Danièle Spehner

A matrix-less measles virus is infectious and elicits extensive cell fusion: consequences for propagation in the brain

Measles viruses (MV) can be isolated from the brains of deceased subacute sclerosing panencephalitis patients only in a cell‐associated form. These viruses are often defective in the matrix (M) protein and always seem to have an altered fusion protein cytoplasmic tail. We reconstituted a cell‐free, infectious M‐less MV (MV‐ΔM) from cDNA. In comparison with standard MV, MV‐ΔM was considerably more efficient at inducing cell‐to‐cell fusion but virus titres were reduced ∼250‐fold. In MV‐ΔM‐induced syncytia the ribonucleocapsids and glycoproteins largely lost co‐localization, confirming the role of M protein as the virus assembly organizer. Genetically modified mice were inoculated with MV‐ΔM or with another highly fusogenic virus bearing glycoproteins with shortened cytoplasmic tails (MV‐Δtails). MV‐ΔM and MV‐Δtails lost acute pathogenicity but penetrated more deeply into the brain parenchyma than standard MV. We suggest that enhanced cell fusion may also favour the propagation of mutated, assembly‐defective MV in human brains.

SAGA and a novel Drosophila export complex anchor efficient transcription and mRNA export to NPC

SAGA/TFTC‐type multiprotein complexes play important roles in the regulation of transcription. We have investigated the importance of the nuclear positioning of a gene, its transcription and the consequent export of the nascent mRNA. We show that E(y)2 is a subunit of the SAGA/TFTC‐type histone acetyl transferase complex in Drosophila and that E(y)2 concentrates at the nuclear periphery. We demonstrate an interaction between E(y)2 and the nuclear pore complex (NPC) and show that SAGA/TFTC also contacts the NPC at the nuclear periphery. E(y)2 forms also a complex with X‐linked male sterile 2 (Xmas‐2) to regulate mRNA transport both in normal conditions and after heat shock. Importantly, E(y)2 and Xmas‐2 knockdown decreases the contact between the heat‐shock protein 70 (hsp70) gene loci and the nuclear envelope before and after activation and interferes with transcription. Thus, E(y)2 and Xmas‐2 together with SAGA/TFTC function in the anchoring of a subset of transcription sites to the NPCs to achieve efficient transcription and mRNA export.

Role of B cell receptor Igα and Igβ subunits in MHC class II-restricted antigen presentation

Abstract The ability of the B cell antigen receptors (BCRs) to enhance MHC class II-restricted antigen presentation was ascribed to mlg-associated Igα/Igβ heterodimers. The relative role of Igα and Igβ subunits in antigen presentation was investigated by fusing their cytoplasmic tails to the extracellular and transmembrane domains of Fc receptors. Igα and Igβ chimera mediate antigen internalization and increase the efficiency of antigen presentation, but they drive antigens to different endosomal compartments. Furthermore, antigens internalized by either chimera are degraded and presented with different kinetics. The cytoplasmic tail of Igα targets antigen towards a major population of newly synthesized MHC class II located in class II-rich compartments. In contrast, Igβ targets antigen towards a minor population of recycling MHC class II molecules, located in transferrin receptor-containing endosomes. Altogether, our data indicate that the composition of BCR could be therefore an important way to modulate the immune response.

Construction of vaccinia virus recombinants expressing several measles virus proteins and analysis of their efficacy in vaccination of mice

