Measles virus (MV), a member of the family Paramyxoviridae and an exclusively human pathogen, is among the most infectious viruses. A progressive fatal neurodegenerative complication, subacute sclerosing panencephalitis (SSPE), occurs during persistent MV infection of the CNS and is associated with biased hypermutations of the viral genome. The observed hypermutations of A-to-G are consistent with conversions catalyzed by the adenosine deaminase acting on RNA (ADAR1). To evaluate the role of ADAR1 in MV infection, we selectively disrupted expression of the IFN-inducible p150 ADAR1 isoform and found it caused embryonic lethality at embryo day (E) 11-E12. We therefore generated p150-deficient and WT mouse embryo fibroblast (MEF) cells stably expressing the MV receptor signaling lymphocyte activation molecule (SLAM or CD150). The p150 −/− but not WT MEF cells displayed extensive syncytium formation and cytopathic effect (CPE) following infection with MV, consistent with an anti-MV role of the p150 isoform of ADAR1. MV titers were 3 to 4 log higher in p150 −/− cells compared with WT cells at 21 h postinfection, and restoration of ADAR1 in p150 −/− cells prevented MV cytopathology. In contrast to infection with MV, p150 disruption had no effect on vesicular stomatitis virus, reovirus, or lymphocytic choriomeningitis virus replication but protected against CPE resulting from infection with Newcastle disease virus, Sendai virus, canine distemper virus, and influenza A virus. Thus, ADAR1 is a restriction factor in the replication of paramyxoviruses and orthomyxoviruses. M easles virus (MV), a member of the family Paramyxoviridae, infects more than 10 million persons worldwide each year, resulting in several hundred thousand deaths (1, 2). A serious complication is the persistent infection of the CNS known as subacute sclerosing panencephalitis (SSPE) that occurs at a frequency of 4-11 cases per 100,000 cases of MV infection. SSPE is a progressive fatal neurodegenerative disease with characteristic features of replication of MV in neurons in the presence of high titers of MV antibodies, modest infiltration of T and B cells into the CNS, and replication of defective MV in the CNS with biased mutation of U-to-C and A-to-G in the viral genome (3-5). These hypermutations occur primarily in the matrix (M) gene but are also observed to a lesser extent in the fusion (F) and hemagglutinin (H) genes (3, 4). A transgenic mouse model that expresses the human MV receptor CD46 recapitulates all the features of SSPE on infection with MV, with biased hypermutations of U-to-C and A-to-G accounting for more than 95% of point mutations in the M gene (3,4,6). Interestingly, these biased hypermutations play a direct role in the pathogenesis of SSPE by facilitating a significant prolongation of MV persistence within the CNS, as opposed to mere accumulation as a result of persistent infection (7). In support of a direct role of M gene hypermutations in the establishment of SSPE, MV generated by reverse genetics and containing a hypermutated M...

BackgroundInfluenza virus remains a significant health and social concern in part because of newly emerging strains, such as avian H5N1 virus. We have developed a prototype H5N1 vaccine using a recombinant, replication-competent Adenovirus serotype 4 (Ad4) vector, derived from the U.S. military Ad4 vaccine strain, to express the hemagglutinin (HA) gene from A/Vietnam/1194/2004 influenza virus (Ad4-H5-Vtn). Our hypothesis is that a mucosally-delivered replicating Ad4-H5-Vtn recombinant vector will be safe and induce protective immunity against H5N1 influenza virus infection and disease pathogenesis.Methodology/Principal FindingsThe Ad4-H5-Vtn vaccine was designed with a partial deletion of the E3 region of Ad4 to accommodate the influenza HA gene. Replication and growth kinetics of the vaccine virus in multiple human cell lines indicated that the vaccine virus is attenuated relative to the wild type virus. Expression of the HA transgene in infected cells was documented by flow cytometry, western blot analysis and induction of HA-specific antibody and cellular immune responses in mice. Of particular note, mice immunized intranasally with the Ad4-H5-Vtn vaccine were protected against lethal H5N1 reassortant viral challenge even in the presence of pre-existing immunity to the Ad4 wild type virus.Conclusions/SignificanceSeveral non-clinical attributes of this vaccine including safety, induction of HA-specific humoral and cellular immunity, and efficacy were demonstrated using an animal model to support Phase 1 clinical trial evaluation of this new vaccine.

