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Genomic medicine holds great promise for improving health care, but integrating searchable and actionable genetic data into electronic health records (EHRs) remains a challenge. Here we describe Neptune, a system for managing the interaction between a clinical laboratory and an EHR system during the clinical reporting process. We developed Neptune and applied it to two clinical sequencing projects that required report customization, variant reanalysis, and EHR integration. Neptune has been applied for the generation and delivery of over 15,000 clinical genomic reports. This work spans two clinical tests based on targeted gene panels that contain 68 and 153 genes respectively. These projects demanded customizable clinical reports that contained a variety of genetic data types including single-nucleotide variants (SNVs), copy-number variants (CNVs), pharmacogenomics, and polygenic risk scores. Two variant reanalysis activities were also supported, highlighting this important workflow. Methods are needed for delivering structured genetic data to EHRs. This need extends beyond developing data formats to providing infrastructure that manages the reporting process itself. Neptune was successfully applied on two high-throughput clinical sequencing projects to build and deliver clinical reports to EHR systems. The software is open source and available at https://gitlab.com/bcm-hgsc/neptune .

Xia-Gibbs syndrome is a rare mendelian disorder characterized by Development Delay (DD), intellectual disability (ID), and hypotonia. Individuals with XGS typically harbor de novo protein-truncating mutations in the AT-Hook DNA binding motif containing 1 (AHDC1) gene, although some missense mutations can also cause XGS. Large de novo heterozygous deletions that encompass the AHDC1 gene have also been ascribed as diagnostic for the disorder, without substantial evidence to support their pathogenicity. We analyzed 19 individuals with large contiguous deletions involving AHDC1, along with other genes. One individual bore the smallest known contiguous AHDC1 deletion (∼350 Kb), encompassing eight other genes within chr1p36.11 (Feline Gardner-Rasheed, IFI6, FAM76A, STX12, PPP1R8, THEMIS2, RPA2, SMPDL3B) and terminating within the first intron of AHDC1. The breakpoint junctions and phase of the deletion were identified using both short and long read sequencing (Oxford Nanopore). Quantification of RNA expression patterns in whole blood revealed that AHDC1 exhibited a mono-allelic expression pattern with no deficiency in overall AHDC1 expression levels, in contrast to the other deleted genes, which exhibited a 50% reduction in mRNA expression. These results suggest that AHDC1 expression in this individual is compensated by a novel regulatory mechanism and advances understanding of mutational and regulatory mechanisms in neurodevelopmental disorders.

Background Oncolytic adenoviruses are promising new treatments against solid tumors, particularly for glioblastoma (GBM), and preclinical models are required to evaluate the mechanisms of efficacy. However, due to the species selectivity of adenovirus, there is currently no single animal model that supports viral replication, tumor oncolysis, and a virus-mediated immune response. To address this gap, we took advantage of the Syrian hamster to develop the first intracranial glioma model that is both adenovirus replication-permissive and immunocompetent. Methods We generated hamster glioma stem-like cells (hamGSCs) by transforming hamster neural stem cells with hTERT, simian virus 40 large T antigen, and h-RasV12. Using a guide-screw system, we generated an intracranial tumor model in the hamster. The efficacy of the oncolytic adenovirus Delta-24-RGD was assessed by survival studies, and tumor-infiltrating lymphocytes (TILs) were evaluated by flow cytometry. Results In vitro, hamGSCs supported viral replication and were susceptible to Delta-24-RGD mediated cell death. In vivo, hamGSCs consistently developed into highly proliferative tumors resembling high-grade glioma. Flow cytometric analysis of hamster gliomas revealed significantly increased T-cell infiltration in Delta-24-RGD infected tumors, indicative of immune activation. Treating tumor-bearing hamsters with Delta-24-RGD led to significantly increased survival compared to hamsters treated with phosphate buffered saline (PBS). Conclusions This adenovirus-permissive, immunocompetent hamster glioma model overcomes the limitations of previous model systems and provides a novel platform to study the interactions between tumor cells, the host immune system, and oncolytic adenoviral therapy; understanding of which will be critical to implementing oncolytic adenovirus in the clinic.

