Construction and properties of pseudorabies virus recombinants with altered control of immediate-early gene expression.

To investigate how altered control of expression of the essential immediate-early (IE) gene of pseudorabies virus influences virus replication and virulence, we replaced the IE promoter with the tissue-specific promoters of the bovine cytokeratin IV gene (CKIV), the bovine cytokeratin VIb gene (CKVIb), or the inducible promoter of Drosophila heat shock gene HSP70. We compared expression of the IE gene of the wild-type virus and recombinant viruses in different cell types and at different temperatures and found that IE expression had become cell type or temperature dependent. When a recombinant virus was titrated on nonpermissive cells or was titrated at nonpermissive temperatures in vitro, the plating efficiency was reduced by more than 99%. Mice were inoculated subcutaneously (s.c.), intraperitoneally (i.p.), or intranasally (i.n.) with a dose equal to 100 times the 50% lethal dose of the wild-type virus. After inoculation with temperature-sensitive recombinant N-HSP, two (s.c.), two (i.p.), and four (i.n.) of five mice died. However, at this dose, recombinant N-CKIV, which contains a promoter specific for stratified epithelial tissue of the tongue mucosa, was not lethal when inoculated s.c. or i.p. but killed four mice when inoculated i.n. Recombinant N-CKVIb, which contains a promoter specific for the suprabasal layers of the epidermis, was not lethal after inoculation by any of the three routes. In explant cultures of nasal mucosa of pigs, replication of N-CKIV and N-CKVIb was not markedly reduced in the epithelium. However, in contrast to results obtained with wild-type virus, infection of the stroma was not observed. We conclude that the replicative ability and virulence of pseudorabies virus can be influenced by altering control of expression of the IE gene.     

DNA sequence of the gene for pseudorabies virus gp50, a glycoprotein without N-linked glycosylation.

The DNA sequence was determined for a region of the pseudorabies virus (PRV) genome to which a mutation defining resistance to a monoclonal antibody has been mapped (M. W. Wathen and L. M. K. Wathen, J. Virol., 51:57-62, 1984). This sequence was found to contain an open reading frame that did not include an amino acid sequence directing N-linked glycosylation. This open reading frame was expressed in uninfected Chinese hamster ovary cells to produce the PRV glycoprotein gp50. When PRV-infected Vero cells were incubated in the presence of tunicamycin, the gp50 that was produced had an identical molecular weight to that produced in the absence of drug. When infected cells were incubated in the presence of monensin, the molecular weight of gp50 was reduced from 60,000 to 45,000, but was not sensitive to endo-beta-N-acetylglucosaminidase H. These observations led to the conclusion that gp50 does not contain N-linked carbohydrate, as predicted from the DNA sequence. A region of the amino acid sequence and the positions of the cysteine residues of PRV gp50 are homologous to glycoprotein D of herpes simplex virus.     

DNA sequence of an immediate-early gene (IEmRNA-5) of herpes simplex virus type I

We describe a 2560 base pair herpes simplex virus type 1 (HSV-1) DNA sequence containing the entire immediate-early mRNA-5 (IEmRNA-5) gene. The 3' and 5' termini of IEmRNA-5 were mapped within this DNA sequence by single-strand specific endonuclease protection experiments. The IEmRNA-5 gene contains DNA sequences from both the unique (Us) and reiterated (TRs/IRs) regions of the HSV-1 DNA short component and is interrupted by a single intron mapping in TRs/IRs. A search of the transcribed DNA sequence revealed no initiator codon within TRs/IRs. The first ATG was located 6 bases into Us sequences and this reading frame (316 codons) was also observed in the 3' transcribed region. The oligonucleotide sequences adjacent to the IEmRNA-5 termini are discussed in relation to those of the HSV-1 thymidine kinase gene and other genes transcribed by RNA polymerase II.     

Complete nucleotide sequence of the nucleoprotein gene of influenza B virus.

A DNA copy of influenza B/Singapore/222/79 viral RNA segment 5, containing the gene coding for the nucleoprotein (NP), has been cloned in Escherichia coli plasmid pBR322, and its nucleotide sequence has been determined. The influenza B NP gene contains 1,839 nucleotides and codes for a protein of 560 amino acids with a molecular weight of 61,593. Comparison of the influenza B NP amino acid sequence with that of influenza A NP (A/PR/8/34) reveals 37% direct homology in the aligned regions, indicating a common ancestor. However, influenza B NP has an additional 50 amino acids at its N-terminal end. As is the case with influenza A NP, influenza B NP is a basic protein, with its charged residues relatively evenly distributed rather than clustered. The structural homology suggests functional similarity between the NP of influenza A and B viruses.     

Cloning and nucleotide sequence of the gene encoding the Ecal DNA methyltransferase.

The gene coding for the GGTNACC specific Ecal DNA methyltransferase (M.Ecal) has been cloned in E. coli from Enterobacter cloacae and its nucleotide sequence has been determined. The ecalM gene codes for a protein of 452 amino acids (Mr: 51,111). It was determined that M.Ecal is an adenine methyltransferase. M.Ecal shows limited amino acid sequence similarity to other adenine methyltransferases. A clone that expresses Ecal methyltransferase at high level was constructed.     

The nucleotide sequence of cloned wheat dwarf virus DNA

Restriction analysis and cloning of virus-specific double-stranded DNA isolated from plants infected with wheat dwarf virus (WDV) indicated that the virus genome, like that of maize streak virus (MSV), consists of a single DNA circle. The complete nucleotide sequence of cloned WDV DNA (2749 nucleotides) has been determined. Comparison of the potential coding regions in WDV DNA with those in the DNA of two strains of MSV suggests that these viruses encode at least two functional proteins, the coat protein read in the virion (+) DNA sense and a composite protein, formed from two open reading regions, in the complementary (-) DNA sense. Although WDV and MSV are serologically unrelated their coat proteins showed 35% direct amino acid sequence and their DNAs showed 46% nucleotide sequence homology. There was too little homology between the DNAs of WDV and those of two geminiviruses with bipartite genomes, cassava latent virus (CLV) and tomato golden mosaic virus (TGMV), to align the sequences. However comparison of the amino acid sequences of predicted proteins of WDV, MSV, TGMV and CLV revealed clear relationships between these viruses and suggested that the monopartite and the bipartite geminiviruses have a common ancestral origin. Four inverted repeat sequences which have the potential to form hairpin structures of deltaG >/= -14 kcal/mol were detected in WDV DNA. The sequence TAATATTAC present in the loop of one of these hairpins is conserved in similar putative structures in MSV DNA and in both DNA components of CLV and TGMV and may function as a recognition sequence for a protein involved in virus DNA replication.     

Nucleotide sequence analysis of DNA. II. Complete nucleotide sequence of the cohesive ends of bacteriophage lambda DNA

At the ends of bacteriophage λ DNA, the 5′-terminated strands are 12 nucleotides longer than the 3′-terminated strands. The complete sequence of deoxynucleotides in both the protruding 5′-terminated single strands of λ DNA has been determined by partial repair and by complete repair followed by sequencing of isolated oligonucleotides. Starting from the 5′-end of the left-hand cohesive end, the 12 nucleotides are in the sequence dpGpGpGpCpGpGpCpGpApCpCpT. The sequence from the right-hand cohesive end is exactly complementary to that from the left-hand end.     

Nucleotide sequence analysis of DNA. X. Complete nucleotide sequence of the cohesive ends of bacteriophage phi80 DNA

The base sequence of the cohesive ends of bacteriophage φ80 DNA has been shown to be identical to the base sequence of the cohesive ends of bacteriophage lambda DNA.