The RNA-dependent protein kinase PKR plays important roles in the antiviral and antiproliferative actions of IFN. The IFN-inducible promoter of the human PKR gene contains a 15-bp DNA element designated KCS. The KCS element is located 4 bp upstream of the interferon-stimulated response element (ISRE) and is required for both basal and IFN-inducible transcription. We have examined the effect of insertion mutations between the KCS and the ISRE elements, as well as altered orientation of the KCS element relative to the ISRE element, to assess a possible functional interaction between them. Large insertions (>or=93 bp) between the KCS and ISRE elements significantly reduced both basal and IFN-inducible promoter activity. The function of the KCS element was dependent on the orientation of KCS relative to the ISRE element. Multimerization of the KCS element increased both basal and IFN-inducible transcription. Electrophoretic mobility shift analyses (EMSA) identified IFN-inducible protein complex formation that required both the KCS and the ISRE DNA element sequences. The novel IFN-inducible protein complexes contained the transcription factor STAT1, as shown by supershift analyses and by their presence in extracts prepared from STAT1 wild-type but not from STAT1-/- null cells. These results, taken together, strongly suggest that the KCS and ISRE elements of the human PKR promoter represent a functional unit.

The protein kinase regulated by RNA (PKR) is an important mediator of the antiviral and antiproliferative actions of interferon (IFN). The promoter of the PKR gene contains a novel 15-bp element designated KCS that is required for both basal and IFN-inducible transcription, with KCS function dependent upon both position and orientation relative to the ISRE element. Novel inducible protein complexes (iKIBP1, iKIBP2) that require both the KCS and the ISRE element sequences for their formation have been identified and characterized. Transcription factors Sp1 and Sp3 were found to be KCS-binding proteins by electrophoretic mobility shift analyses (EMSA) and Sepharose bead-KCS oligonucleotide pull-down assays. However, only Sp3 but not Sp1 was a constituent of the inducible iKIBP complexes. EMSA also identified STAT1, STAT2, and IRF-9 as components of the iKIBP complexes, indicating that ISGF-3 participates in iKIBP complex formation. Proteins bound at the KCS element in the absence of ISRE were able to recruit both STAT1 and STAT2 to the KCS element; recruitment was dependent upon IFN-alpha treatment. Chromatin immunoprecipitation assays revealed that the binding of Sp3, similar to STAT1 and STAT2, at the PKR promoter in vivo was IFN-dependent, but that Sp1 binding was not dependent upon IFN treatment. These results, taken together, strongly suggest a role for Sp1 in basal and Sp3 in inducible transcription of PKR and that a potential function of the KCS element is to facilitate the recruitment of ISGF-3 complex components to the PKR promoter to stimulate transcription.

The protein kinase regulated by RNA (PKR) is interferon (IFN)-inducible and plays important roles in many cellular processes, including virus multiplication, cell growth, and apoptosis. The TATA-less PKR promoter possesses a novel 15-bp DNA element (kinase conserved sequence (KCS)) unique to the human and mouse PKR genes that is conserved in sequence and position. We found that Sp1 and Sp3 of the Sp family of transcription factors bind at the KCS element. Their involvement was analyzed in the activation of basal and IFN-inducible PKR promoter activity. Both the small and large isoforms of Sp3 co-purified with KCS protein binding activity (KBP) by using nuclear extracts from HeLa cells not treated with IFN. Two forms of the KCS-binding protein complex were demonstrated by electrophoretic mobility shift assay analysis; one contained Sp1 and the other Sp3. In mouse cells null for all Sp3 isoforms, PKR expression was reduced to ϳ50% that of wild-type cells in the absence of IFN. The IFN-inducible expression of PKR, however, was Sp3-independent but STAT1-and JAK1-dependent. Overexpression of Sp1 in human U cells resulted in increased PKR promoter activity. In Drosophila SL2 cells lacking Sp proteins, both Sp1 and Sp3 large but not small isoforms activated PKR promoter expression, with the Sp1-mediated activation dominant. Mutational analysis of the PKR promoter region indicated a cooperative interaction between two different Sp sites, one of which is within the KCS element. These results establish that, in the absence of IFN treatment, activation of PKR basal expression is mediated by Sp1 and Sp3 proteins in a cooperative manner.

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Acute promyelocytic leukemia (APL) results from a chromosomal translocation that gives rise to the leukemogenic fusion protein PML-RARa (promyelocytic leukemia-retinoic acid a receptor). Differentiation of leukemic cells and complete remission of APL are achieved by treatment of patients with pharmacological doses of all-trans retinoic acid (ATRA), making APL a model disease for differentiation therapy. However, because patients are resistant to further treatment with ATRA on relapse, it is necessary to develop alternative treatment strategies to specifically target APL. We therefore sought to develop a treatment strategy based on lentiviral vector-mediated delivery of small interfering RNA (siRNA) that specifically targets the breakpoint region of PML-RARa. Unlike treatment with ATRA, which resulted in differentiation of leukemic NB4 cells, delivery of siRNA targeting PML-RARa into NB4 cells resulted in both differentiation and apoptosis, consistent with the specific knockdown of PML-RARa. Intraperitoneal injection of NB4 cells transduced with lentiviral vectors delivering PML-RARa-specific siRNA but not control siRNA prevented development of disease in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Taken together, these results indicate that development of PML-RARa-specific siRNA may represent a promising treatment strategy for ATRA-resistant APL.

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