Linker Insertion Mutations Research Articles

Herpes simplex virus type 1 glycoprotein E domains involved in virus spread and disease.

Herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) functions as an immunoglobulin G (IgG) Fc binding protein and is involved in virus spread. Previously we studied a gE mutant virus that was impaired for IgG Fc binding but intact for spread and another that was normal for both activities. To further evaluate the role of gE in spread, two additional mutant viruses were constructed by introducing linker insertion mutations either outside the IgG Fc binding domain at gE position 210 or within the IgG Fc binding domain at position 380. Both mutant viruses were impaired for spread in epidermal cells in vitro; however, the 380 mutant virus was significantly more impaired and was as defective as gE null virus. gE mutant viruses were inoculated into the murine flank to measure epidermal disease at the inoculation site, travel of virus to dorsal root ganglia, and spread of virus from ganglia back to skin to produce zosteriform lesions. Disease at the inoculation and zosteriform sites was reduced for both mutant viruses, but more so for the 380 mutant virus. Moreover, the 380 mutant virus was highly impaired in its ability to reach the ganglia, as demonstrated by virus culture and real-time quantitative PCR. The results indicate that the domain surrounding amino acid 380 is important for both spread and IgG Fc binding and suggest that this domain is a potential target for antiviral therapy or vaccines.

Characterization of a panel of insertion mutants in human cytomegalovirus glycoprotein B.

Glycoprotein B (gB; gpUL55) of human cytomegalovirus (HCMV) plays a critical role in virus entry and cell-to-cell spread of infection. To define the structure-function relationships in gB, a panel of linker-insertion mutations was generated throughout the coding region. This strategy yielded a panel of 22 mutants with four amino acid insertions and 3 large truncation mutants. Assessment of the mutant proteins' biosynthetic properties and folding patterns analyzed in context with predicted secondary features revealed novel insights into gB's structure and trafficking properties. All of the insertion mutants were able to assemble into oligomers, suggesting that oligomerization is tolerant of small insertions and/or that multiple regions of the protein may be involved. Computer algorithm predictions of gB's secondary structure indicate that the furin-recognized cleavage site falls within an exposed loop. This loop may be particularly sensitive to structural alterations, since insertions upstream and downstream of the cleavage site rendered the mutant proteins cleavage defective. In addition, a strong correlation existed between terminal folding and cleavage of gB. Interestingly, terminal folding was not correlated with delivery to the cell surface but may influence the rate of transport to the cell surface. Nine mutants, containing insertions in both the extracellular and intracellular portions of gB, retained wild-type structural properties. This panel of characterized gB mutants, the first of this type for an HCMV protein, will be a useful tool in dissecting the role of gB during HCMV infection.

Rb Independent Inhibition of Cell Growth by p15INK4B

The INK4 cyclin dependent kinase inhibitors (CDKI), such as p15INK4B and p16INK4A, block cell cycle progression from G to S phase. This is mediated by inhibition of phosphorylation of proteins, including the retinoblastoma susceptibility protein (Rb), by cyclin dependent kinases. Ectopic over-expression of the p16INK4A CDKI can inhibit growth of cell lines depending on Rb status. Cell lines lacking Rb, with few exceptions, are resistant to growth inhibition by p16INK4A. The effects of ectopic over-expression of p15INK4B in cell lines with and without wild type Rb were examined by measuring cell recovery. Proliferation was inhibited in cells lacking Rb as well as in cells with wild type Rb expression. Experiments analyzing the effectiveness of chimeric p15INK4B/p16INK4A proteins indicated that the Rb independent growth inhibition required N-terminal residues of p15INK4B. Linker insertion mutation of p15INK4B showed that the inhibition was dependent on intact ankyrin structures. Double staining flow cytometry found that the growth inhibition correlated with a decrease in cells in G2/M phases of the cell cycle. These findings are consistent with Rb independent inhibition of the progression from G1 to S caused by overexpression of p15INK4B.

Functional analysis of carboxy-terminal deletion mutants of c-Myb.

