The understanding of HTLV-induced disease is hampered by the lack of a suitable animal model allowing the study of both viral replication and leukemogenesis in vivo. Although valuable information has been obtained in different species, such as rabbits, mice, rats, and monkeys, none of these systems was able to conciliate topics as different as viral infectivity, propagation within the host, and generation of leukemic cells. An alternate strategy is based on the understanding of diseases induced by viruses closely related to HTLV-1, like bovine leukemia virus (BLV). Both viruses indeed belong to the same subfamily of retroviruses, harbor a similar genomic organization, and infect and transform cells of the hematopoietic system. The main advantage of the BLV system is that it allows direct experimentation in two different species, cattle and sheep.
Background The aim was to investigate safety and immunogenicity of vaccine formulations against respiratory syncytial virus (RSV) containing the stabilized prefusion conformation of RSV fusion protein (RSVPreF3). Methods This phase I/II, randomized, controlled, observer-blind study enrolled 48 young adults (YA; 18–40 years) and 1005 older adults (OA; 60–80 years) between January and August 2019. Participants were randomized into equally sized groups to receive two doses of unadjuvanted (YA and OA) or AS01-adjuvanted (OA) vaccine or placebo two months apart. Vaccine safety and immunogenicity were assessed until one (YA) or 12 months (OA) after second vaccination. Results The RSVPreF3 vaccines boosted humoral (RSVPreF3-specific IgG and RSV-A neutralizing antibody) responses, which increased in an antigen-concentration-dependent manner and were highest post-dose one. Compared to pre-vaccination, the geometric mean frequencies of polyfunctional CD4+ T-cells increased after each dose and were significantly higher in adjuvanted than unadjuvanted vaccinees. Post-vaccination immune responses persisted until end of follow-up. Solicited adverse events (AEs) were mostly mild-to-moderate and transient. Despite a higher observed reactogenicity of AS01-containing vaccines, no safety concerns were identified for any assessed formulation. Conclusions Based on safety and immunogenicity profiles, the AS01E-adjuvanted vaccine containing 120 μg of RSVPreF3 was selected for further clinical development. Trial registration ClinicalTrials.gov NCT03814590; URL: https://clinicaltrials.gov/ct2/show/NCT03814590
Tax, through its interactions with the TTP repressor, indirectly increases TNF-alpha expression. This observation is of importance for the cell transformation process induced by leukemogenic retroviruses, because TNF-alpha overexpression plays a central role in pathogenesis.
The Rep78 and Rep68 proteins of adeno-associated virus (AAV) type 2 are involved in DNA replication, regulation of gene expression, and targeting site-specific integration. They bind to a specific Rep recognition sequence (RRS) found in both the viral inverted terminal repeats and the AAVS1 integration locus on human chromosome 19. Previous in vitro studies implied that an N-terminal segment of Rep is involved in DNA recognition, while additional domains might stabilize binding and mediate multimerization. In order to define the minimal requirements for Rep to recognize its target site in the human genome, we developed one-hybrid assays in which DNA-protein interactions are detected in vivo. Chimeric proteins consisting of the N terminus of Rep fused to different oligomerization motifs and a transcriptional activation domain were analyzed for oligomerization, DNA binding, and activation of reporter gene expression. Expression of reporter genes was driven from RRS motifs cloned upstream of minimal promoters and examined in mammalian cells from transfected plasmids and in Saccharomyces cerevisiae from a reporter cassette integrated into the yeast genome. Our results show for the first time that chimeric proteins containing the amino-terminal 244 residues of Rep are able to target the RRS in vitro and in vivo when incorporated into artificial multimers. These studies suggest that chimeric proteins may be used to harness the unique targeting feature of AAV for gene therapy applications.Adeno-associated virus (AAV) type 2 is a nonpathogenic human parvovirus that relies upon a helper virus for efficient replication (5). Under conditions that are not permissive for replication, AAV infection results in integration of the viral genome into the host chromosome (6,30,44). A unique characteristic of AAV integration is that in human cell lines it can be targeted in about 70% of cases to a specific site on chromosome 19q13. 3-qter (24, 25, 45). The preintegration locus, termed AAVS1, has been cloned and sequenced (23). Sitespecific integration by AAV into the preintegration locus requires cis-acting sequences within the viral origin of replication and AAVS1 as well as the DNA-binding activity of the Rep proteins (31, 60). It would be particularly attractive to harness this unique targeting feature for gene transfer vehicles, since this would decrease the chances of insertional mutagenesis associated with random integration.The AAV genome is a single-stranded linear DNA molecule with inverted terminal repeats (ITRs) that fold into hairpin structures and serve as the origins for DNA replication (5). The ITR is involved in regulation of gene expression, initiation of DNA replication, packaging of the viral genome, site-specific integration, and rescue from the integrated state. The genome contains two open reading frames (ORFs) encoding nonstructural (Rep) and structural (Cap) proteins. Expression is regulated by three viral promoters, p5, p19, and p40. The rep gene encodes four overlapping multifunctional Rep proteins, named a...
