected gene expression. Our data indicate strain variations in PA gene products, which play a major role in suppression of host protein synthesis

PB2 Residue 271 Plays a Key Role in Enhanced Polymerase Activity of Influenza A Viruses in Mammalian Host Cells▿

The direct infection of humans with highly pathogenic avian H5N1 influenza viruses has suggested viral mutation as one mechanism for the emergence of novel human influenza A viruses. Although the polymerase complex is known to be a key component in host adaptation, mutations that enhance the polymerase activity of avian viruses in mammalian hosts are not fully characterized. The genomic comparison of influenza A virus isolates has identified highly conserved residues in influenza proteins that are specific to either human or avian viruses, including 10 residues in PB2. We characterized the activity of avian polymerase complexes containing avian-to-human mutations at these conserved PB2 residues and found that, in addition to the E627K mutation, the PB2 mutation T271A enhances polymerase activity in human cells. We confirmed the effects of the T271A mutation using recombinant WSN viruses containing avian NP and polymerase genes with wild-type (WT) or mutant PB2. The 271A virus showed enhanced growth compared to that of the WT in mammalian cells in vitro. The 271A mutant did not increase viral pathogenicity significantly in mice compared to that of the 627K mutant, but it did enhance the lung virus titer. Also, cell infiltration was more evident in lungs of 271A-infected mice than in those of the WT. Interestingly, the avian-derived PB2 of the 2009 pandemic H1N1 influenza virus has 271A. The characterization of the polymerase activity of A/California/04/2009 (H1N1) and corresponding PB2 mutants indicates that the high polymerase activity of the pandemic strain in mammalian cells is, in part, dependent on 271A. Our results clearly indicate the contribution of PB2 amino acid 271 to enhanced polymerase activity and viral growth in mammalian hosts

Amino acid differences between classical swine, Eurasian swine and the 2009 pH1N1 M1 proteins.

<p>Number of isolates containing the indicated residues/total number of isolates is indicated.</p

TR swine virus but not the pH1N1 strain induces filament formation from infected human lung and swine kidney cells.

<p>Human A549 and swine LLC-PK1 cells infected with the ACal-04/09 or AWisc05 were processed for visualization of viral surface proteins 18 hpi by IF microscopy using anti-H1N1 mouse serum followed by anti-mouse IgG-Texas Red.</p

Electron microscopy analysis of TR or pH1N1-infected A549 cells.

<p>(<b>A</b>) Mock infected A549 cells, and A549 cells infected with AWisc05 or ACal-04/09 were fixed at 18 h and processed for scanning electron microscopy. Bars measure 10 µm (top) and 1 µm (bottom). Arrows point to virions budding from infected cell surfaces. (<b>B</b>) Electron micrograph of negatively stained ACal-04/09 virion released from infected A549 cells.</p

Substitutions at positions 30, 207 and 209 in the ACal-04/09 M1 protein resulted in filament formation in infected cells.

<p>A549 cells were infected with ACal-04/09 viruses containing single or multiple substitutions at positions 30, 142, 207, and 209 in the M1 protein as indicated. Cells were fixed and processed for visualization of viral surface proteins 18 h by IF microscopy as described for Fig. 1.</p

Mutations of ACal-04/09 M1 at residues 30 or 207 and 209 reduced VLP production from transfected cells.

<p>(A) 293T cells were transfected with expression plasmids containing WT or mutant ACal-04/09, or other M1 genes. Purified radiolabeled VLPs in culture supernatants were analyzed by SDS-PAGE and total M1 in cell lysates was determined by Western blot analysis. (B) Band intensities were quantified using BioRad software. VLP production is shown as the amount of M1 released into cell culture supernatant normalized to M1 produced in cell lysates. ACal-04/09 WT was set to 1. Data shown are an average of three individual experiments. Error bars represent standard deviation.</p