Smart grid with great flexibility requirements will not accommodate high proportion of distributed generation with uncertainty in the future. Hence, it becomes indispensable to research scheduling strategy of distributed generation, aiming to reduce serious curtailment of renewable energy. This paper firstly proposes a virtual power supply, which consists of wind power (WP), photovoltaic (PV) power and pumped storage (PS) power. Based on that, a scheduling strategy structure considering stochastic characteristics caused by WP and PV is designed. After that, according to probability distribution of WP and PV, typical virtual power scenarios are obtained via scenario prediction and scenario reduction method. Furthermore, based on flexibility of virtual power and guidance of time-sharing electricity price mechanism, a coordinated scheduling model with optimization objective that maximizes profit is established. Finally, in simulation, validity of proposed model is verified via comparing with different scheduling models. Besides, profits of system under various peak-valley prices are analyzed, which can provide guidance for electricity pricing. The results illustrate that the proposed scheduling strategy can efficiently balance economy and flexibility in optimizing WP and PV consumption and profits of system are increased with 28% at most.

Summary Xia-Gibbs syndrome (XGS; MIM: 615829) is a phenotypically heterogeneous neurodevelopmental disorder (NDD) caused by newly arising mutations in the AT-Hook DNA-Binding Motif-Containing 1 (AHDC1) gene that are predicted to lead to truncated AHDC1 protein synthesis. More than 270 individuals have been diagnosed with XGS worldwide. Despite the absence of an independent assay for AHDC1 protein function to corroborate potential functional consequences of rare variant genetic findings, there are also reports of individuals with XGS-like trait manifestations who have de novo missense AHDC1 mutations and who have been provided a molecular diagnosis of the disorder. To investigate a potential contribution of missense mutations to XGS, we mapped the missense mutations from 10 such individuals to the AHDC1 conserved protein domain structure and detailed the observed phenotypes. Five newly identified individuals were ascertained from a local XGS Registry, and an additional five were taken from external reports or databases, including one publication. Where clinical data were available, individuals with missense mutations all displayed phenotypes consistent with those observed in individuals with AHDC1 truncating mutations, including delayed motor milestones, intellectual disability (ID), hypotonia, and speech delay. A subset of the 10 reported missense mutations cluster in two regions of the AHDC1 protein with known conserved domains, likely representing functional motifs. Variants outside the clustered regions score lower for computational prediction of their likely damaging effects. Overall, de novo missense variants in AHDC1 are likely diagnostic of XGS when in silico analysis of their position relative to conserved regions is considered together with disease trait manifestations.

Purpose Cardiovascular disease (CVD) is the leading cause of death in adults in the United States, yet the benefits of genetic testing are not universally accepted. Methods We developed the "HeartCare" panel of genes associated with CVD, evaluating high-penetrance Mendelian conditions, coronary artery disease (CAD) polygenic risk, LPA gene polymorphisms, and specific pharmacogenetic (PGx) variants. We enrolled 709 individuals from cardiology clinics at Baylor College of Medicine, and samples were analyzed in a CAP/CLIA-certified laboratory. Results were returned to the ordering physician and uploaded to the electronic medical record. Results Notably, 32% of patients had a genetic finding with clinical management implications, even after excluding PGx results, including 9% who were molecularly diagnosed with a Mendelian condition. Among surveyed physicians, 84% reported medical management changes based on these results, including specialist referrals, cardiac tests, and medication changes. LPA polymorphisms and high polygenic risk of CAD were found in 20% and 9% of patients, respectively, leading to diet, lifestyle, and other changes. Warfarin and simvastatin pharmacogenetic variants were present in roughly half of the cohort. Conclusion Our results support the use of genetic information in routine cardiovascular health management and provide a roadmap for accompanying research.