Bovine leukemia virus (BLV)taxHuman T-cell leukemia virus type 1 (HTLV-1) 1 and bovine leukemia virus (BLV) are members of the Deltaretrovirus genus in the Retroviridae family (1-4). In addition to the structural proteins Gag, Pol, and Env, these viruses also encode a series of regulatory proteins: Tax, Rex, p12/R3, and p13/G4. The Tax protein is a transcriptional activator, which increases the synthesis of viral proteins acting on a triplicate 21-bp element located in the 5Ј long terminal repeat (LTR) (5-7). Tax does not interact directly with DNA but rather acts via cellular factors, such as members of the CREB/ATF family of basic leucine zipper proteins (8 -11). The tax gene is believed to be essential because its presence is absolutely required for infectivity in vivo (12). Besides its role in the regulation of transcription, the Tax protein also exhibits an oncogenic potential (13). Tax behaves as an immortalizing oncogene because it is able to cooperate with the Ha-ras oncoprotein to fully transform primary rat embryo fibroblasts (14, 15). Both transactivation and immortalizing functions of Tax can be dissociated by mutations in specific regions of the protein. For example, mutation of the phosphorylation sites at serines 106 and 293 of BLV Tax abrogates immortalization potential in vitro but maintains transcriptional activity and viral oncogenicity in vivo (16,17). Conversely, the transactivation of the LTR promoter is not required for Tax to transform primary cells in vitro (14). The Tax protein of HTLV-1 (Tax1) is known to activate several cellular genes including IL-2, IL-2R␣, IL-3, TNF-␣, and . Tax1 is also involved in cell cycle regulation by direct activation of cyclin D3 and cyclin kinases cdk4 and cdk6 (21), or by inactivating the cyclin-dependent kinase inhibitor p16 INK4A (22). In fact, protein-protein interactions with cellular factors are crucial for Tax1 to perturb the regulation of many cellular pathways (23). These HTLV-1 Tax binding factors include the human mitotic checkpoint protein HsMAD1 (24), MEKK1 (25), the IB kinase (26), or the PCAF protein (27). In contrast, little is known about the cellular partners of the BLV Tax protein. In this study, we describe a functional interaction between the homeobox protein MSX2 and the BLV Tax oncoprotein. EXPERIMENTAL PROCEDURESPlasmids-Plasmids pSGMSX2 and pEGFPMSX2 were constructed by subcloning the human MSX2-cDNA (kindly offered by Dr. T. Iimura, Tokyo Medical and Dental University, Japan) into pSG5 (Stratagene) and pEGFP-C1 (Clontech), respectively. Plasmids pcDNAMSX2-c-Myc, pcDNAMSX2⌬N-c-Myc, and pcDNAMSX2⌬C-c-Myc, which express respectively, C-terminal c-Myc-tagged full-length, amino acids 80 -267 and amino acids 1-200 of the human MSX-2 protein, were constructed by inserting the human MSX2 cDNA, or PCR-derived MSX2 truncation mutants into pcDNA3.1/myc-HisB (Invitrogen). Plasmid pcDNARAP74-flag was obtained by subcloning the human RAP74 cDNA (kindly offered by Dr. Z. Burton, Michigan State University) into pcDNA3.1Flag (Invitrogen).The pLTRL...
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