PI3-kinase deletion promotes myelodysplasia by dysregulating autophagy in hematopoietic stem cells.

Myelodysplastic syndrome (MDS) is a clonal malignancy arising in hematopoietic stem cells (HSCs). The mechanisms of MDS initiation in HSCs are still poorly understood. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is frequently activated in acute myeloid leukemia, but in MDS, PI3K/AKT is often down-regulated. To determine whether PI3K down-regulation can perturb HSC function, we generated a triple knockout (TKO) mouse model with Pik3ca, Pik3cb, and Pik3cd deletion in hematopoietic cells. Unexpectedly, PI3K deficiency caused cytopenias, decreased survival, and multilineage dysplasia with chromosomal abnormalities, consistent with MDS initiation. TKO HSCs exhibit impaired autophagy, and pharmacologic autophagy induction improved HSC differentiation. Using intracellular LC3 and P62 flow cytometry and transmission electron microscopy, we also observed abnormal autophagic degradation in patient MDS HSCs. Therefore, we have uncovered an important protective role for PI3K in maintaining autophagic flux in HSCs to preserve the balance between self-renewal and differentiation and to prevent MDS initiation.

Zinc-finger antiviral protein (ZAP) is a restriction factor for replication of modified vaccinia virus Ankara (MVA) in human cells.

Modified vaccinia virus Ankara (MVA) is an approved smallpox vaccine and a promising vaccine vector for other pathogens as well as for cancer therapeutics with more than 200 current or completed clinical trials. MVA was derived by passaging the parental Ankara vaccine virus hundreds of times in chick embryo fibroblasts during which it lost the ability to replicate in human and most other mammalian cells. Although this replication deficiency is an important safety feature, the genetic basis of the host restriction is not understood. Here, an unbiased human genome-wide RNAi screen in human A549 cells revealed that the zinc-finger antiviral protein (ZAP), previously shown to inhibit certain RNA viruses, is a host restriction factor for MVA, a DNA virus. Additional studies demonstrated enhanced MVA replication in several human cell lines following knockdown of ZAP. Furthermore, CRISPR-Cas9 knockout of ZAP in human A549 cells increased MVA replication and spread by more than one log but had no effect on a non-attenuated strain of vaccinia virus. The intact viral C16 protein, which had been disrupted in MVA, antagonized ZAP by binding and sequestering the protein in cytoplasmic punctate structures. Studies aimed at exploring the mechanism by which ZAP restricts MVA replication in the absence of C16 showed that knockout of ZAP had no discernible effect on viral DNA or individual mRNA or protein species as determined by droplet digital polymerase chain reaction, deep RNA sequencing and mass spectrometry, respectively. Instead, inactivation of ZAP reduced the number of aberrant, dense, spherical particles that typically form in MVA-infected human cells, suggesting that ZAP has a novel role in interfering with a late step in the assembly of infectious MVA virions in the absence of the C16 protein.

Human Host Range Restriction of the Vaccinia Virus C7/K1 Double Deletion Mutant Is Mediated by an Atypical Mode of Translation Inhibition.

Replication of vaccinia virus in human cells depends on the viral C7 or K1 protein. A previous human genome-wide short interfering RNA (siRNA) screen with a C7/K1 double deletion mutant revealed SAMD9 as a principal host range restriction factor along with additional candidates, including WDR6 and FTSJ1. To compare their abilities to restrict replication, the cellular genes were individually inactivated by CRISPR/Cas9 mutagenesis. The C7/K1 deletion mutant exhibited enhanced replication in each knockout (KO) cell line but reached wild-type levels only in SAMD9 KO cells. SAMD9 was not depleted in either WDR6 or FTSJ1 KO cells, suggesting less efficient alternative rescue mechanisms. Using the SAMD9 KO cells as controls, we verified a specific block in host and viral intermediate and late protein synthesis in HeLa cells and demonstrated that the inhibition could be triggered by events preceding viral DNA replication. Inhibition of cap-dependent and -independent protein synthesis occurred primarily at the translational level, as supported by DNA and mRNA transfection experiments. Concurrent with collapse of polyribosomes, viral mRNA was predominantly in 80S and lighter ribonucleoprotein fractions. We confirmed the accumulation of cytoplasmic granules in HeLa cells infected with the C7/K1 deletion mutant and further showed that viral mRNA was sequestered with SAMD9. RNA granules were still detected in G3BP KO U2OS cells, which remained nonpermissive for the C7/K1 deletion mutant. Inhibition of cap-dependent and internal ribosome entry site-mediated translation, sequestration of viral mRNA, and failure of PKR, RNase L, or G3BP KO cells to restore protein synthesis support an unusual mechanism of host restriction.IMPORTANCE A dynamic relationship exists between viruses and their hosts in which each ostensibly attempts to exploit the other's vulnerabilities. A window is opened into the established condition, which evolved over millennia, if loss-of-function mutations occur in either the virus or host. Thus, the inability of viral host range mutants to replicate in specific cells can be overcome by identifying and inactivating the opposing cellular gene. Here, we investigated a C7/K1 host range mutant of vaccinia virus in which the cellular gene SAMD9 serves as the principal host restriction factor. Host restriction was triggered early in infection and manifested as a block in translation of viral mRNAs. Features of the block include inhibition of cap-dependent and internal ribosome entry site-mediated translation, sequestration of viral RNA, and inability to overcome the inhibition by inactivation of protein kinase R, ribonuclease L, or G3 binding proteins, suggesting a novel mechanism of host restriction.