Glioma-Parvovirus Interactions: Molecular Insights and Therapeutic Potential

This work was supported by grants from the Spanish Ministerio de Ciencia e Innovación (SAF2008-03238) and Comunidad de Madrid (S-SAL/0185/2006) to the laboratory of J.M.A.The Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM) is in part supported by an institutional grant from Fundación Ramón Areces.Peer reviewe

Integrase defective lentiviral vector as a vaccine platform for delivering HIV-1 antigens

Despite improvements in the therapy of HIV infection allow for controlling AIDS pandemic spread, to date no effective preventive HIV‐1 vaccine is available. To be effective, a global HIV‐vaccine will have to elicit potent and durable immune responses against the enormous diversity of HIV variants. Vaccine delivery systems play a pivotal role for a successful vaccine. In this context, self-inactivating, non-replicating and non-integrating IDLVs (Integrase Defective Lentiviral Vectors) represent attractive tools for gene delivery into target cells with high efficiency, inducing both humoral and cellular immune response in mice and NHP animal models. Here we describe the development of IDLV for the delivery of two novel rationally designed HIV-1 antigens: HTI (HIVACAT T‐cell Immunogen), a mosaic antigen containing conserved regions in the gag, pol, vif and nef genes and including beneficial epitopes, targeted by functional T-cells in patients with low viral load; and UFO (Uncleaved pre‐Fusion Optimized gp140 Trimers ), a consensus sequence of Env from HIV group M, modified to increase native-like trimer production and exposure of bNAbs epitopes. In particular, HIV- or SIV-based IDLV-HTI were able to induce specific cell-mediated immune response in BALB/c mice. Moreover, SIV-based IDLV expressing UFO antigens induced high titres of specific Abs in BALB/c mice and tier 1 NAbs in NHP

The Control of Herpesvirus Immediate Early Gene Expression

The experiments performed in this study investigate the regulation of the immediate early (IE) genes of herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV). Transcription mediated by an HSV-1 IE gene promoter and upstream regulatory sequences is stimulated by a structural virion component (Post et al., 1981). In order to identify the trans-inducing factor (TIF) involved, a series of HSV-1 genomic clones were transfected into baby hamster kidney (BHK) cells together with chimaeric plasmids which contained the thymidine kinase (TK) gene under IE control. Fragments, EcoRI i (0.62 to 0.72 map units), EcoRI b (0.72 to 0.87map units) and BamHI f (0.64 to 0.69 map units), a sub fragment of EcoRI i, were found to elevate TK expression in this assay. The stimulatory sequences were localised to to a 2. 6kb fragment, contained within the plasmid, pMCl. According to the mapping data of Hall et al. (1981) this region contained the complete sequences only of a 1.7kb transcript. The stimulatory effect was abolished by an 8bp linker insertion, which disrupted the reading frame of this gene, but not by other insertions within BamHI f. Induction mediated by the BamHI f fragment was confined to genes under the control of IE sequences, showing it to have the same specificity as the virion TIF, whereas EcoRI b mediated a general stimulatory effect. These experiments indicated that the 1.7kb gene within BamHI f encoded the TIF and that polypeptides encoded by EcoRI b were unlikely to be involved. Hybridisation of pMC6 (a subclone of pMCl) to infected cell RNA, prior to its translation in vitro, prevented the synthesis of a polypeptide of molecular weight 65,000. Immunoprecipitation of translated samples with monoclonal antibody identified this species as Vmw65, a major structural polypeptide. The gene encoding this polypeptide was found to possess an efficient promoter which could direct efficient expression in the absence of viral trans-activating polypeptides. The studies performed identify the virion factor responsible for trans-induction of IE genes as Vmw65, a major structural polypeptide, located in the tegument of the virion. Expression of HSV-1 regulated genes is not increased by infection with PRV, a related herpesvirus (Batterson and Roizman, 1983). Experiments were therefore carried out to examine the control of the PRV major IE gene. The 5' terminus of the raRNA was mapped by S1 nuclease analysis and hybrid plasmids, which contained IE upstream sequences linked to the HSV-1 TK gene, were constructed. Gene expression under the control of PRV IE or HSV-1 IE gene 3 upstream regions was compared using transient assays. It was found that infection with UV-irradiated PRV did not stimulate expression from PRV IE or from HSV-1 IE gene 3 upstream regions in BHK or pig kidney cells, indicating that PRV did not possess an effective TIF. Infection with UV-treated HSV-1, or cotransfection with pMCl (which encodes Vmw65) stimulated expression from both PRV and HSV IE gene upstream regions. However, co-infection with PRV and HSV-1 did not result in increased synthesis of the PRV IE polypeptide. The nucleotide sequence of the 5' end of the PRV transcript and its upstream region was determined. This region was unlike the upstream regions of HSV IE genes in overall structure, but showed a strong similarity to the enhancers of human and murine cytomegaloviruses (HCMV and MCMV). In particular, a reiterated 15bp element of the PRV upstream region was homologous to a conserved, repeated sequence element found in both HCMV and MCMV enhancer regions, and was also related to the "TAATGARATTC" motif found upstream of all HSV IE genes. In order to facilitate future investigations concerning the mechanism of action of the HSV-1 TIF, Vmw65, preliminary purification and characterisation were carried out. A polypeptide of molecular weight, 65,500 was partially purified from HSV-1 virions by treatment with NP40. Immunoprecipitation studies identified this species as the TIF, and also showed that a 65,000 molecular weight binding protein isolated from infected cells was unrelated. Vmw65 has no affinity for double-stranded calf thymus DNA, suggesting that it does not mediate transcriptional regulation by direct interaction with IE upstream sequences

