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-Δ tails ). MV-ΔM and MV-Δ tails 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.

Rabies is one of the oldest diseases know to man, but its successful control has remained elusive. Although effective vaccines of tissue culture origin against rabies do exist, such preparations are expensive. Live vaccinia virus (VV) recombinants expressing influenza or hepatitis B antigens have recently been used to immunize against these diseases. We have now used this approach to produce a novel rabies vaccine. We first altered the rabies glycoprotein cDNA by site-directed mutagenesis and removed the poly(dG) tail. We then aligned the modified cDNA with an early VV promoter sequence inserted within a cloned copy of the vaccinia thymidine kinase gene and transfected this plasmid into VV-infected cells. Recombination between the virus and the plasmid resulted in a recombinant virus harbouring the rabies glycoprotein cDNA. Inoculation of rabbits with the live recombinant virus induced high titres of rabies virus-neutralizing antibodies, and scarification with the recombinant VV protected mice against challenge with street rabies virus.

A vaccinia virus late gene coding for a major structural polypeptide of 11 kDa was sequenced. Although the 5' flanking gene region is very A+T rich, it shows little homology either to the corresponding region of vaccinia early genes or to consensus sequences characteristic of most eukaryotic genes. Three DNA fragments (100, 200, and 500 base pairs, respectively), derived from the flanking region and including the late gene mRNA start site, were inserted into the coding sequence of the vaccinia virus thymidine kinase (TK) early gene by homologous in vivo recombination. Recombinants were selected on the basis of their TK-phenotype. Cells were infected with the recombinant viruses and RNA was isolated at 1-hr intervals. Transcripts initiating either from the TK early promoter, or from the late gene promoter at its authentic position, or from the translocated late gene promoters within the early gene were detected by nuclease S1 mapping. Early after infection, only transcripts from the TK early promoter were detected. Later in infection, however, transcripts were also initiated from the translocated late promoters. This RNA appeared at the same time and in similar quantities as the RNA from the late promoter at its authentic position. No quantitative differences in promoter efficiency between the 100-, 200-, and 500-base-pair insertions were observed. We conclude that all necessary signals for correct regulation of late-gene expression reside within only 100 base pairs of 5' flanking sequence.Vaccinia virus, a member of the poxvirus family, contains a large double-stranded DNA genome of 180 kilobase pairs. Expression of this large amount of genetic information is temporally well-regulated. Early genes are transcribed shortly after penetration of the virus particles into the host cell. After DNA replication, late genes encoding predominantly structural polypeptides are expressed. The molecular basis for this temporal regulation is not understood.In contrast to other animal DNA viruses, which replicate in the nucleus of the infected cells and which use the host cell RNA polymerase to transcribe their genes, vaccinia replicates in the cytoplasm and utilizes its own transcription system. A multisubunit RNA polymerase (1, 2) and enzymes involved in modification of RNA (3, 4) have been isolated from purified virus particles. The mRNAs made by these enzymes are not spliced (5, 6) but have cap structures (7) and poly(A) tails (8), which are characteristic features of eukaryotic mRNAs. As might be expected, recent evidence suggests that vaccinia virus has evolved its own regulatory signals for gene expression, First, vaccinia genes are transcribed in cell-free extracts prepared from infected cells but not in extracts from non-infected cells (9), which suggests that the host-cell transcriptional machinery does not recognize vaccinia promoter elements. Second, the 5' flanking regions of four early genes that have been sequenced (10-13) lack the regulatory elements characteristic of the corresponding regions of most cel...

The in vitro interactions between vaccinia virus (VV) and monocyte-derived human dendritic cells (DC) have been studied to gain a better understanding of the mechanisms involved in the induction of an immune response by VV. This work showed that VV binds to DC less efficiently than to HeLa cells (HeLa). Capping of viral antigens on the DC surface and electron microscopic examinations suggested that VV enters into DC mainly by endocytosis instead of fusion as for HeLa. Early viral-encoded proteins were expressed in DC but late viral proteins and viral DNA synthesis did not occur. Nevertheless, when successfully infected, DC expressed a similar amount of a foreign, viral-encoded protein, as HeLa, if the early component of the p7.5 promoter was used. VV infection did not lead to DC maturation as determined by following the level of several cell surface markers associated with maturation, but an inhibition of the expression of the costimulatory molecule CD80 was noticed. The proliferation of allogeneic peripheral blood lymphocytes (PBL) was stimulated by VV-infected DC or inhibited depending on the particular donor lymphocytes employed. PBL from VV-vaccinated individuals with good memory responses to VV antigens proliferated in the presence of infected autologous DC. PBL from individuals with poor memory responses to VV and one unvaccinated individual also proliferated, albeit to a lower level, in the presence of infected autologous DC. These results suggest that VV-infected DC could both stimulate memory cells and prime naive cells in vitro.