Stimulation of baculovirus transcriptome expression in mammalian cells by baculoviral transcriptional activators

Autographa californica multiple n ucleopolyhedro virus (AcMNPV), the type species of the family Baculoviridae, is an insect-specific virus that can enter a variety of mammalian cells. The potential of this versatile virus for protein expression or gene therapy in mammalian cells has become the focus of many studies. In most mammalian cells, transduced AcMNPV genes are either not expressed or expressed at an extremely low level. Here, we studied the effects of the two major AcMNPV trans-activators, IE1 and IE2, on the activation of AcMNPV genome in Vero E6 cells. Microarray analysis showed that when IE1 was overexpressed, it significantly activated genes gp64 and pe38, and upregulated ie2, he65, pcna, orf16, orf17 and orf25. Although, there were only two genes, pe38 and orf 17, that were activated by IE2, we discovered interestingly that the combination of IE1 and IE2 factors had a synergistic effect on activation of the AcMNPV genome in mammalian cells, and activated around 38%, or 59 out of the 155 genes placed on the microarray. This is the first detailed study of baculoviral transcription regulation in mammalian cells, and it shows that the baculoviral genome can be activated in a mammalian system, and also that the two major trans-activators, IE1 and IE2, play a central role in this activation

A Non-coding RNA of Insect HzNV-1 Virus Establishes Latent Viral Infection through MicroRNA

Heliothis zea nudivirus-1 (HzNV-1) is an insect virus previously known as Hz-1 baculovirus. One of its major early genes, hhi1, is responsible for the establishment of productive viral infection; another gene, pag1, which expresses a non-coding RNA, is the only viral transcript detectable during viral latency. Here we showed that this non-coding RNA was further processed into at least two distinct miRNAs, which targeted and degraded hhi1 transcript. This is a result strikingly similar to a recent report that herpes simplex virus produces tightly-regulated latent specific miRNAs to silence its own key early transcripts. Nevertheless, proof for the establishment of viral latency by miRNA is still lacking. We further showed that HzNV-1 latency could be directly induced by pag1-derived miRNAs in cells infected with a pag1-deleted, latency-deficient virus. This result suggests the existence of a novel mechanism, where miRNAs can be functional for the establishment of viral latency

Heliothis zea Nudivirus 1 Gene hhi1 Induces Apoptosis Which Is Blocked by the Hz-iap2 Gene and a Noncoding Gene, pag1

Heliothis zea nudivirus 1 (HzNV-1 or Hz-1 virus), previously regarded as a nonoccluded baculovirus, recently has been placed in the Nudivirus genus. This virus generates HzNV-1 HindIII-I 1 (hhi1) and many other transcripts during productive viral infection; during latent viral infection, however, persistency-associated gene 1 (pag1) is the only gene expressed. In this report, we used transient expression assays to show that hhi1 can trigger strong apoptosis in transfected cells, which can be blocked, at least partially, by the inhibitor of apoptosis genes Autographa californica iap2 (Ac-iap2) and H. zea iap2 (Hz-iap2). In addition to these two genes, unexpectedly, pag1, which encodes a noncoding RNA with no detectable protein product, was found to efficiently suppress hhi1-induced apoptosis. The assay of pro-Sf-caspase-1 processing by hhi1 transfection did not detect the small P12 subunit at any of the time intervals tested, suggesting that hhi1 of HzNV-1 induces apoptosis through alternative caspase pathways

Cooperation of ie1 and p35 genes in the activation of baculovirus AcMNPV and HzNV-1 promoters

HzNV-1 is a non-occluded virus belongs to the family of the baculovirus. One of the first detectable transcripts expressed by HzNV-1 virus infection is a 6.2 kb gene, hhi1, located in the HindIII-I fragment of the viral genome. Here we show that infection of baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) could activate the expression of the hhi1 promoter. By using constructs containing progressive deletions of the upstream regulatory regions of the hhi 1 gene, we demonstrated that the most highly activated area was located between nucleotides -62 to +277 of the hhi1 promoter. We subsequently searched the entire 130 kb AcMNPV genome and identified two baculovirus genes, ie1 and p35, that their cooperation is required for the activation of the hhi I promoter. Further, by taking advantages of a baculovirus DNA chip and low background baculovirus gene expressions in the mammalian cells, we went on to identify a specific set of baculoviral genes, including orf21 and orf25, that could be specifically activated by the combination of ie1 and p35 genes. We conclude that a unique cooperative mechanism of ie1 and p35 exists in the genome of AcMNPV, which can activate the expression of a specific set of AcMNPV and HzNV-1 promoters. (C) 2008 Elsevier B.V. All rights reserved

A Roadmap for Human Liver Differentiation from Pluripotent Stem Cells

10.1016/j.celrep.2018.01.087Cell Reports2282190-220

Exome chip meta-analysis identifies novel loci and East Asian-specific coding variants that contribute to lipid levels and coronary artery disease.

Most genome-wide association studies have been of European individuals, even though most genetic variation in humans is seen only in non-European samples. To search for novel loci associated with blood lipid levels and clarify the mechanism of action at previously identified lipid loci, we used an exome array to examine protein-coding genetic variants in 47,532 East Asian individuals. We identified 255 variants at 41 loci that reached chip-wide significance, including 3 novel loci and 14 East Asian-specific coding variant associations. After a meta-analysis including >300,000 European samples, we identified an additional nine novel loci. Sixteen genes were identified by protein-altering variants in both East Asians and Europeans, and thus are likely to be functional genes. Our data demonstrate that most of the low-frequency or rare coding variants associated with lipids are population specific, and that examining genomic data across diverse ancestries may facilitate the identification of functional genes at associated loci