Herpes Simplex Virus Ribonucleotide Reductase DNA and Requirements for mRNA 3' End Formation

This study was undertaken to examine the organisation and structures of mRNAs mapping at the herpes simplex virus type 2 (HSV-2) ribonucleotide reductase locus. From comparisons between equivalent HSV-2 and HSV-1 nucleotide sequences, putative transcription control signals were identified, and functional analysis of certain control elements was performed. The positions of translated regions within the mRNAs encoding ribonucleotide reductase were identified as were conserved amino acid domains between viral and cellular reductases. During the course of these comparisons, a conserved DNA sequence, YGTGTTYY (Y = pyrimidine), located 30nuc downstream from the polyadenylation (AATAAA) signal of both viral and cellular genes, was identified. Functional tests showed that the YGTGTTYY signal was required for efficient formation of mRNA 3' termini. Arrangement of mRNAs and DNA Sequence Comparisons at the HSV-2 Ribonucleotide Reductase Locus. Four overlapping mRNAs have been identified at the ribonucleotide reductase locus. These mRNAs are unspliced and share common 5' and 3' termini. Two early mRNAs with sizes of 4.5kb and 1.2kb have a common 3' terminus and encode HSV-2 polypeptides Vmw138 and Vmw38 respectively which are almost certainly components of the viral ribonucleotide reductase. The other two mRNAs are late, 5' co-terminal species with sizes of 6.4kb and 1.7kb which appear to encode an identical 54,000 mol. wt. protein; the 6.4kb transcript is 3' co-terminal with the early mRNAs. As a consequence of this mRNA arrangement, the 5' termini of the 4.5kb and 1.2kb mRNAs and the 3' terminus of the 1.7kb mRNA are located within larger, overlapping transcripts. The genome region adjacent to the 6.4kb and 1.7kb mRNAs specifies three late transcripts which are synthesised in the opposite orientation; the 5' terminal region of one of these species overlaps with the 6.4kb and 1.7kb mRNAs. The 3' terminus of the late 1.7kb mRNA is located within the transcribed region of the 6. 4kb species. Previously, it has been proposed that read-through of HSV poly A sites is due to inefficient processing at certain 3' termini. The ability of sequences flanking the 1.7kb mRNA 3' terminus to produce functional mRNA was tested using a plasmid containing the bacterial chloramphenicol acetyltransferase (CAT) protein coding sequences fused to an HSV-2 immediate early promoter. CAT activities produced in HeLa cells by a plasmid carrying the 1.7kb mRNA 3' terminal sequences were comparable to activities obtained with a plasmid containing the 3' processing signals from a non-internal poly A site. Read-through of HSV poly A sites may therefore reflect a general reduction in mRNA 3' processing efficiency rather than differences in the 3' processing signals of individual genes. The HSV-1 transcripts equivalent to the 4.5kb and 1.2kb mRNAs have sizes of 5. 0kb and 1. 2kb and encode HSV-1 polypeptides Vmw136 and Vmw38 respectively. Nucleotide sequence comparisons between the 5' flanking regions of these equivalent HSV-2 and HSV-1 mRNAs have revealed the following homologies: 1) upstream from the 4.5kb and 5.0kb mRNA 5' termini, blocks of conserved sequences are present which resemble transcription control signals at the promoter regions of other genes; TATA box homologues, C-rich tracts and A+C-rich elements have been identified. (Abstract shortened by ProQuest.)