Effect of Transgene Location, Transcriptional Control Elements and Transgene Features in Armed Oncolytic Adenoviruses

Clinical results with oncolytic adenoviruses (OAds) used as antitumor monotherapies show limited efficacy. To increase OAd potency, transgenes have been inserted into their genome, a strategy known as “arming OAds”. Here, we review different parameters that affect the outcome of armed OAds. Recombinant adenovirus used in gene therapy and vaccination have been the basis for the design of armed OAds. Hence, early region 1 (E1) and early region 3 (E3) have been the most commonly used transgene insertion sites, along with partially or complete E3 deletions. Besides transgene location and orientation, transcriptional control elements, transgene function, either virocentric or immunocentric, and even the codons encoding it, greatly impact on transgene levels and virus fitness.

Download Full-text

Adenovirus Modulates Toll-Like Receptor 4 Signaling by Reprogramming ORP1L-VAP Protein Contacts for Cholesterol Transport from Endosomes to the Endoplasmic Reticulum

Journal of Virology ◽

10.1128/jvi.01904-16 ◽

2017 ◽

Vol 91 (6) ◽

Cited By ~ 13

Author(s):

Nicholas L. Cianciola ◽

Stacey Chung ◽

Danny Manor ◽

Cathleen R. Carlin

Keyword(s):

Innate Immunity ◽

Endoplasmic Reticulum ◽

Membrane Protein ◽

Lipid Droplets ◽

Cholesterol Transport ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Human Adenoviruses ◽

Early Region ◽

Early Region 3

ABSTRACT Human adenoviruses (Ads) generally cause mild self-limiting infections but can lead to serious disease and even be fatal in high-risk individuals, underscoring the importance of understanding how the virus counteracts host defense mechanisms. This study had two goals. First, we wished to determine the molecular basis of cholesterol homeostatic responses induced by the early region 3 membrane protein RIDα via its direct interaction with the sterol-binding protein ORP1L, a member of the evolutionarily conserved family of oxysterol-binding protein (OSBP)-related proteins (ORPs). Second, we wished to determine how this interaction regulates innate immunity to adenovirus. ORP1L is known to form highly dynamic contacts with endoplasmic reticulum-resident VAP proteins that regulate late endosome function under regulation of Rab7-GTP. Our studies have demonstrated that ORP1L-VAP complexes also support transport of LDL-derived cholesterol from endosomes to the endoplasmic reticulum, where it was converted to cholesteryl esters stored in lipid droplets when ORP1L was bound to RIDα. The virally induced mechanism counteracted defects in the predominant cholesterol transport pathway regulated by the late endosomal membrane protein Niemann-Pick disease type C protein 1 (NPC1) arising during early stages of viral infection. However, unlike NPC1, RIDα did not reconstitute transport to endoplasmic reticulum pools that regulate SREBP transcription factors. RIDα-induced lipid trafficking also attenuated proinflammatory signaling by Toll-like receptor 4, which has a central role in Ad pathogenesis and is known to be tightly regulated by cholesterol-rich “lipid rafts.” Collectively, these data show that RIDα utilizes ORP1L in a way that is distinct from its normal function in uninfected cells to fine-tune lipid raft cholesterol that regulates innate immunity to adenovirus in endosomes. IMPORTANCE Early region 3 proteins encoded by human adenoviruses that attenuate immune-mediated pathology have been a particularly rich source of information regarding intracellular protein trafficking. Our studies with the early region 3-encoded RIDα protein also provided fundamental new information regarding mechanisms of nonvesicular lipid transport and the flow of molecular information at membrane contacts between different organelles. We describe a new pathway that delivers cholesterol from endosomes to the endoplasmic reticulum, where it is esterified and stored in lipid droplets. Although lipid droplets are attracting renewed interest from the standpoint of normal physiology and human diseases, including those resulting from viral infections, experimental model systems for evaluating how and why they accumulate are still limited. Our studies also revealed an intriguing relationship between lipid droplets and innate immunity that may represent a new paradigm for viruses utilizing these organelles.

Download Full-text

Abstract 1422: Candesartan-induced Upregulation Of Transcriptional Regulator Yin Yang 1 Blocks Leukocyte-endothelial Interaction Under Physiological Flow.

Circulation ◽

10.1161/circ.116.suppl_16.ii_292-c ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Hideto Ishii ◽

Daisuke Mori ◽

Mayuko Sato ◽

Masayuki Yoshida

Keyword(s):