The c-myb gene is implicated in the differentiation and proliferation of hematopoietic cells. Truncations of the N and/or C terminus of c-Myb, found in v-Myb, can potentiate its transforming ability. Two negative regulatory subregions, located in the C terminus, were mapped previously by using GAL4-c-Myb fusion proteins in transient transfection assays for the transcriptional activation of a GAL4-responsive reporter gene. To dissect the C terminus of c-Myb in terms of its involvement in transcriptional activation and oncogenic transformation, a series of C-terminal deletion mutants of c-Myb were analyzed. In addition, linker insertion mutants within the transactivation domain and/or heptad leucine repeat of c-Myb were examined along with those deletion mutants. In this study, we demonstrated that the removal of both of the two previously mapped negative regulatory subregions from the native form of c-Myb not only supertransactivates a Myb-responsive reporter gene but also potentiates its transforming ability in culture. However, in contrast to previous results, cells transformed by all of the mutants analyzed here except v-Myb itself exhibited the same phenotype as those transformed by c-Myb. The proliferating cells were bipotenial and differentiated into both the granulocytic and monocytic lineages. This result implies that the C terminus of c-Myb alone has no effect on the lineage determination. Finally, the transactivation activities of these mutants correlated with their transforming activities when a mim-1 reporter gene was used but not when a model promoter containing five tandem Myb-binding sites was used. In particular, a very weakly transforming mutant with a linker insertion in the heptad leucine repeat superactivated the model promoter but not the mim-1 reporter gene.

Insertion mutagenesis of XpsD, an outer-membrane protein involved in extracellular protein secretion in Xanthomonas campestris pv. campestris.

XpsD is an outer-membrane protein required for extracellular protein secretion in Xanthomonas campestris pv. campestris. Cross-linking and gelfiltration chromatography analyses have suggested that it forms a multimer. To determine its structure-function relationship, linker-insertion mutants were constructed in an xpsD gene carried on a plasmid. To assay for secretion function, each mutant gene was introduced into an xpsD::Tn5 mutant strain (XC1708) and assayed for alpha-amylase secretion on starch plates. To test whether the mutant genes exerted a dominant-negative effect, each was introduced into the parental strain XC1701 and examined for secretion interference. Nine functional, one semi-functional and eleven non-functional mutants were obtained. All the non-functional mutants, except two for which the mutant proteins were undetectable on immunoblots, showed interference of normal secretion. The insertion sites in the different mutant proteins are randomly distributed throughout the entire sequence of the XpsD protein. All the permissive insertion sites are located where beta-turn or coiled secondary structure is predicted. Over half of the non-permissive sites are located within predicted helical or beta-sheet regions. By pretreating total membranes of XC1701 in SDS at 50 degrees C, an immunoreactive band with high molecular mass (HMM) could be detected that remained in the stacking gel during SDS-PAGE. The semi-functional and all functional mutant proteins formed HMM complexes that were as SDS-resistant as those of the wild-type, whereas all except three of the non-functional mutant proteins formed HMM structures that were less resistant to SDS than the wild-type. By analysing the appearance of SDS-resistant HMM complexes, we were able to detect conformational alterations in XpsD that are too subtle to be detected by other assays.

Mutational Analysis of Stem-Loops in the RNA Packaging Signal of the Moloney Murine Leukemia Virus

The retroviral RNA genome is targeted for incorporation into the nascent virion particle by the ψ region, a specific block of RNA sequences near the 5′ end. A number of deletions and linker insertion mutations were introduced into the ψ region of cloned DNA of the Moloney murine leukemia virus, and the mutants were introduced into cells in culture and tested for their ability to direct the assembly of virions and the packaging of viral RNA. Only a small portion of the ψ region was important for packaging, containing the so-called stem-loops C and D. Additional mutants were used to demonstrate that the base pairing of stem D, and the sequence of loop D, were essential for normal packaging of the RNA. Two mutants with alterations near the 5′ splice donor were also replication-defective, probably due to effects on gene expression. The results allow a high-resolution definition of the RNA structures required during virus replication in culture.

Truncation of the C-terminal acidic transcriptional activation domain of herpes simplex virus VP16 produces a phenotype similar to that of the in1814 linker insertion mutation.

