Stable Deletion Mutants Research Articles

Biallelic Deleterious Germline SH2B3 Variants Cause a Novel Clinical Syndrome of Myeloproliferation and Multi-Organ Autoimmunity

SH2B3 is a negative regulator of cytokine receptor signalling pathways in hematopoietic tissue. Acquired deleterious SH2B3 variants resulting in increased JAK/STAT signalling are observed in myeloproliferative neoplasms (MPN) however the clinical implications and phenotype of deleterious germline variants in SH2B3 are less clear. A single consanguineous kindred has been previously described with homozygous deleterious germline SH2B3 variants presenting with hepatosplenomegaly, autoimmune hepatitis, developmental delay, autoimmune thyroiditis and acute lymphoblastic leukemia (Perez-Garcia, A., et al, Blood, 2013), however a clinically valid gene-disease pair according to formal criteria has not been met to date for SH2B3. We identified two probands from unrelated kindred with germline homozygous deleterious SH2B3 variants. The first patient presented with splenomegaly, thrombocytosis, neutrophilia and a leukoerythroblastic blood film at 3 months old. He developed alopecia areata at 2 years and autoimmune hypothyroidism at 6 years. He was otherwise developmentally normal without any detected liver abnormalities or thrombotic episodes. Whilst his splenomegaly and neutrophil count gradually improved throughout life, his platelet count remained markedly elevated (~1000x109/L at 18 years of age). Targeted NGS identified a homozygous frameshift variant in SH2B3 (NM_005475.3:c.441_468del; p.(Arg148Profs*40)). Both parents were heterozygous carriers of this variant. The second patient was a male born to consanguineous parents and noted shortly after birth to have isolated splenomegaly. He was diagnosed with autoimmune hypothyroidism and Raynaud syndrome at 12 years and was noted to have a persistent thrombocytosis ranging from 550 - 780 x109/L. He developed autoimmune hepatitis and insulin dependent diabetes at 17 years and around the same time he also suffered a right-sided middle cerebral artery territory infarct with a right carotid artery thrombus. Whole genome sequencing detected a homozygous missense variant in SH2B3 (NM_005475.3:c.1204G>A; p.(Val402Met)) in the patient with both parents being heterozygous carriers. In order to understand these variants further we modelled the human p.(Arg148Profs*40) variant from patient 1 in zebrafish. CRISPR-Cas9 gene editing was targeted to the analogous region of zebrafish sh2b3 to create assorted carboxyl-truncating mutations in F0 crispants. On-target gene-editing was verified by Sanger sequencing and NGS. F0 crispants had a significantly increased number of macrophages and CD41-expressing cells (HSCs and thrombocytes), replicating the patient phenotype. Treatment of the sh2b3 crispant fish with the JAK inhibitor ruxolitinib intercepted the myeloproliferative phenotype. The myeloproliferative phenotype was not observed in stable deletion mutants, likely reflecting genetic compensation not present in crispants. Skin-derived fibroblasts from patient 2 who carried the Val402Met homozygous variant, showed increased phosphorylation of JAK2, STAT5 and STAT3 by immunoblotting after stimulation with IL3, GH, GM-CSF and EPO compared to healthy controls. In conclusion, these newly described kindreds showing striking phenotypic similarity to each other as well as the previously described kindred and in conjunction with our functional data provide sufficient evidence for biallelic homozygous deleterious variants in SH2B3 to be considered a valid gene-disease association with a clinical syndrome of bone marrow myeloproliferation and multi-organ autoimmune manifestations. Notably, neither of our patients had evidence of acute lymphoblastic leukemia or overt hematological malignancy and further affected individuals will need to be described in order to fully understand the breadth of the phenotype. Moreover, our data supports ruxolitinib as being a potential therapeutic option for patients with this rare novel immune dysregulatory syndrome.

CR4 Signaling Contributes to a DC-Driven Enhanced Immune Response Against Complement-Opsonized HIV-1.

Dendritic cells (DCs) possess intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. In turn, HIV-1 has evolved strategies to evade innate immune sensing by DCs resulting in suboptimal maturation and poor antiviral immune responses. We previously showed that complement-opsonized HIV-1 (HIV-C) was able to efficiently infect various DC subsets significantly higher than non-opsonized HIV-1 (HIV) and therefore also mediate a higher antiviral immunity. Thus, complement coating of HIV-1 might play a role with respect to viral control occurring early during infection via modulation of DCs. To determine in detail which complement receptors (CRs) expressed on DCs was responsible for infection and superior pro-inflammatory and antiviral effects, we generated stable deletion mutants for the α-chains of CR3, CD11b, and CR4, CD11c using CRISPR/Cas9 in THP1-derived DCs. We found that CD11c deletion resulted in impaired DC infection as well as antiviral and pro-inflammatory immunity upon exposure to complement-coated HIV-1. In contrast, sole expression of CD11b on DCs shifted the cells to an anti-inflammatory, regulatory DC type. We here illustrated that CR4 comprised of CD11c and CD18 is the major player with respect to DC infection associated with a potent early pro-inflammatory immune response. A more detailed characterization of CR3 and CR4 functions using our powerful tool might open novel avenues for early therapeutic intervention during HIV-1 infection.