Measles virus genes encoding the haemagglutinin (HA), fusion protein (F) or nucleoprotein (NP) have been inserted into the vaccinia virus genome either alone or in various combinations. In each case the measles virus genes were expressed from the 7.5K promoter and were incorporated into the thymidine kinase (tk) or K1L loci of the Copenhagen strain of vaccinia virus. Cells infected by the recombinants synthesized measles virus proteins indistinguishable from those induced in measles virus-infected cells. However, in some instances the level of expression in cells infected by recombinants expressing more than one measles virus gene was reduced when compared to those encoding a single gene. The sera from mice immunized with recombinants containing either HA, HA.F, HA.NP or HA.F.NP had similar levels of measles virus neutralizing antibodies which remained constant throughout a 7 month period. Analysis of these sera by immunoprecipitation of radiolabelled measles virus confirmed the presence of specific antibody to each of the antigens where appropriate. The introduction of the measles virus genes into the K1L and the tk sites despite attenuating the virus for mice by 10-fold and 1000-fold respectively did not affect the vaccination efficiency, i.e. ability to induce measles virus antibody and protect mice. Vaccination of BALB/c (H2d) mice with HA and F, but not NP, recombinants completely protected the animals against a lethal measles virus challenge. In contrast, although the HA recombinant protected CBA (H2k) mice, the F recombinant did so poorly. However, by immunizing CBA mice with a recombinant expressing both F and NP, protection was increased to more than 75%. Our findings demonstrate the ability of three measles virus antigens expressed from the vaccinia virus genome alone or in combination to contribute to protective immunity against measles virus infection of mice. They also suggest that the association of measles virus antigens in a single recombinant DNA vaccine could be beneficial to overcome host-related restriction of the immune response to particular antigens.

Handbook of Cryo-Preparation Methods for Electron Microscopy

Cryo-Fixation Methods. Macromolecular Assemblies. Cryo-ultramicrotomy. Low-Temperature Embedding. Freeze-Fracture and Metal Shadowing. Immunolabeling, Electron Tomography.

Heat shock proteins 70 and 60 share common receptors which are expressed on human monocyte-derived but not epidermal dendritic cells

Priming of CTL by means of heat shock proteins (hsp) is dependent on antigen‐presenting cells (APC), which present the hsp‐associated peptides, via their cell surface MHC class I molecules, toCD8+ T cells. It has not yet been established how human (hu) hsp70 interacts with the major (hu)APC, the dendritic cells (DC). Here we show that (hu)hsp70 is specifically internalized intoCD14–, Toll‐like receptor 4– monocyte‐derived (hu)DC by receptor‐mediated endocytosis. We further demonstrate that (hu)hsp70 and (hu)hsp60 share the same receptors on (hu)monocyte‐derived DC. Both molecules as well as MHC class I molecules are spontaneously internalized and reach the MHC class II‐enriched compartments. Finally, freshly isolated (hu) epidermal Langerhans cells (LC), the DC of the skin, as well as CD34+‐derived LC do not bind hsp60 or hsp70. Given the likely importance of the internalization of hsp70 by APC in the induction of the immune responses, the finding that hsp60 and hsp70 are internalized through the same receptor(s) may explain why microbial hsp60 represents a major T cell antigen. This may rationalize the use of microbial hsp60 to primeimmune responses against microbes. The lack of hsp60/70 receptors on epidermal LC raises the crucial question as to whether absence of priming of the skin and mucosal immune systems by hsp‐polypeptide complexes could account for some tissue‐specific diseases. This work also points to a potential advantage of using monocyte‐derived DC in human immunotherapeutic applications of hsp60/70.

Similar genetic organization between a region of fowlpox virus DNA and the vaccinia virus HindIII J fragment despite divergent location of the thymidine kinase gene

DNA from Fowlpox virus, a member of the Avipoxvirus genus, has been found to hybridize to DNA from vaccinia virus, a member of the Orthopoxvirus genus. The greatest homology detected was around the region containing the vaccinia virus thymidine kinase locus. A 3.1-kbp fowlpox virus fragment that hybridizes to the vaccinia virus HindIII J fragment has been cloned and its sequence determined. Comparison of the nucleotide and deduced amino acid sequence to the cross hybridizing vaccinia fragment revealed extensive conservation of six open reading frames as well as a similar organization along the genome. Nevertheless a fowlpox virus gene corresponding to the vaccinia virus thymidine kinase gene was apparently lacking within the region studied and is probably located elsewhere in the genome. Despite this intriguing divergence, our results indicate that the Avipoxviruses are more closely related to the Orthopoxviruses than previously suspected.

Physical mapping of vaccinia virus temperature-sensitive mutations.