We examined the phenotype of a herpes simplex virus (HSV) type 1 mutant (V422) in which the C-terminal acidic activation domain of the virion transactivator VP16 is truncated at residue 422. The efficiency of plaque formation by V422 on Vero cells was boosted by approximately 100-fold by including hexamethylene bis-acetimide (HMBA) in the growth medium, as previously observed with the in1814 VP16 linker insertion mutant isolated by Preston and colleagues. V422 displayed severely reduced levels of the immediate-early transcripts encoding ICP0 and ICP4 during infection in the presence of cycloheximide, and this defect was partially overcome by the addition of HMBA. The defect in plaque formation exhibited by V422 and in 1814 was efficiently complemented in U2OS osteosarcoma cells, which had previously been shown to complement ICP0 null mutations. Taken in combination, these data confirm the key role of VP16 in triggering the onset of the HSV lytic cycle.

Site-directed and linker insertion mutagenesis of herpes simplex virus type 1 glycoprotein H.

The gH-gL complex of herpes simplex virus type 1 (HSV-1) is essential for virion infectivity and virus-induced cell fusion, but functional domains of the gH molecule remain to be defined. We have addressed this question by mutagenesis. A set of linker insertion mutants in HSV-1 gH was generated and tested in transient assays for their ability to complement a gH-negative virus. Insertions at three sites in the C-terminal third of the external domain affected the ability of gH to function in cell-cell fusion and virus entry, while insertions at six sites in the N-terminal half of the external domain induced conformational changes in gH such that it was not recognized by monoclonal antibody LP11, although expression at the cell surface was unchanged. A recombinant virus in which a potential integrin-binding motif, RGD, in gH was changed to the triplet RGE entered cells as efficiently as the wild type, indicating that HSV-1 entry is not mediated by means of the gH-RGD motif binding to cell surface integrins. Furthermore, mutagenesis of the glycosylation site which is positionally conserved in all herpesvirus gH sequences in close proximity to the transmembrane domain generated a recombinant virus that grew in vitro with wild-type single-step kinetics.

Structural analysis of the adenovirus type 5 E1B 55-kilodalton-E4orf6 protein complex.

The adenovirus type 5 (Ad5) early 1B (E1B) 55-kDa (E1B-55kDa)-E4orf6 protein complex has been implicated in the selective modulation of nucleocytoplasmic mRNA transport at late times after infection. Using a combined immunoprecipitation-immunoblotting assay, we mapped the domains in E1B-55kDa required for the interaction with the E4orf6 protein in lytically infected A549 cells. Several domains in the 496-residue 55-kDa polypeptide contributed to a stable association with the E4orf6 protein in E1B mutant virus-infected cells. Linker insertion mutations at amino acids 180 and 224 caused reduced binding of the E4orf6 protein, whereas linker insertion mutations at amino acid 143 and in the central domain of E1B-55kDa eliminated the binding of the E4orf6 protein. Earlier work showing that the central domain of E1B-55kDa is required for binding to p53 and the recent observation that the E4orf6 protein also interacts with the tumor suppressor protein led us to suspect that p53 might play a role in the E1B-E4 protein interaction. However, coimmunoprecipitation assays with extracts prepared from infected p53-negative H1299 cells established that p53 is not needed for the E1B-E4 protein interaction in adenovirus-infected cells. Using two different protein-protein interaction assays, we also mapped the region in the E4orf6 protein required for E1B-55kDa interaction to the amino-terminal 55 amino acid residues. Interestingly, both binding assays established that the same region in the E4orf6/7 protein can potentially interact with E1B-55kDa. Our results demonstrate that two distinct segments in the 55-kDa protein encoding the transformation and late lytic functions independently interact with p53 and the E4orf6 protein in vivo and provide further insight by which the multifunctional 55-kDa EIB protein can exert its multiple activities in lytically infected cells and in adenovirus transformation.

Mapping regions of herpes simplex virus type 1 glycoprotein I required for formation of the viral Fc receptor for monomeric IgG.