Xia-Gibbs syndrome (XGS) is a rare Mendelian disease typically caused by de novo stop-gain or frameshift mutations in the AT-hook DNA binding motif containing 1 (AHDC1) gene. Patients usually present in early infancy with hypotonia and developmental delay and later exhibit intellectual disability (ID). The overall presentation is variable, however, and the emerging clinical picture is still evolving. A detailed phenotypic analysis of 34 XGS individuals revealed five core phenotypes (delayed motor milestones, speech delay, low muscle tone, ID, and hypotonia) in more than 80% of individuals and an additional 12 features that occurred more variably. Seizures and scoliosis were more frequently associated with truncations that arise before the midpoint of the protein although the occurrence of most features could not be predicted by the mutation position. Transient expression of wild type and different patient truncated AHDC1 protein forms in human cell lines revealed abnormal patterns of nuclear localization including a diffuse distribution of a short truncated form and nucleolar aggregation in mid-protein truncated forms. Overall, both the occurrence of variable phenotypes and the different distribution of the expressed protein reflect the heterogeneity of this syndrome.

Treatment with the oncolytic adenovirus, Delta-24-RGD, resulted in dramatic tumor response in 20% of recurrent malignant glioma patients in a recent Phase I clinical trial. Studies in immunocompetent mice corroborate the patient data indicating that Delta-24-RGD effects are due to both direct tumor cell lysis and viral-mediated anti-tumor immune response. However, it is unclear why only a fraction of patients responds in this manner. Due to poor adenoviral replication in immunocompetent mouse models, the mechanisms by which Delta-24-RGD elicits an effective anti-tumor immune response remain poorly understood. Therefore, we sought to develop a syngeneic Syrian hamster glioma model that is both adenovirus replication-permissive and immunocompetent. We transformed hamster neural stem cells with hTERT, simian virus 40 large T antigen, and h-RasV12 and re-implanted the transformed cells into hamster brains where they developed into tumors. Hamster glioma stem cells (GSCs) were isolated from the resulting tumors and were re-implanted into naive hamster brains using a guide-screw system. In vitro, hamster GSCs supported viral replication and were susceptible to Delta-24-RGD mediated cell death. In vivo, hamster GSCs consistently developed into highly proliferative tumors resembling high-grade glioma. Following delivery of Delta-24-RGD by intratumoral injection, immunohistochemistry for viral proteins demonstrated viral infectivity and replication in hamster gliomas. Flow cytometric analysis of hamster gliomas revealed increased T-cell infiltration in Delta-24-RGD infected tumors. Delta-24-RGD treatment of tumor-bearing hamsters led to significantly increased survival compared to hamsters treated with PBS. In summary, we have developed an adenovirus-permissive, immunocompetent hamster glioma model that provides a novel platform in which to study the interactions between tumor cells, the host immune system, and oncolytic adenoviral therapy.

Myxoma virus (MYXV) is a novel oncolytic virus that has been shown to replicate in pancreatic cancer cells, but its efficacy in animal models of pancreatic cancer has not been determined. The efficacy of MYXV as monotherapy or in combination with gemcitabine was evaluated in intraperitoneal dissemination (IPD) models of pancreatic cancer. The effects of an intact immune system on the efficacy of MYXV therapy was tested by comparing immunodeficient versus immunocompetent murine models and combination therapy with gemcitabine was also evaluated. In cell culture, MYXV replication was robust in a broad range of pancreatic cancer cells and also showed increased oncolysis in combination with gemcitabine. In animal models, MYXV treatment conferred survival benefits over control or gemcitabine-treated cohorts regardless of the cell line or animal model used. MYXV monotherapy was most effective in an immunocompetent IPD model, and resulted in 60% long-term survivors. In Pan02 engrafted immunocompetent IPD models, sequential treatment in which MYXV was administered first, followed by gemcitabine, was the most effective and resulted in 100% long-term survivors. MYXV is an effective oncolytic virus for pancreatic cancer and can be combined with gemcitabine to enhance survival, particularly in the presence of an intact host immune system.

