Biomedical subjects

N A DeLuca

Publications and source records attributed to N A DeLuca.

45 records · Page 3Linked to original sources

PubMed1989

Separation of primary structural components conferring autoregulation, transactivation, and DNA-binding properties to the herpes simplex virus transcriptional regulatory protein ICP4.

A truncated ICP4 peptide which contains the amino-terminal 774 amino acids of the 1,298-amino-acid polypeptide is proficient for DNA binding, autoregulation, and transactivation of some viral genes (N. A. DeLuca and P. A. Schaffer, J. Virol. 62:732-743, 1988) and hence exhibits many of the properties characteristic of intact ICP4. To define the primary sequence important for the activities inherent in the amino-terminal half of the ICP4 molecule, insertional and deletion mutagenesis of the sequences encoding these residues were conducted. The DNA-binding activity of the molecule as assayed by the association with a consensus binding site was sensitive to insertional mutagenesis in two closely linked regions of the molecule. One region between amino acids 445 and 487 is critical for DNA binding and may contain a helix-turn-helix motif. The second region between amino acids 263 and 338 reduces the binding activity to a consensus binding site. When analyzed in the viral background, the DNA-binding activity of a peptide containing an insertion at amino acid 338 to a consensus binding site was reduced while the association with an alternative sequence was eliminated, suggesting a possible mechanism by which ICP4 may recognize a broader range of sequence elements. Mutations which eliminated DNA binding also eliminated or reduced both transactivation and autoregulation, supporting the requirement for DNA binding for these activities. Peptides that retained the deduced DNA-binding domain but lacked amino acids 143 through 210 retained the ability to associate with the consensus site and autoregulatory activity but were deficient for transactivation, demonstrating that the structural requirements for transactivation are greater than those required for autoregulation.

DNA↗

PubMed1988

Physical and functional domains of the herpes simplex virus transcriptional regulatory protein ICP4.

A characteristic common to DNA animal viruses is the expression early in infection of viral proteins that act in trans to regulate subsequent RNA polymerase II-dependent transcription of the remainder of the viral genome. The predominant transcriptional regulatory protein specified by herpes simplex virus type 1 is the immediate-early protein ICP4. ICP4 is a complex multifunctional protein required for the activation of many herpes simplex virus type 1 transcriptional units and for repression of its own transcription. In the present study we have introduced nonsense and deletion mutations into both genome copies of the ICP4 gene such that the resulting mutants express only defined subsets of the primary ICP4 amino acid sequence. The partial peptides retain activities and physical properties of the intact ICP4 molecule, permitting one to attribute individual activities and properties to defined amino acid sequences.

Amino Acid Sequence↗

PubMed1987

Activities of herpes simplex virus type 1 (HSV-1) ICP4 genes specifying nonsense peptides.

Synthetic oligonucleotide linkers containing translational termination codons in all possible reading frames were inserted at various positions in the cloned gene encoding the herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein, ICP4. It was determined that the amino-terminal 60 percent of the ICP4 gene was sufficient for trans-induction of a thymidine kinase promoter-CAT chimera (pTKCAT) and negative regulation of an ICP4 promoter-CAT chimera (pIE3CAT); however, it was relatively inefficient in complementing an ICP4 deletion mutant. The amino-terminal ninety amino acids do not appear to be required for infectivity as reflected by the replication competence of a mutant virus containing a linker insertion at amino acid 12. The size of the ICP4 molecule expressed from the mutant virus was consistent with translational restart at the next methionine codon corresponding to amino acid 90 of the deduced ICP4 amino acid sequence.

Amino Acid Sequence↗

PubMed1987

Linker-insertion nonsense and restriction-site deletion mutations of the gB glycoprotein gene of herpes simplex virus type 1.

To study the effects of missense, nonsense, and deletion mutations of the gB glycoprotein gene of herpes simplex virus type 1, a gB-transformed cell line was isolated that, after virus infection, would express sufficient quantities of gB from the cellular chromosome to complement temperature-sensitive gB mutants. The transformed cell line was then used as a permissive cell to transfer two gB mutations from plasmid to viral DNA. One of the mutants, K082, harbored an HpaI linker insertion that introduced one new amino acid and a chain terminator codon within amino acid residue 43. The other mutant contained a 969-base-pair deletion in a part of the gene that includes the membrane-spanning region; a correspondingly shorter gB polypeptide was detected by sodium dodecyl sulfate-gel electrophoresis after immunoprecipitation of infected-cell extracts with four pooled monoclonal antibodies. No polypeptide was observed from K082-infected cells. The shortened gB polypeptide was efficiently processed and secreted into the growth medium. Each of the four monoclonal antibodies precipitated full-length gB, and three of the four precipitated the shortened polypeptide. Enveloped virus particles could be purified after infection of nonpermissive cells with either mutant virus. Virus particles appeared to possess normal polypeptide and glycopeptide profiles except for the absence of gB. Therefore, the presence of gB is not essential for viral assembly, including envelopment. Recombinants in virus stocks grown on the gB-transformed cells occurred at frequencies on the order of 10(-7) to 10(-5), compared with a frequency of approximately 10(-2) in mixed infections with the two mutants.