Generation of a stable thrombin-activatable fibrinolysis inhibitor deletion mutant exerting full carboxypeptidase activity without activation

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a zymogen that can be activated to form activated TAFI (TAFIa) (Ala93-Val401) through removal of the N-terminal activation peptide (Phe1-Arg92). TAFIa is thermally unstable, and the role of the activation peptide in the activity and stability of TAFI zymogen remains unclear. To better understand the role of the activation peptide in the activity and stability of TAFI. We constructed a deletion mutant, TAFI-CIIYQ-∆1-73 , in which the first 73 amino acids of the activation peptide are absent. The intrinsic activity and functional stability were determined with a chromogenic assay. The activation of TAFI-CIIYQ-∆1-73 by TAFI activators was evaluated with western blot analysis. In comparison with TAFI-CIIYQ, the deletion mutant exerted high intrinsic activity ('full' apparent TAFIa activity) without cleavage by TAFI activators. TAFI-CIIYQ-∆1-73 was cleavable by thrombin. However, in the presence of thrombomodulin, the thrombin-mediated cleavage of TAFI-CIIYQ-∆1-73 was not accelerated. TAFI-CIIYQ-∆1-73 showed a similar functional stability profile to that of TAFI-CIIYQ. Full cleavage by thrombin did not affect the apparent carboxypeptidase activity of TAFI-CIIYQ-∆1-73 , but resulted in a significant loss of functional stability. A stable deletion mutant of TAFI with full carboxypeptidase activity without activation is described. The segment Ala74-Arg92 in the activation peptide contributes significantly to the role of the activation peptide in stabilization of the catalytic moiety in TAFI zymogen.

Expression of the highly conserved vaccinia virus E6 protein is required for virion morphogenesis

The vaccinia virus E6R gene (VACVWR062) is conserved in all members of the poxvirus family and encodes a protein associated with the mature virion. We confirmed this association and provided evidence for an internal location. An inducible mutant that conditionally expresses E6 was constructed. In the absence of inducer, plaque formation and virus production were severely inhibited in several cell lines, whereas some replication occurred in others. This difference could be due to variation in the stringency of repression, since we could not isolate a stable deletion mutant even in the more “permissive” cells. Under non-permissive conditions, viral late proteins were synthesized but processing of core proteins was inefficient, indicative of an assembly block. Transmission electron microscopy of sections of cells infected with the mutant in the absence of inducer revealed morphogenetic defects with crescents and empty immature virions adjacent to dense inclusions of viroplasm. Mature virions were infrequent and cores appeared to have lucent centers.

The Sulfolobus solfataricus AAA protein Sso0909, a homologue of the eukaryotic ESCRT Vps4 ATPase

Sso0909 is a protein of the thermo-acidophilic crenarchaeon Sulfolobus solfataricus, annotated as a p60 katanin-like ATPase. We present here results supporting the hypothesis that Sso0909 is an orthologue of the eukaryotic ESCRT (endosomal sorting complex required for transport) ATPase Vps4 (vacular protein sorting 4). The spectrum of Sso0909 homologues is limited to several orders of Crenarchaea and to three euryarchaeal Thermoplasmata species, where they were presumably acquired by lateral gene transfer. Almost invariably, Sso0909 homologues occur in the genomic vicinity of homologues of eukaryotic ESCRT-III components, which are the targets of disassembly by Vps4, as well as with a creanarchaeal-specific coiled-coil protein. S. solfataricus sso0909 is constitutively expressed under normal growth conditions and appears to be essential, as judged by the failure to obtain stable deletion mutants. We expressed Sso0909 in Escherichia coli and S. solfataricus, but have not obtained preparations with ATPase activity so far.

Stable deletions arising in the readthrough region of Soil-borne wheat mosaic virus RNA2 define the 5′ limit of the functional promoter for the p19 subgenomic RNA

The appearance of de novo deletion mutations in the readthrough (RT) region (nucleotide positions 861-2591) downstream of the capsid protein (CP) gene of a Japanese strain of Soil-borne wheat mosaic virus RNA2 was examined using infectious transcripts. Mutant RNA2s with different deletions predominated in independent serial passage experiments but all best-adapted mutants retained the 3'-terminal portion of the RT gene in frame with the CP gene. The longest best-adapted mutation deleted the 1434 nucleotides between positions 1061 and 2494. When the RT protein was truncated by insertion of a termination codon plus an additional nucleotide to give a +1 frame-shift, after serial passages the progeny viruses regained the ability to express the C-terminal region of RT by an internal deletion. The 5' terminus of the p19 subgenomic RNA was identified at position 2598 and an essential transcription signal for this mRNA mapped between positions 2534 and 2563. A mutant in which this essential promoter element has been deleted cannot transcribe the p19 subgenomic RNA and has lost infectivity in planta. These results indicate that the 3'-terminal region of the RT gene has a major function in cis for expression of p19, which is essential for infecting plants. A reason for retaining the RT C-terminal region in stable deletion mutants is still unknown.

