The complete genomic DNA sequence of the highly attenuated vaccinia strain modified vaccinia Ankara (MVA) was determined. The genome of MVA is 178 kb in length, significantly smaller than that of the vaccinia Copenhagen genome, which is 192 kb. The 193 open reading frames (ORFs) mapped in the MVA genome probably correspond to 177 genes, 25 of which are split and/or have suffered mutations resulting in truncated proteins. The left terminal genomic region of MVA contains four large deletions and one large insertion relative to the Copenhagen strain. In addition, many ORFs in this region are fragmented, leaving only eight genes structurally intact and therefore presumably functional. The inserted DNA codes for a cluster of genes that is also found in the vaccinia WR strain and in cowpox virus and includes a highly fragmented gene homologous to the cowpox virus host range gene, providing further evidence that a cowpox-like virus was the ancestor of vaccinia. Surprisingly, the central conserved region of the genome also contains some fragmented genes, including ORF F5L, encoding a major membrane protein, and ORFs F11L and O1L, encoding proteins of 39.7 and 77.6 kDa, respectively. The right terminal genomic region carries three large deletions all classical poxviral immune evasion genes and all ankyrin-like genes located in this region are fragmented except for those encoding the interleukin-1 beta receptor and the 68-kDa ankyrin-like protein B18R. Thus, the attenuated phenotype of MVA is the result of numerous mutations, particularly affecting the host interactive proteins, including the ankyrin-like genes, but also involving some structural proteins.

Transcription of the immunoglobulin kappa light-chain genes depends on the presence of a TATA box upstream of the leader gene segment and is regulated by an enhancer sequence in the large intron. In studying a rearranged mouse kappa light-chain gene we have now found that sequences between--90 and--160 base pairs (bp) upstream of the coding region are essential for correct transcription in gene transfer experiments. This region contains the deca- and pentadecanucleotide sequences TNATTTGCAT and TGCAGCCTGTGNCCAG, which we call dc and pd, respectively. Sequences related to dc and pd were found upstream of all human and mouse kappa-chain variable region (Vk) genes, upstream of lambda-chain variable region (V lambda) genes, and within the mouse heavy-chain enhancer. An inverted and complementary form of the dc element (ATGCAAATNA, called cd) occurs upstream of all heavy-chain variable region (VH) genes. The newly defined sequences may be involved in the control of immunoglobulin gene transcription.

A general method for constructing and selecting recombinant vaccinia viruses with insertions, deletions, or mutations in any gene that is similar in principle to one originally devised for Saccharomyces cerevisiae (S. Scherer and R. W. Davis, Proc. Natl. Acad. Sci. USA 76:4951-4955, 1979) is described. The selectable marker used, Escherichia coli guanine phosphoribosyltransferase, is not retained within the final recombinant virus, and hence, this procedure may be used serially to introduce several foreign genes or to make multiple site-directed mutations.

We have previously shown that an Escherichia coli-expressed, denatured spike (S) protein fragment of the severe acute respiratory coronavirus, containing residues 1029 to 1192 which include the heptad repeat 2 (HR2) domain, was able to induce neutralizing polyclonal antibodies (C. The virus-cell membrane fusion event is an essential step in the entry process of all enveloped animal viruses, including important human pathogens such as influenza virus, human immunodeficiency virus (HIV) (8, 23), and the newly emerged severe acute respiratory syndrome coronavirus (SARS-CoV) (9). Following the binding to their receptors on the cell surface, virus-encoded membrane fusion proteins mediate the fusion process. In many but not all cases, the viral fusion proteins are proteolytically processed by host proteases into 2 subunits that remain closely associated with each other: a surface subunit with a receptor-binding site and a transmembrane subunit with a fusion peptide consisting of two or more heptad repeat domains. Upon interaction of the fusion protein with a cellular receptor, the buried fusion peptide is exposed and inserted into the membrane of the target cell. A series of conformational changes trigger virus-cell fusion activity (9) and lead to the unloading of the viral genome into cells. Additionally, many viral fusion proteins also induce cell-cell fusion, i.e., the formation of multinucleated syncytia, facilitating the rapid spread of virus infection.TThe spike (S) protein of coronaviruses is responsible for receptor binding and membrane fusion. It shares similarity with class I virus fusion proteins (2, 3). Typically, it is a type I integral membrane protein, which is N-glycosylated and trimerized in the endoplasmic reticulum. The N-terminal S1 protein contains the receptor-binding site (10,18,22,34). The C-terminal S2 protein is a fusion subunit and anchors on the viral envelope through a transmembrane domain. The S2 protein ectodomain contains two 4,3 hydrophobic heptad repeats (HR1 and HR2) and a putative, internal fusion peptide (3, 23). For the SARS-CoV S protein, the HR2 is located adjacent to the transmembrane domain, whereas the HR1 is 140 to 170 residues upstream of the HR2.Crystallographic, biophysical, and biochemical analysis of the fusion core of SARS-CoV S protein (2,12,19,27,30,35) and other class I fusion proteins (8, 25) supports a model of membrane fusion probably adopted by these enveloped viruses. After the attachment of the receptor-binding subunit to the receptor, the HR1 and HR2 domains in the membrane fusion subunit interact with each other and form a six-helix bundle, a complex consisting of a homotrimeric HR1 coiled coil surrounded by three HR2 helices. The spacer domain (or link, or interhelical domain) between HR1 and HR2 forms a loop and reverses the direction of the polypeptide chain so that the HR2 helices pack against the HR1 coiled coil in an antiparallel manner. This conformational change results in a close apposition of the fusion peptide, already exposed and inserted into th...

Mycophenolic acid, an inhibitor of purine metabolism, was shown to block the replication of vaccinia virus in normal cell lines. This observation led to the development of a dominant one-step plaque selection system, based on expression of the Escherichia coli gpt gene, for the isolation of recombinant vaccinia viruses. Synthesis of xanthine-guanine phosphoribosyltransferase enabled only the recombinant viruses to form large plaques in a selective medium containing mycophenolic acid, xanthine, and hypoxanthine. To utilize the selection system efficiently, we constructed a series of plasmids that contain the E. coli gpt gene and allow insertion of foreign genes into multiple unique restriction endonuclease sites in all three reading frames between the translation initiation codon of a strong late promoter and synthetic translation termination sequences. The selection-expression cassette is flanked by vaccinia virus DNA that directs homologous recombination into the virus genome. The new vectors allow high-level expression of complete or partial open reading frames and rapid construction of recombinant viruses by facilitating the cloning steps and by simplifying their isolation. The system was tested by cloning the E. coli beta-galactosidase gene; in 24 h, this enzyme accounted for approximately 3.5% of the total infected-cell protein.