Base Sequence↗

PubMed1985

Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate-early regulatory protein ICP4.

Using Vero cells transformed with the wild-type gene for ICP4 as the permissive host cell, we isolated herpes simplex virus type 1 (HSV-1) mutants containing deletions in both copies of the ICP4 gene. The mutants, d120 and d202, contained deletions of 4.1 and 0.5 kilobases, respectively, in each copy of ICP4. ICP4 mRNA synthesized in d202-infected Vero cells was 0.5 kilobases smaller than that synthesized in cells infected with the wild-type virus. No ICP4 mRNA was detected in d120-infected Vero cells. d120 and d202 specified polypeptides that reacted with ICP4 antiserum and were smaller than the wild-type ICP4 polypeptide by 130 and 30 kilodaltons, respectively. The only other HSV-1 gene products detectable on infection of Vero cells with d120 and d202 were ICP6 (of the early kinetic class of HSV-1 polypeptides), ICP0 (immediate early), ICP22 (immediate early), and ICP27 (immediate early). Immediate-early polypeptides ICP0 and ICP27 were expressed to a higher level in Vero cells infected with an ICP4 temperature-sensitive (ts) mutant (tsB32) at 39 degrees C, indicating immediate-early stimulatory activity associated with the ts ICP4 polypeptide. In addition, the patterns of complementation of d120, d202, and tsB32 in ICP4-transformed cells also demonstrated inhibitory activity associated with the ts form of the ICP4 polypeptide.

Cell Transformation, Viral↗

PubMed1985

Activation of immediate-early, early, and late promoters by temperature-sensitive and wild-type forms of herpes simplex virus type 1 protein ICP4.

To better define the activities on herpes simplex virus type 1 gene expression of temperature-sensitive and wild-type forms of the transcriptional regulatory protein ICP4, regulatory sequences from immediate-early, early, and late herpes simplex virus genes were fused to the gene for chloramphenicol acetyltransferase (CAT). These constructs were used in trans induction and cotransfection experiments with wild-type and temperature-sensitive mutant alleles of ICP4. The ICP4 genes used in this study were cloned from the KOS strain (wild type) and two phenotypically distinct temperature-sensitive ICP4 mutants, tsB32 and tsL14 (DeLuca et al., J. Virol. 52:767-776, 1984), both alone and in conjunction with three other immediate-early genes. The latter series of plasmids was used to assess the influence of additional immediate-early gene products on gene expression in the presence of a given ICP4 allele. The results of this study demonstrate that the phenotypes of these ICP4 mutants observed in cell culture at the nonpermissive temperature were determined in part by activities associated with the mutant ICP4 polypeptides and that these activities differed from those of wild-type ICP4. Low levels of wild-type ICP4 had a marginal but reproducible stimulatory effect on immediate-early CAT gene expression, especially the pIE4/5CAT chimera. This effect was diminished with increasing quantities of ICP4, suggesting an inhibitory role for the wild-type form of the protein. The ICP4 mutants had a strong stimulatory effect on immediate-early CAT expression, consistent with their phenotypes at 39 degrees C. The mutant forms of the ICP4 polypeptide differed in their ability to induce CAT activity from an early chimeric gene. Thus, the tsL14 form of ICP4 was effective in early gene induction (i.e., ptkCAT was induced), whereas the ICP4 derived from tsB32 was slightly inhibitory. Cotransfection of tsB32 ICP4 simultaneously with other immediate-early genes resulted in a marginal increase in ptkCAT induction. This induction was enhanced when the gene for ICP4 was inactivated by restriction enzyme cleavage, substantiating the inhibitory effect of the tsB32 form of ICP4. The two mutant ICP4 genes (tsB32 and tsL14) were unable to trans-activate either of the late CAT constructs (p5CAT and pL42CAT) tested. Cotransfecting tsL14 ICP4 with the other immediate-early genes resulted in activation of p5CAT but not pL42CAT. Taken together, these studies demonstrate that (i) low levels of wild-type ICP4 have stimulatory effect on immediate-early promoters and that higher concentrations of wild-type ICP4 have an inhibitory effect on these promoters, (ii) isolated mutant form of ICP4 exhibit activities that reflect the phenotypes of the mutants from which they were isolated, and (iii) immediate-early gene products other than ICP4 are involved in determining the distinct phenotypes of the two mutants at 39 degrees Celsius.

