Role for the phosphoprotein P subunit of the paramyxovirus polymerase in limiting induction of host cell antiviral responses

Six amino acid substitutions in the shared N-terminal region of the P subunit of the viral polymerase and the accessory V protein convert the noncytopathic paramyxovirus simian virus 5 (SV5), which is a poor inducer of host cell responses, into a P/V mutant (P/V-CPI-) that induces high levels of apoptosis, interferon-beta (IFN-beta), and proinflammatory cytokines. In this study, we addressed the question of whether these new mutant phenotypes are due to the presence of an altered P protein or of an altered V protein or of both proteins. By the use of the P/V-CPI- mutant as a backbone, new mutant viruses were engineered to express the wild-type (WT) V protein (+V-wt) or WT P protein (+P-wt) from an additional gene inserted between the HN and L genes. In human epithelial cell lines, the +V-wt virus showed reduced activation of apoptosis and lower secretion of IFN-beta and proinflammatory cytokines compared to the parental P/V-CPI- virus. The presence of a V protein lacking the C-terminal cysteine-rich domain (corresponding to the SV5 I protein) did not reduce these host cell responses to P/V-CPI- infection. Unexpectedly, the +P-wt virus, which expressed a WT P subunit of the viral polymerase, also induced much lower levels of host cell responses than the parental P/V-CPI- mutant. For both +V-wt and +P-wt viruses, reduced levels of IFN-beta synthesis correlated with reduced IRF-3 dimerization and nuclear localization of IRF-3 and NF-kappaB, suggesting that the WT P and V proteins acted at an early stage in antiviral pathways. Host cell responses induced by the various P/V mutants directly correlated with levels of viral mRNA accumulation but not with steady-state levels of genomic RNA. Our results support the hypothesis that WT P and V proteins limit induction of antiviral responses by controlling the production of key viral inducers. A model is presented for the mechanism by which both the P subunit of the viral polymerase and the V accessory protein contribute to the ability of a paramyxovirus to limit activation of antiviral responses.     

Transposon Tn7 directs transposition into the genome of filamentous bacteriophage M13 using the element-encoded TnsE protein

The bacterial transposon Tn7 has a pathway of transposition that preferentially targets conjugal plasmids. We propose that this same transposition pathway recognizes a structure or complex found during filamentous bacteriophage replication, likely by targeting negative-strand synthesis. The ability to insert into both plasmid and bacteriophage DNAs that are capable of cell-to-cell transfer would help explain the wide distribution of Tn7 relatives.     

Ancient population genomics and the study of evolution

Recently, the study of ancient DNA (aDNA) has been greatly enhanced by the development of second-generation DNA sequencing technologies and targeted enrichment strategies. These developments have allowed the recovery of several complete ancient genomes, a result that would have been considered virtually impossible only a decade ago. Prior to these developments, aDNA research was largely focused on the recovery of short DNA sequences and their use in the study of phylogenetic relationships, molecular rates, species identification and population structure. However, it is now possible to sequence a large number of modern and ancient complete genomes from a single species and thereby study the genomic patterns of evolutionary change over time. Such a study would herald the beginnings of ancient population genomics and its use in the study of evolution. Species that are amenable to such large-scale studies warrant increased research effort. We report here progress on a population genomic study of the Adélie penguin (Pygoscelis adeliae). This species is ideally suited to ancient population genomic research because both modern and ancient samples are abundant in the permafrost conditions of Antarctica. This species will enable us to directly address many of the fundamental questions in ecology and evolution.     

Biogenesis and cytotoxicity of APOL1 renal risk variant proteins in hepatocytes and hepatoma cells

Two APOL1 gene variants, which likely evolved to protect individuals from African sleeping sickness, are strongly associated with nondiabetic kidney disease in individuals with recent African ancestry. Consistent with its role in trypanosome killing, the pro-death APOL1 protein is toxic to most cells, but its mechanism of cell death is poorly understood and little is known regarding its intracellular trafficking and secretion. Because the liver appears to be the main source of circulating APOL1, we examined its secretory behavior and mechanism of toxicity in hepatoma cells and primary human hepatocytes. APOL1 is poorly secreted in vitro, even in the presence of chemical chaper­ones; however, it is efficiently secreted in wild-type transgenic mice, suggesting that APOL1 secretion has specialized requirements that cultured cells fail to support. In hepatoma cells, inducible expression of APOL1 and its risk variants promoted cell death, with the G1 variant displaying the highest degree of toxicity. To explore the basis for APOL1-mediated cell toxicity, endoplasmic reticulum stress, pyroptosis, autophagy, and apoptosis were examined. Our results suggest that autophagy represents the predominant mechanism of APOL1-mediated cell death. Overall, these results increase our understanding of the basic biology and trafficking behavior of circulating APOL1 from the liver.     

Detecting non-allelic homologous recombination from high-throughput sequencing data

Non-allelic homologous recombination (NAHR) is a common mechanism for generating genome rearrangements and is implicated in numerous genetic disorders, but its detection in high-throughput sequencing data poses a serious challenge. We present a probabilistic model of NAHR and demonstrate its ability to find NAHR in low-coverage sequencing data from 44 individuals. We identify NAHR-mediated deletions or duplications in 109 of 324 potential NAHR loci in at least one of the individuals. These calls segregate by ancestry, are more common in closely spaced repeats, often result in duplicated genes or pseudogenes, and affect highly studied genes such as GBA and CYP2E1.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0633-1) contains supplementary material, which is available to authorized users.     

Excessive hepatic mitochondrial TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease

Approximately one-third of the U.S. population has nonalcoholic fatty liver disease (NAFLD), a condition closely associated with insulin resistance and increased risk of liver injury. Dysregulated mitochondrial metabolism is central in these disorders, but the manner and degree of dysregulation are disputed. This study tested whether humans with NAFLD have abnormal in vivo hepatic mitochondrial metabolism. Subjects with low (3.0%) and high (17%) intrahepatic triglyceride (IHTG) were studied using (2)H and (13)C tracers to evaluate systemic lipolysis, hepatic glucose production, and mitochondrial pathways (TCA cycle, anaplerosis, and ketogenesis). Individuals with NAFLD had 50% higher rates of lipolysis and 30% higher rates of gluconeogenesis. There was a positive correlation between IHTG content and both mitochondrial oxidative and anaplerotic fluxes. These data indicate that mitochondrial oxidative metabolism is ~2-fold greater in those with NAFLD, providing a potential link between IHTG content, oxidative stress, and liver damage.