Glycoprotein E (gE) and glycoprotein I (gI) of herpes simplex virus type 1 (HSV-1) form a complex that binds the Fc domain of monomeric IgG. In this study, we used two approaches to map the regions of gI-1 required for formation of the HSV-1 Fc receptor for monomeric IgG. First, we constructed six plasmids encoding gD-1/gI-1 fusion proteins. Each fusion protein contains a large gI-1 peptide inserted into the ectodomain of gD-1. gD-1/gI-1 fusion proteins were coexpressed with gE-1 using a transfection-infection assay in which cells were transfected with individual fusion protein constructs and then infected with a gE+/gI- virus. Cells were then assayed for monomeric IgG binding using immunofluorescence microscopy. Transfection-infection with two of six fusion proteins conferred monomeric IgG binding activity to cells, whereas cells infected with gE+/gI- virus alone failed to bind IgG monomers. The smallest gI-1 peptide to confer monomeric IgG binding activity contained amino acids 43 to 192. To more precisely map the region of gI-1 required for monomeric IgG binding, we constructed a panel of 10 gI-1 linker insertion mutants. Transfection-infection studies identified two mutants containing linker insertions at gI-1 amino acids 128 and 145, which failed to bind monomeric IgG. The other eight mutants demonstrated wild-type IgG binding activity. Taken together, these results indicate that the region of gI-1 between amino acids 128 and 145 is required for formation of the HSV-1 Fc receptor for monomeric IgG.

The role of Gag in human immunodeficiency virus type 1 virion morphogenesis and early steps of the viral life cycle.

The phenotypes of a series of mutant human immunodeficiency virus type 1 proviruses with linker insertion and deletion mutations within the gag coding region were characterized. These mutants, with mutations in the matrix, capsid, and p2 coding regions, produced replication-defective virion particles with defects in the early steps of the viral life cycle. To investigate this phenotype further, the abilities of mutant virion particles to enter T cells, initiate and complete reverse transcription, and transport the newly transcribed proviral DNA were investigated. Only 4 of 10 of the mutants appeared to make wild-type levels of viral DNA. Biochemical analyses of the mutants revealed the middle region of CA as being important in determining virion particle density and sedimentation in velocity gradients. This region also appears to be critical in determining the morphology of mature virion particles by electron microscopy. Particles with aberrant morphology were uninfectious, and only those mutants which displayed cone-shaped cores were capable of carrying out the early steps of the viral life cycle. Thus, the normal morphology of human immunodeficiency virus type 1 appears to be critical to infectivity.

Linker insertion scanning of regA, an activator of exotoxin A production in Pseudomonas aeruginosa.

RegA is a transcriptional activator that controls exotoxin A (ETA) production in Pseudomonas aeruginosa. To date, functional assays performed with the purified protein have not clearly defined the molecular mechanism of action of RegA. In this study, we sought to identify important coding regions of regA by analysing the sequences around linker insertion mutations in regA that affected toxA transcription. First, we constructed a strain with the regAB locus deleted from the chromosome, PA103 delta regAB::Gm. toxA transcription was obliterated in strain PA103 delta regAB::Gm, demonstrating that the regAB locus is essential for ETA production. Next, we constructed a series of 6 bp linker insertion mutations distributed throughout regA. These regA linker insertion mutants were sequenced and screened in PA103 delta regAB::Gm for their effects on regulation of ETA production. Six linker insertion mutations occurring between amino acids (aa) 53 and 163 of RegA were isolated that resulted in depression of toxA transcription to varying levels relative to the parental regAB locus. One of these linker insertion mutations (pTR53), resulted in a lack of iron-regulated ETA production and occurred directly upstream from a predicted transmembrane alpha-helix. The other five linker mutations (pTR88, pTR124, pTR132, pTR132-2 and pTR163) occurred within or flanked a region of RegA between aa 87-142 with similarity to the transcriptional activation domains of ToxR, VirG and OmpR. These data suggest the presence of a previously unidentified transcriptional activation domain in RegA between aa 87-142 and implicate the predicted transmembrane alpha-helix in the N-terminus as being involved in sensory transduction.

Influenza virus polymerase basic protein 1 interacts with influenza virus polymerase basic protein 2 at multiple sites

Three polymerase proteins of influenza type A virus interact with each other to form the active polymerase complex. Polymerase basic protein 1 (PB1) can interact with PB2 in the presence or absence of polymerase acidic protein. In this study, we investigated the domains of PB1 involved in complex formation with PB2 in vivo, using coexpression and coimmunoprecipitation of the PB1-PB2 complex with monospecific antibodies. Results show that PB1 possesses at least two regions which can interact independently and form stable complexes with PB2. Both of these regions are located at the NH2 terminus of PB1; the COOH-terminal half of PB1 is not involved in interacting with PB2. Deletion analysis further demonstrated that the interacting regions of PB1 encompass amino acids (aa) 48 to 145 and aa 251 to 321. Linker insertions throughout the PB1 sequences did not affect complex formation with PB2. Deletion and linker-insertion mutants of PB1 were tested for polymerase activity in vivo. For this analysis, we developed a simplified assay for viral polymerase activity that uses a reporter chloramphenicol acetyltransferase gene containing the 5' and 3' ends of influenza viral promoter and nontranslating regions (minus sense) of the NS gene joined to a hepatitis delta virus ribozyme at its 3' end. This assay demonstrated that all deletion mutants of PB1 exhibited either background or greatly reduced polymerase activity irrespective of the ability to interact with PB2 and that all linker-insertion mutants except one at the extreme COOH end (L-746) of PB1 were also negative for viral polymerase activity. These results show that compared with complex formation of PB1 with PB2, the polymerase activity of PB1 was extremely sensitive to structural perturbation.