Acetyltransferases↗

PubMed1984

Nucleotide sequence of a region of the herpes simplex virus type 1 gB glycoprotein gene: mutations affecting rate of virus entry and cell fusion.

The tsB5 isolate of herpes simplex virus type I (HSV-1) enters host cells more rapidly than does KOS, an independent isolate of HSV-1, and this rate-of-entry determinant is located between prototypic map coordinates 0.350 and 0.360 (1). The nucleotide sequence of strain tsB5 has now been determined between prototypic map coordinates 0.347 and 0.360. Comparison of the tsB5 sequence to the homologous KOS sequence revealed that the rate-of-entry difference between these two HSV-1 strains may be due to the single amino acid difference observed within these sequences (0.350 to 0.360). A cell fusion determinant in tsB5 is located between coordinates 0.345 and 0.355 and to the left of the rate-of-entry determinant (1). Nucleotide sequence analysis revealed a second amino acid difference between tsB5 and KOS at coordinate 0.349. The cell fusion determinant was tentatively assigned to this location.

Amino Acid Sequence↗

PubMed1984

Nucleotide sequence specifying the glycoprotein gene, gB, of herpes simplex virus type 1.

The nucleotide sequence thought to specify the glycoprotein gene, gB, of the KOS strain of herpes simplex virus type 1 (HSV-1) has been determined. A 3.1-kilobase (kb), viral-specified RNA was mapped to the left half of the BamHI-G fragment (0.345 to 0.399 map units). TATA, CAT-box, and possible mRNA start sequences characteristic of HSV-1 genes are found near 0.368 map units. The first available ATG codon is at 0.366 and the first in-phase chain terminator at 0.348 map units. A polyA-addition signal (AATAAA) occurs 17 nucleotides past the chain terminator. Translation of these sequences would yield a 100.3-kilodalton (kDa) polypeptide characterized by a 5' signal sequence, nine N-linked saccharide addition sites, a strongly hydrophobic membrane-spanning sequence, and a highly charged 3' cytoplasmic anchor sequence. Two mutants of KOS, tsJ12 and tsJ20, that are temperature-sensitive for viral growth and for the production of gB, have been physically mapped to 0.357 to 0.360 and 0.360 to 0.364 map units, respectively (DeLuca et al., in preparation). The nucleotide sequence of the mutants was determined in these regions. In both cases a single amino acid replacement within the 100.3-kDa polypeptide is predicted from the sequence analysis.

Amino Acid Sequence↗

PubMed1984

Temperature-sensitive mutants in herpes simplex virus type 1 ICP4 permissive for early gene expression.

A large number of temperature-sensitive (ts) mutants of herpes simplex virus type 1 (HSV-1) in the gene encoding the immediate-early transcriptional regulatory protein, ICP4, have been isolated and characterized with respect to expression of the immediate-early, early, and late viral gene products. The hallmark of these mutants is the overproduction of immediate-early gene products and the underproduction of early and late gene products. The present study involves the preliminary genetic and molecular characterization of two unique regulatory mutants of HSV-1, ts48 and ts303. Genetically, both mutants exhibit inefficient complementation with eight ts mutants in complementation group 1-2, which defines the gene for ICP4, and marker rescue experiments place the mutations in both mutants in the 3' portion of the coding sequence for ICP4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of ts48- and ts303-infected cell polypeptides synthesized at the nonpermissive temperature demonstrates that immediate-early polypeptides ICP4 and ICP27 are overproduced, with the simultaneous production of early polypeptides ICP6, ICP8, gB, and others. Immediate-early polypeptides are resynthesized upon temperature shift-up early in infection; however, shift-up late in infection does not result in the resynthesis of immediate-early polypeptides. Late gene products are either absent or underrepresented under long-term labeling conditions. To examine the effects of the mutations in ts48, ts303, and other ICP4 mutants specifically on early gene expression, trans-induction experiments were performed in cells transfected with the gene for chloramphenicol acetyltransferase under early gene control (tk) and superinfected with KOS, tsB32, ts48, and ts303. Mutant tsB32 did not induce chloramphenicol acetyltransferase activity above the basal level; however, ts48 and ts303 induced chloramphenicol acetyltransferase activity nearly equal to wild-type levels. Fifteen to fifty percent of wild-type levels of viral DNA are synthesized at the nonpermissive temperature in ts48- and ts303-infected cells, indicating that immediate-early and early gene functions are intact (or nearly so) and that the block in ts48 and ts303 is in a regulatory event subsequent to that exhibited by other mutants in complementation group 1-2 which are DNA-.

Chromosome Mapping↗