We present genetic evidence that an in vivo role of α-synuclein (α-syn) is to inhibit phospholipase D2 (PLD2), an enzyme that is believed to participate in vesicle trafficking, membrane signaling, and both endo- and exocytosis. Overexpression of PLD2 in rat substantia nigra pars compacta (SNc) caused severe neurodegeneration of dopamine (DA) neurons, loss of striatal DA, and an associated ipsilateral amphetamine-induced rotational asymmetry. Coexpression of human wild type α-syn suppressed PLD2 neurodegeneration, DA loss, and amphetamine-induced rotational asymmetry. However, an α-syn mutant defective for inhibition of PLD2 in vitro also failed to inhibit PLD toxicity in vivo. Further, reduction of PLD2 activity in SNc, either by siRNA knockdown of PLD2 or overexpression of α-syn, both produced an unusual contralateral amphetamine-induced rotational asymmetry, opposite to that seen with overexpression of PLD2, suggesting that PLD2 and α-syn were both involved in DA release or reuptake. Finally, α-syn coimmunoprecipitated with PLD2 from extracts prepared from striatal tissues. Taken together, our data demonstrate that α-syn is an inhibitor of PLD2 in vivo, and confirm earlier reports that α-syn inhibits PLD2 in vitro. Our data also demonstrate that it is possible to use viral-mediated gene transfer to study gene interactions in vivo.

Two small-interfering RNAs (siRNAs) targeting alpha-synuclein (alpha-syn) and three control siRNAs were cloned in an adeno-associated virus (AAV) vector and unilaterally injected into rat substantia nigra pars compacta (SNc). Reduction of alpha-syn resulted in a rapid (4 week) reduction in the number of tyrosine hydroxylase (TH) positive cells and striatal dopamine (DA) on the injected side. The level of neurodegeneration induced by the different siRNAs correlated with their ability to downregulate alpha-syn protein and mRNA in tissue culture and in vivo. Examination of various SNc neuronal markers indicated that neurodegeneration was due to cell loss and not just downregulation of DA synthesis. Reduction of alpha-syn also resulted in a pronounced amphetamine induced behavioral asymmetry consistent with the level of neurodegeneration. In contrast, none of the three control siRNAs, which targeted genes not normally expressed in SNc, showed evidence of neurodegeneration or behavioral asymmetry, even at longer survival times. Moreover, co-expression of both rat alpha-syn and alpha-syn siRNA partially reversed the neurodegenerative and behavioral effects of alpha-syn siRNA alone. Our data show that alpha-syn plays an important role in the rat SNc and suggest that both up- and downregulation of wild-type alpha-syn expression increase the risk of nigrostriatal pathology.

It is not known how influenza A viruses, important human pathogens, counter PKR activation, a crucial host antiviral response. Here we elucidate this mechanism. We show that the direct binding of PKR to the NS1 protein in vitro that results in inhibition of PKR activation requires the NS1 123–127 amino acid sequence. To establish whether such direct binding of PKR to the NS1 protein is responsible for inhibiting PKR activation in infected cells, we generated recombinant influenza A/Udorn/72 viruses expressing NS1 proteins in which amino acids 123/124 or 126/127 are changed to alanines. In cells infected with these mutant viruses, PKR is activated, eIF-2α is phosphorylated and viral protein synthesis is inhibited, indicating that direct binding of PKR to the 123–127 sequence of the NS1 protein is necessary and sufficient to block PKR activation in influenza A virus-infected cells. Unexpectedly, the 123/124 mutant virus is not attenuated because reduced viral protein synthesis is offset by enhanced viral RNA synthesis at very early times of infection. These early viral RNAs include those synthesized predominantly at later times during wild-type virus infection, demonstrating that wild-type temporal regulation of viral RNA synthesis is absent in 123/124 virus-infected cells. Enhanced early viral RNA synthesis after 123/124 virus infection also occurs in mouse PKR−/− cells, demonstrating that PKR activation and deregulation of the time course of viral RNA synthesis are not coupled. These results indicate that the 123/124 site of the NS1A protein most likely functionally interacts with the viral polymerase to mediate temporal regulation of viral RNA synthesis. This interaction would occur in the nucleus, whereas PKR would bind to NS1A proteins in the cytoplasm prior to their import into the nucleus.