Identification of the Receptor-Binding Regions of pb5 Proteins of Bacteriophages T5 and BF23

The receptor-binding protein pb5T5of bacteriophage T5, when expressed from theoadgene cloned in pVK88 under the control of the phage T7 promoter/polymerase system, has been shown to bind to its FhuA receptor on the surface ofE. coli,where it blocks FhuA for subsequent adsorption of T5 (Mondigleret al., FEMS Microbiol. Lett.,130, 293–300, 1995). In the present study the blocking assay has been applied to analyze the effects of several mutations withinoadon the FhuA-binding properties of corresponding pb5 derivatives. Three classes of mutations were tested: (i)oaddeletion derivatives, (ii) theoadmutation known to interfere with FhuA-binding of T5 (Heller and Bryniok,J. Virol.,49, 20–25, 1984), and (iii) linker-insertion mutations at a site very close to theoadmutation. Of the corresponding pb5 derivatives only one, a deletion derivative lacking the 153 C-terminal amino acids, was as active in the blocking assay as wild-type pb5T5. All other derivatives were inactive or almost inactive. Isolation and molecular characterization of phenotypic revertants of T5oadshowed that all revertants were true genotypic revertants of theoadmutation. Theoadmutation has been identified as a G to T exchange resulting in a substitution of Gly for Trp at position 166 of pb5T5. DNA sequencing of thehrsgene of bacteriophage BF23 and comparing the deduced amino acid sequence of pb5BF23with that of pb5T5revealed distinct regions of similarity and nonsimilarity. We propose that the receptor-binding region of pb5T5(pb5BF23) is formed by the region of nonsimilarity extending from amino acid position 89 (88) to position 305 (283).

Human adenovirus type 5 variants with sequence alterations flanking the E2A gene: effects on E2 expression and DNA replication.

The human adenovirus type 5 (Ad5) E2 transcription unit is divided into a promoter-proximal region, E2A, and a distal region, E2B, each with its own polyadenylation site. Together these regions encode the three virus-derived proteins necessary for genome replication. Ad5 variants were produced that carried linker insertion mutations immediately 5' and/or 3' to the coding sequence for the E2A gene DNA binding protein (DBP). Two variants carrying solely a 5' lesion showed decreased usage of the adjacent 3' splice site, via which the DBP mRNA is produced, and an increased usage of the alternative downstream splice sites in the E2B region, wherein viral DNA polymerase and terminal protein precursor are encoded; these viruses showed somewhat reduced growth. A variant carrying a 3' lesion showed a marginal increase in DBP expression and slightly accelerated growth. When lesions 5' and 3' to the DBP coding sequence were combined in cis, the resulting virus was severely defective for growth and expressed E2B products to the virtual exclusion of E2A DBP. These data indicate that interactions must occur between the E2A 3' splice site and polyadenylation site before this region can be treated as an exon by the RNA processing machinery, and that a sequence alteration at the polyadenylation site that alone has only minor effects on the pattern of RNA processing can drastically affect terminal exon usage when placed in cis with a mutation that reduces splicing efficiency at the upstream 3' splice site. The data further indicate that, in vivo, Ad5 DNA replication is limited by prevailing DBP levels rather than by levels of polymerase or terminal protein precursor.