Transcriptional Control ◽

Recombinant Adenovirus ◽

Recent Observation ◽

Umbilical Vein ◽

Dominant Role ◽

Transcriptional Regulator ◽

Potential Contribution ◽

Flow Conditions ◽

Yin Yang 1 ◽

Yin Yang

[Objective] Transcriptional control of inflammation-relevant genes plays an important role in atherosclerosis. We have reported a dominant role of Yin Yang 1 (YY1), a transcriptional regulator, in proliferation of vascular smooth muscle cells (Santiago FS, Ishii H, et al. Circ Res in press). However, its effect in vascular endothelium remains unclear. Recent observation points a novel “genomic” effect of Angiotensin II to influence inflammation. In this study, we examined a potential contribution of YY1 in angiotensin type II receptor blocker (ARB)-mediated modulation of leukocyte-endothelial adhesion under flow condition. [Methods and Results] Human umbilical vein endothelial cells (HUVEC) were co-incubated in the presence or absence of candesartan, an ARB (1 uM) for 4 hours with TNFα (5 ng/ml). Treatment with candesartan dramatically inhibited THP-1 cell adhesion to TNFα-activated HUVEC under static (p <0.01) and flow conditions (p <0.05). We documented that upregulation of YY1 mRNA after candesartan treatment. To examine a role for YY1 in leukocyte-endothelial interaction, YY1 was overexpressed via recombinant adenovirus. The expression levels of VCAM-1 was significantly decreased in HUVEC treated with adenovirus YY1. In addition, adenovirus YY1 blocked THP-1 adhesion to TNFα-activated HUVEC under flow conditions (TNFα, 8.40±0.64/HPF; TNFα+candesartan, 1.93±0.54/HPF; p <0.0001). The expression level of phosphorylated-JNK, as determined by western blotting analysis, was reduced following YY1 transfection. In contrast, YY1 inhibition by YY1 siRNA did not influence leukocyte-endothelial adhesion and the expression of adhesion molecules. [Conclusion] We demonstrated that candesartan treatment upregulated YY1 in HUVEC and inhibited leukocyte-endothelial adhesion by inhibition of VCAM-1 expression. Our findings suggest a novel anti-inflammatory effect of ARB via YY1-dependent mechanisms.

Download Full-text

Shuffling adenovirus promoters: a viral recombinant with early region 1A under late transcriptional control.

The EMBO Journal ◽

10.1002/j.1460-2075.1983.tb01512.x ◽

1983 ◽

Vol 2 (6) ◽

pp. 845-851 ◽

Cited By ~ 4

Author(s):

D. Solnick

Keyword(s):

Transcriptional Control ◽

Early Region

Download Full-text

Adenovirus Early Region 3 Transgenes Expressed in β Cells Prevent Autoimmune Diabetes in Nonobese Diabetic Mice: Effects of Deleting the Adenovirus Death Protein 11.6K

Journal of Virology ◽

10.1128/jvi.79.1.619-621.2005 ◽

2005 ◽

Vol 79 (1) ◽

pp. 619-621 ◽

Cited By ~ 1

Author(s):

Melissa A. Pierce ◽

Anton Svetlanov ◽

Marshall S. Horwitz ◽

David V. Serreze

Keyword(s):

Autoimmune Diabetes ◽

Gene Cassette ◽

Diabetic Mice ◽

Β Cells ◽

Early Region ◽

Nonobese Diabetic ◽

Proapoptotic Protein ◽

Nonobese Diabetic Mice ◽

Early Region 3

ABSTRACT The incidence of type 1 diabetes (T1D) is decreased in nonobese diabetic mice expressing the complete cassette of adenovirus early region 3 (E3) immunomodulating genes in pancreatic β cells. Embedded among the antiapoptotic E3 genes is one encoding an adenovirus death protein (ADP), which contributes to release of virion particles by promoting cell lysis. Because removal of this proapoptotic protein might have further enhanced the ability of E3 proteins to prevent T1D, an ADP-inactivated E3 construct was tested. Significantly, deletion of ADP did not improve the diabetes-protective effect of an E3 gene cassette.

Download Full-text

E4orf1 Limits the Oncolytic Potential of the E1B-55K Deletion Mutant Adenovirus

Journal of Virology ◽

10.1128/jvi.01972-08 ◽

2009 ◽

Vol 83 (6) ◽

pp. 2406-2416 ◽

Cited By ~ 17

Author(s):

Michael A. Thomas ◽

Robin S. Broughton ◽

Felicia D. Goodrum ◽

David A. Ornelles

Keyword(s):

Protein Synthesis ◽

Deletion Mutant ◽

Mutant Virus ◽

Nucleotide Exchange ◽

Viral Protein Synthesis ◽

Early Region ◽

Oncolytic Adenoviruses ◽

P70 S6k ◽

Ribosomal S6 Kinase ◽

Tumor Selective

ABSTRACT Clinical trials have shown oncolytic adenoviruses to be tumor selective with minimal toxicity toward normal tissue. The virus ONYX-015, in which the gene encoding the early region 1B 55-kDa (E1B-55K) protein is deleted, has been most effective when used in combination with either chemotherapy or radiation therapy. Therefore, improving the oncolytic nature of tumor-selective adenoviruses remains an important objective for improving this form of cancer therapy. Cells infected during the G1 phase of the cell cycle with the E1B-55K deletion mutant virus exhibit a reduced rate of viral late protein synthesis, produce fewer viral progeny, and are less efficiently killed than cells infected during the S phase. Here we demonstrate that the G1 restriction imposed on the E1B-55K deletion mutant virus is due to the viral oncogene encoded by open reading frame 1 of early region 4 (E4orf1). E4orf1 has been reported to signal through the phosphatidylinositol 3′-kinase pathway leading to the activation of Akt, mTOR, and p70 S6K. Evidence presented here shows that E4orf1 may also induce the phosphorylation of Akt and p70 S6K in a manner that depends on Rac1 and its guanine nucleotide exchange factor Tiam1. Accordingly, agents that have been reported to disrupt the Tiam1-Rac1 interaction or to prevent phosphorylation of the ribosomal S6 kinase partially alleviated the E4orf1 restriction to late viral protein synthesis and enhanced tumor cell killing by the E1B-55K mutant virus. These results demonstrate that E4orf1 limits the oncolytic nature of a conditionally replicating adenovirus such as ONYX-015. The therapeutic value of similar oncolytic adenoviruses may be improved by abrogating E4orf1 function.

Download Full-text