Activation of the latent protein kinase, PKR, by extracellular stresses and triggering of resultant cellular apoptosis are mediated by the protein, PACT, which itself gets phosphorylated in stressed cells. We have analyzed the underlying biochemical mechanism by carrying out alanine-scanning mutagenesis of the PKR activation domain of PACT. Among the indispensable residues identified were two serine residues, whose phosphorylation was essential for the cellular actions of PACT. Two-dimensional gel analysis, Western analysis using phosphoamino acid-specific antiserum, and in vivo 32P labeling of PACT demonstrated that constitutive phosphorylation of one of the two residues, Ser246, was required for stress-induced phosphorylation of the other, Ser287. Substitution of either of them by threonine or aspartic acid, but not alanine, was tolerated. Substitution of both residues with the phosphoserine mimetic, aspartic acid, produced a mutant PACT that, unlike the wild-type protein, caused PKR activation and apoptosis, even in unstressed cells. These results indicate that phosphorylation of specific serine residues in the activation domain of PACT is the major mode of transmission of cellular stress response to PKR.

The mammalian protein kinase PKR is a critical component of the innate immune response against virus infection. Its cellular actions are mediated by modulating cell signaling and translational regulation. To be enzymatically active, latent PKR needs to be activated by binding to one of its activators, dsRNA or PACT protein. Although the structures of the N-terminal dsRNA-binding domain and the C-terminal kinase domain of PKR have been separately determined, the mode of activation of the enzyme remains unknown. To address this problem, we used biochemical, genetic, and NMR analyses to identify the PACT-binding motif (PBM) located in the kinase domain and demonstrated an intramolecular interaction between PBM and dsRNA-binding domain. This interaction is responsible for keeping PKR in an inactive conformation, because its disruption by point mutations of appropriate residues produced constitutively active PKR. Furthermore, a short decoy peptide, representing PBM, was able to activate PKR by interfering with the intramolecular interaction. These observations suggest a model for PKR activation upon binding of dsRNA or PACT.