DNA restriction fragments from the Copenhagen strain of vaccinia virus have been molecularly cloned into the plasmid pAT 153. Eleven fragments obtained with SalI and nine fragments with HindIII together overlap about 90% of the 190 kb genome. The positioning of SalI and XhoI restriction sites demonstrated that vaccinia Copenhagen DNA differs in a number of sites from other vaccinia strains. Vaccinia temperature-sensitive mutants in 17 different complementation groups have been mapped to specific regions on the viral genome by marker rescue with cloned restriction fragments of the wild type strain. As a rule, the physical order of the mutations corresponded to the genetic order previously established from recombination data. The results have thus enabled the proper alignment of the genetic and physical maps and provided a means of comparing intervals expressed on these two scales. All ts mutations that have been mapped so far lie within a central region that spans approximately 60% of the genome. This clustering of mutations confirms and extends previous observations suggesting that the majority of the genes required for viral multiplication under in vitro conditions are located in the highly conserved central region of the orthopoxvirus genome.

In vivo chromatin organization of mouse rod photoreceptors correlates with histone modifications.

Background The folding of genetic information into chromatin plays important regulatory roles in many nuclear processes and particularly in gene transcription. Post translational histone modifications are associated with specific chromatin condensation states and with distinct transcriptional activities. The peculiar chromatin organization of rod photoreceptor nuclei, with a large central domain of condensed chromatin surrounded by a thin border of extended chromatin was used as a model to correlate in vivo chromatin structure, histone modifications and transcriptional activity. Methodology We investigated the functional relationships between chromatin compaction, distribution of histone modifications and location of RNA polymerase II in intact murine rod photoreceptors using cryo-preparation methods, electron tomography and immunogold labeling. Our results show that the characteristic central heterochromatin of rod nuclei is organized into concentric domains characterized by a progressive loosening of the chromatin architecture from inside towards outside and by specific combinations of silencing histone marks. The peripheral heterochromatin is formed by closely packed 30nm fibers as revealed by a characteristic optical diffraction signal. Unexpectedly, the still highly condensed most external heterochromatin domain contains acetylated histones, which are usually associated with active transcription and decondensed chromatin. Histone acetylation is thus not sufficient in vivo for complete chromatin decondensation. The euchromatin domain contains several degrees of chromatin compaction and the histone tails are hyperacetylated, enriched in H3K4 monomethylation and hypo trimethylated on H3K9, H3K27 and H4K20. The transcriptionally active RNA polymerases II molecules are confined in the euchromatin domain and are preferentially located at the vicinity of the interface with heterochromatin. Conclusions Our results show that transcription is located in the most decondensed and highly acetylated chromatin regions, but since acetylation is found associated with compact chromatin it is not sufficient to decondense chromatin in vivo. We also show that a combination of histone marks defines distinct concentric heterochromatin domains.

Detection of a protein encoded by the vaccinia virus C7L open reading frame and study of its effect on virus multiplication in different cell lines

Vaccinia virus encodes several proteins, the activity of which is essential for multiplication in different cell types. Both the C7L and K1L open reading frames (ORFs) have been characterized as viral determinants for multiplication in human cells. To confirm and extend these findings we inserted the C7L ORF into the genome of a mutant virus unable to multiply in human cells and showed that this virus recovered its ability to replicate. Deletion of C7L from a wild-type viral genome did not adversely affect virus multiplication in human cells but it did reduce replication in hamster Dede cells. When both C7L and K1L were deleted from the vaccinia virus genome only poor or no viral yields were obtained from various human cell lines. Recombinant viruses were also constructed to facilitate the study of C7L protein synthesis during infection. One virus in which the lacZ ORF was fused downstream and in-frame with the C7L ORF enabled us to characterize the C7L protein as an early gene product. Another recombinant virus was constructed so that the carboxy terminus of the C7L ORF product contained an additional 28 amino acids from the carboxy terminus of K1L. Tagging of C7L in this way allowed us to detect the fusion protein by immunoprecipitation with antibodies against the K1L protein. Furthermore, the hybrid protein retained its biological properties. The recombinant viruses constructed in this work should be useful for studies of the molecular basis of the activity of viral host range proteins.