A case of total pseudoamnesia]

A synthetic vaccinia virus promoter (Psel) was constructed based upon sequences which increase activity of the P7.5 early/late promoter. Comparison of luciferase activity in lysates from cells infected with recombinant vaccinia viruses expressing the luciferase gene either under the control of the P7.5 promoter or Psel, demonstrated significantly enhanced activity mediated by Psel at both early and late times post infection. This promoter may be of considerable benefit in the construction of recombinant poxviruses where early foreign gene expression is important for generating a protective immune response in vaccinated animals, or in reporter/target gene expression in vitro.     

Self-assembly of human papillomavirus type 1 capsids by expression of the L1 protein alone or by coexpression of the L1 and L2 capsid proteins

Vaccinia virus vectors were used to express the major (L1) and minor (L2) capsid proteins of human papillomavirus type 1 (HPV-1) with the vaccinia virus early (p7.5K) or late (pSynth, p11K) promoters. All constructs expressed the appropriate-sized HPV proteins, and both L1 and L2, singly or in combination, localized to the nucleus. Capsids were purified by cesium chloride density gradient centrifugation from nuclei of cells infected with a vaccinia virus-L1 (vac-L1) recombinant or a vac-L1-L2 recombinant but not from vac-L2-infected cells. Electron microscopy showed that the particles were 55 nm in diameter and had icosahedral symmetry. Immunogold-labeled antibodies confirmed the presence of the L1 and L2 proteins in the HPV-1 capsids. Capsids containing L1 alone were fewer and more variable in size and shape than capsids containing the L1 and L2 proteins. The L1-plus-L2 capsids were indistinguishable in appearance from HPV-1 virions obtained from plantar warts. The ability to produce HPV capsids in vitro will be useful in many studies of HPV pathogenicity.     

Addition of the MSA1 signal and anchor sequences to the malaria merozoite surface antigen 1 C-terminal region enhances immunogenicity when expressed by recombinant vaccinia virus

Genes encoding four different C-terminal fragments of a Plasmodium falciparum merozoite surface antigen were generated: MSA1C-(Si,A), containing signal and anchor regions of MSA1; MSA1C-(Si,nA), containing the signal but not the anchor; MSA1C-(nSi,A), containing the anchor but not the signal, and MSA1C-(nSi,nA) containing neither the signal nor the anchor region. Each gene was inserted into the thymidine kinase region of vaccinia virus, under the control of a synthetic strong early/ late promoter. When the plasmodial genes were expressed in cells infected by the recombinant vaccinia virus, the two proteins containing the signal region were transported to the surface of infected cells. Infection of mice and rabbits with the latter recombinant viruses stimulated C-terminal-specific antibody levels that were 10-80-fold higher than those induced by the two recombinant viruses without the signal region. The combination of the signal and anchor regions with the C-terminal MSA1 protein also generated the most effective neutralization in a P. falciparum invasion assay.     

Advantages of continuous hyperpolarized arrest with pinacidil over St. Thomas'' Hospital solution during prolonged ischemia

vlf-1 is a baculovirus gene that regulates very late gene expression (J. R. McLachlin and L. K. Miller, J. Virol., 68, 7746-7756, 1994) and also plays a crucial role in the replication of the budded form of Autographa californica nuclear polyhedrosis virus (AcMNPV) (S. Yang and L. K. Miller, "Expression and mutational analysis of the baculovirus very late factor 1 (vlf-1) gene." Virology, 245, 99-109, 1998). To examine the influence of vlf-1 expression on baculovirus infection, we constructed recombinant viruses that expressed only low levels of VLF-1 and recombinants with vlf-1 under the control of different promoters. Viruses with mutant alleles of vlf-1 that produced low levels of VLF-1 replicated the budded form of the virus normally but produced no occlusion bodies. Thus, a higher concentration of VLF-1 was needed to activate very late gene expression than was needed to support budded virus production. By altering the level and/or timing of vlf-1 expression, the timing of polyhedrin gene (polh) expression, which normally occurs very late in infection, could be advanced or delayed. Early overexpression of vlf-1 increased the level of expression from the polh promoter but caused premature cellular disintegration. The data indicate that VLF-1 is the limiting factor in very late gene expression and that the level of VLF-1 controls the onset of occlusion.     

Expression of the Bunyamwera virus M genome segment and intracellular localization of NSm

Bunyamwera (BUN) virus is the prototype of the family Bunyaviridae and contains a trisegmented, single-stranded RNA genome of negative polarity. The medium (M) RNA segment encodes the two virion glycoproteins, G1 and G2, and a nonstructural protein, NSm, in the form of a polyprotein precursor which is cotranslationally cleaved. The gene order of the M segment is 5' G2-NSm-G1 3'. We have raised a monospecific antiserum in rabbits to a branched chain synthetic peptide to a region of the NSm protein which specifically immunoprecipitates NSm from BUN-infected cells. Indirect immunofluorescence experiments on BUN-infected cells using this antiserum gave a perinuclear staining pattern, suggesting that like the viral structural proteins, NSm localizes to the Golgi complex. An essentially full-length M segment cDNA was cloned into a recombinant vaccinia virus under control of bacteriophage T7 promoter and terminator sequences and expressed in cells co-infected with a second recombinant vaccinia virus which synthesizes T7 RNA polymerase. G1, G2, and NSm were detected in cells dually infected with the recombinant vaccina viruses, indicating that processing of the M segment-encoded precursor does not require other BUN proteins. Immunofluorescence experiments showed that the BUN glycoproteins expressed from this recombinant vaccinia virus system localized to the Golgi complex like authentic BUN proteins.     

African swine fever virus gene A179L, a viral homologue of bcl-2, protects cells from programmed cell death

The African swine fever virus (ASFV) open reading frame A179L, which is similar to the human proto-oncogene bcl-2, has been cloned and expressed in vaccinia virus under control of the pEIL synthetic early/late promoter. The A179L gene product prevented cell death in HeLa and BSC-40 cells doubly infected with another recombinant vaccinia virus expressing the interferon-induced double-stranded RNA-activated protein kinase (p68 kinase), which activates a rapid cell death characteristic of apoptosis. This finding suggests that the A179L gene has a function similar to that of bcl-2 in preventing apoptosis and may play an important role during productive ASFV infection.