Glioblastoma is the most malignant type of glioma (World Health Organization grade 4) and has a median survival time of only 14.6 months even after the standard treatment of surgical resection followed by radiotherapy with concomitant and adjuvant temozolomide administration. Although some novel and promising anti-glioma agents have been developed, therapeutic advancement has remained disappointing due to the lack of efficient delivery tools. Here we focused on exosomes derived from mesenchymal stem cells (MSCs) as carriers to deliver biological therapeutic agents for the treatment of gliomas. These exosomes are expected to cross the blood-brain barrier and selectively home to the glioma tissue, similar to the parental cells (MSCs). Additionally, MSCs could produce sufficient amounts of exosomes on a clinically applicable scale. Thus, MSC exosomes present a potentially ideal cell-free delivery vehicle for glioma therapy. We hypothesized that exosomes derived from human bone marrow-derived MSCs (BM-hMSCs) can target gliomas and can be used as delivery vehicles following systemic administration. In this study, we employed microRNA (miR-128) as the therapeutic cargo, and exosomes were isolated from a cultured media of BM-hMSCs using ultracentrifugation. At first, exosomes labeled with fluorescent dye were incubated with glioma cells, and exosomes taken up by glioma cells were evaluated by confocal fluorescence microscopy and flow cytometry. Labeled exosomes were also systemically injected into a glioma xenograft model and the ability to home to glioma tissue was evaluated by an in vivo imaging system and histological study. The results showed that MSC exosomes can be taken up by glioma cells and can home to glioma tissues. Next, we cultured miR-128-enriched MSC exosomes with glioma cells. To check the function of miR-128 delivered to glioma cells by MSC exosomes, the down-regulation of the BMI-1 target gene of miR128 was evaluated using real-time polymerase chain reaction and western blot analyses. We also systemically injected these exosomes into glioma xenograft models and evaluated the function of miR-128 by analyzing the downregulation of BMI-1 at the tumor site and the animal survival time in vivo. We confirmed that miR-128 delivered by MSC exosomes could function at the target site both in vitro and in vivo. These results indicate that MSC exosomes may be an ideal delivery vehicle for the treatment of gliomas, especially for microRNA replacement therapy. Citation Format: Shinji Yamashita, Tal Shahar, Anwar Hossain, Brittany Parker Kerrigan, Joy Gumin, Shoudong Li, Feng Gao, Kouji Yamasaki, Yuzaburo Shimizu, Frederick F. Lang. The exosomes derived from BM-hMSCs home to glioma and deliver synthetic microRNA mimics after systemic administration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2668.

Within the past decade, many oncolytic viruses (OVs) have been studied as potential treatments for pancreatic cancer and some of these are currently under clinical trials. The applicability of certain OVs, such as adenoviruses, herpesviruses and reoviruses, for the treatment of pancreatic cancer has been intensively studied for several years, whereas the applicability of other more recently investigated OVs, such as poxviruses and parvoviruses, is only starting to be determined. At the same time, studies have identified key characteristics of pancreatic cancer biology that provide a better understanding of the important factors or pathways involved in this disease. This review aims to summarise the different replication-competent OVs proposed as therapeutics for pancreatic cancer. It also focuses on the unique biology of these viruses that makes them exciting candidate virotherapies for pancreatic cancer and discusses how they could be genetically manipulated or combined with other drugs to improve their efficacy based on what is currently known about the molecular biology of pancreatic cancer.

We previously identified a set of 50 genes that were differentially transcribed in the hippocampal CA1 region of aged, learning-impaired rats compared to aged, superior learning animals during a Morris water maze paradigm. In the current study, we expressed three of these genes (Pctk1, Tcf12 and Ccnd1), which had shown increased transcription in aged, learning impaired rats, in the hippocampus of young rats using viral gene transfer and tested for learning and memory deficits at age 7–14 months. Pctk1 injected animals displayed a modest deficit in acquiring latency in both the Morris water maze and the reverse Morris maze. In the radial arm water maze paradigm, Pctk1, Tcf12 and Ccnd1 expressing animals all showed significant deficits in spatial working memory compared to controls. Rats injected with Ccnd1 and Tcf12, but not Pctk1, also showed a significant deficit in spatial reference memory in the radial arm water maze. Electrophysiological experiments revealed no difference in LTP in Ccnd1 and Pctk1 animals. However, LTD induced by low frequency stimulation was observed in control and Ccnd1 animals, but not in Pctk1 treated animals. In addition, neither Ccnd1 nor Pctk1 expression produced any detectable neuropathology. In contrast Tcf12 expressing animals displayed significant neurodegeneration in both CA1 and dentate gyrus. Several Tcf12 animals also developed tumors that appeared to be glioblastomas, suggesting that aberrant Tcf12 expression in the hippocampus is tumorigenic. Thus, behavioral experiments suggested that overexpression of Pctk1 and Ccnd1 produce a deficit in learning and memory, but electrophysiological experiments do not point to a simple mechanism. In contrast, the learning and memory deficits in Tcf12 animals are likely due to neuropathology associated with Tcf12 gene expression.