Non-AUG translation initiation in a plant RNA virus: a forty-amino-acid extension is added to the N terminus of the soil-borne wheat mosaic virus capsid protein.

RNA 2 of soil-borne wheat mosaic virus (SBWMV), the type species of the genus Furovirus, encodes a protein previously hypothesized to be initiated at an in-frame non-AUG codon upstream of the AUG initiation codon (nucleotide positions 334 to 336) for the 19-kDa capsid protein. Site-directed mutagenesis and in vitro transcription and translation analysis indicated that CUG (nucleotides 214 to 216) is the initiation codon for a protein with a calculated molecular mass of 25 kDa composed of a 40-amino-acid extension to the N terminus of the 19-kDa capsid protein. A stable deletion mutant, which was isolated after extensive passages of a wild-type SBWMV, contained a mixture of two deleted RNA 2's, only one of which coded for the 25-kDa protein. The amino acid sequence of the N-terminal extension was moderately conserved and the CUG initiation codon was preserved among three SBWMV isolates from Japan and the United States. This amino acid sequence conservation, as well as the retention of expression of the 25-kDa protein in the stable deletion mutant, suggests that the 25-kDa protein is functional in the life cycle of SBWMV. This is the first report of a non-AUG translation initiation in a plant RNA virus genome.

Sequence analysis of wheat and oat furovirus capsid protein genes suggests that oat golden stripe virus is a strain of soil-borne wheat mosaic virus

In northern blots, cDNA probes prepared to soil-borne wheat mosaic virus (SBWMV) RNA-1 and RNA-2 hybridized to RNA-1 and RNA-2, respectively, from a UK isolate of oat golden stripe virus (OGSV), as well as to their homologous RNAs. RT-PCR was used to amplify, clone and sequence a region of about 750 nucleotides spanning the capsid protein gene and part of the readthrough protein on RNA-2 from OGSV, a French isolate of SBWMV and two stable deletion mutants (Lab1 and Okl-7) of SBWMV isolates from Nebraska and Oklahoma respectively. There was very high (96.7–99.1%) nucleotide homology between all these sequences and the wild-type SBWMV sequences from Nebraska and Oklahoma. OGSV was more similar to SBWMV from France and Nebraska than were any of the isolates to SBWMV from Oklahoma. Of the few differences in the deduced amino acid sequences of the capsid proteins from the different isolates, OGSV differed from all SBWMV isolates only in one amino acid (isoleucine for valine at position 88). The high degree of similarity suggests that OGSV may best be classified as an oat strain of SBWMV.

Use of phenotypic suppression for direct selection of loss of aminoglycoside antibiotic resistance determinants.

A strain of Escherichia coli containing a conditional drug dependent arginine auxotrophy was used to select for the loss of plasmid and/or transposon encoded kanamycin (Km) or streptomycin (Sm) resistance determinants. Because these determinants inactivate the corresponding drug thus eliminating drug suppression, loss of the drug-resistance determinants was selected directly by growth on minimal media plates containing sub-lethal dosages of the drug. This method was used to select loss of Km or Sm resistance determinants due to loss of plasmids, transposition from plasmid to chromosome, and education of transposons from the chromosome. Drug suppression was compared to phage PRD1 resistance in selecting for loss of plasmid vehicles during transposition and was found to be 10-1,000 times more efficient. Eighty percent of the eductant clones had undergone imprecise eductions suggesting that this method may be useful in selecting stable deletion mutants. An antibiotic suppressible strain of Pseudomonas stutzeri was obtained implying a broad utility of this selection procedure.

Tumor Antigen Induction by the Envelope-Defective Bryan Strain of Rous Sarcoma Viruses 2

Chicken, quail, and turkey cells were infected and/or transformed by various avian leukosis or sarcoma oncovirus (ALSV) strains as well as by the envelope-defective Bryan high-titer strain of Rous sarcoma virus [BH-RSV(-)]. All fibroblast-transforming avian sarcoma viruses (ASV), including BH-RSV(-), were able to induce the expression of the previously described avian tumor-specific cell-surface antigen(s) (TSSA). Thus TSSA was group-specific for all tested transforming ASV. Since BH-RSV(-) is a stable deletion mutant lacking the capacity to code for the 85 000d major virus envelope glycoprotein (gp85), it seemed unlikely that the group-specific antigen determinants of gp85 that were recently detected on ALSV-infected cells could account for TSSA expression.