Studies have shown that alpha-synuclein (alpha-syn) deposited in Lewy bodies in brain tissue from patients with Parkinson disease (PD) is extensively phosphorylated at Ser-129. We used recombinant Adeno-associated virus (rAAV) to overexpress human wild-type (wt) alpha-syn and two human alpha-syn mutants with site-directed replacement of Ser-129 to alanine (S129A) or to aspartate (S129D) in the nigrostriatal tract of the rat to investigate the effect of Ser-129 phosphorylation state on dopaminergic neuron pathology. Rats were injected with rAAV2/5 vectors in the substantia nigra pars compacta (SNc) on one side of the brain; the other side remained as a nontransduced control. The level of human wt or mutant alpha-syn expressed on the injected side was about four times the endogenous rat alpha-syn. There was a significant reduction of dopaminergic neurons in the SNc and dopamine (DA) and tyrosine hydroxylase (TH) levels in the striatum of all S129A-treated rats as early as 4 wk postinjection. Nigral DA pathology occurred more slowly in the wt-injected animals, but by 26 wk the wt alpha-syn group lost nigral TH neurons equivalent to the mutated S129A group at 8 wk. In stark contrast, we did not observe any pathological changes in S129D-treated animals. Therefore, the nonphosphorylated form of S129 exacerbates alpha-syn-induced nigral pathology, whereas Ser-129 phosphorylation eliminates alpha-syn-induced nigrostriatal degeneration. This suggests possible new therapeutic targets for Parkinson Disease.

A major component of the cellular antiviral system is the latent protein kinase PKR, which is activated by binding to either double-stranded RNA (dsRNA) or the cellular PACT protein. Activated PKR phosphorylates the translation initiation factor eIF2, thereby inhibiting viral and cellular protein synthesis and virus replication. To evade the antiviral effects of PKR, many viruses, including influenza A virus, have evolved multiple mechanisms. For influenza A virus, the non-structural (NS1A) protein plays a major role in blocking activation of PKR during virus infection. The mechanism by which the NS1A protein inhibits PKR activation in infected cells has not been established. In the present study, we first carried out a series of in vitro experiments to determine whether the NS1A protein could utilize a common mechanism to inhibit PKR activation by both PACT and dsRNA, despite their different modes of activation. We demonstrated that the direct binding of the NS1A protein to the N-terminal 230 amino acid region of PKR can serve as such a common mechanism and that this binding does not require the RNA-binding activity of the NS1A protein. The lack of requirement for NS1A RNA-binding activity for the inhibition of PKR activation in vivo was established by two approaches. First, we showed that an NS1A protein lacking RNA-binding activity, like the wild-type (wt) protein, blocked PKR activation by PACT in vivo, as well as the downstream effects of PKR activation in cells, namely, eIF2 phosphorylation and apoptosis. In addition, we demonstrated that PKR activation is inhibited in cells infected with a recombinant influenza A virus expressing NS1A mutant protein that cannot bind RNA, as is the case in cells infected with wild-type influenza A virus.

The cellular protein, PACT, can directly activate protein kinase (PKR) in vitro by the interaction of PACT domain 3 with PKR. In contrast, in vivo, PACT-mediated PKR activation and concomitant inhibition of protein synthesis require additional cellular stresses. We observed that without such stresses, cotransfection of a PACT expression vector with various reporter genes enhances their levels of expression. This effect was promoter and inducer-independent and PACT specific and mediated by PACT domains 1 and 2. PACT did not increase the level of the reporter mRNA but enhanced its translation by suppressing phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) caused by the transfection process. To further examine the phenomenon, we generated cell lines expressing a PACT mutant containing only domains 1 and 2. Reporter gene expression was higher and eIF2alpha phosphorylation was lower in such cell lines compared with the corresponding control cells. Thus, different domains of PACT can either promote or inhibit translation by appropriately modulating the status of eIF2alpha phosphorylation.