Immunocytochemical analysis of hormone mediated nuclear translocation of wild type and mutant glucocorticoid receptors

We have analyzed structural and functional features of the human glucocorticoid receptor (hGR) for their effects on receptor subcellular distribution. COS 1 cells transiently transfected with wild type and mutant hGR cDNAs were assessed immunocytochemically using well-characterized antipeptide antibodies to the hGR. The effect of administration of steroid hormones (and the antiglucocorticoid RU486) on receptor localization was evaluated. Unliganded wild type receptors expressed in COS 1 cells were predominately cytoplasmic. Addition of glucocorticoids or the glucocorticoid receptor antagonist, RU486, resulted in complete translocation of these receptors into the nucleus whereas non-glucocorticoid steroids or dibutyryl cAMP were not effective in promoting nuclear translocation. Thus, nuclear translocation was specific for steroids capable of high affinity binding to the hGR. To elucidate the potential role of receptor domains in receptor localization, COS 1 cells transiently transfected with various receptor cDNA mutants were analyzed in a similar manner. Translocation of an hGR deletion mutant lacking the majority of the amino terminus (deletion of amino acids 77–262) was identical to the wild type receptor despite the absence of a transactivation domain. Receptors in which the DNA binding domain was either partially or totally deleted showed an impaired capacity to undergo hormone-inducible nuclear translocation. Deletion of the hinge region of the hGR (which also contains part of the nuclear localization signal, NL1) resulted in receptor localization in the cytoplasm. Mutants in the ligand binding domain exhibited two localization phenotypes, exclusively nuclear or cytoplasmic. Receptor mutants truncated after amino acid 550 were found in the nucleus in the presence and absence of hormone consistent with the existence of nuclear localization inhibitory sequences in the ligand binding domain of the receptor. However, a linker insertion mutant (at amino acid 582) which results in a receptor deficient in ligand binding did not undergo nuclear translocation indicating that nuclear localization inhibitory sequences were intact in this mutant. The role of receptor phosphorylation on hormone induced nuclear translocation was also examined. Mouse glucocorticoid receptors which contained mutations of certain hormone inducible phosphorylation sites exhibited translocation properties similar to wild type mGR indicating that these phosphorylation sites on the receptor do not play a major role in hormone inducible nuclear translocation.

Mutations that specifically impair the DNA binding activity of the herpes simplex virus protein UL42.

The herpes simplex virus DNA polymerase is a heterodimer consisting of a catalytic subunit and the protein UL42, which functions as a processivity factor. It has been hypothesized that UL42 tethers the catalytic subunit to the DNA template by virtue of DNA binding activity (J. Gottlieb, A. I. Marcy, D. M. Coen, and M. D. Challberg, J. Virol. 64:5976-5987, 1990). Relevant to this hypothesis, we identified two linker insertion mutants of UL42 that were unable to bind to a double-stranded-DNA-cellulose column but retained their ability to bind the catalytic subunit. These mutants were severely impaired in the stimulation of long-chain-DNA synthesis by the catalytic subunit in vitro. In transfected cells, the expressed mutant proteins localized to the nucleus but were nonetheless deficient in complementing the growth of a UL42 null virus. Thus, unlike many other processivity factors, UL42 appears to require an intrinsic DNA binding activity for its function both in vitro and in infected cells. Possible mechanisms for the activity of UL42 and its potential as a drug target are discussed.

Proteus mirabilis urease: operon fusion and linker insertion analysis of ure gene organization, regulation, and function

Urease is an inducible virulence factor of uropathogenic Proteus mirabilis. Although eight contiguous genes necessary for urease activity have been cloned and sequenced, the transcriptional organization and regulation of specific genes within the Proteus gene cluster has not been investigated in detail. The first gene, ureR, is located 400 bp upstream and is oriented in the direction opposite the other seven genes, ureDABCEFG. The structural subunits of urease are encoded by ureABC. Previously, UreR was shown to contain a putative helix-turn-helix DNA-binding motif 30 residues upstream of a consensus sequence which is a signature for the AraC family of positive regulators; this polypeptide is homologous to other DNA-binding regulatory proteins. Nested deletions of ureR linked to either ureD-lacZ or ureA-lacZ operon fusions demonstrated that an intact ureR is required for urea-induced synthesis of LacZ from either ureA or ureD and identified a urea-regulated promoter in the ureR-ureD intergenic region. However, lacZ operon fusions to fragments encompassing putative promoter regions upstream of ureA and ureF demonstrated that no urea-regulated promoters occur upstream of these open reading frames; regions upstream of ureR, ureE, and ureG were not tested. These data suggest that UreR acts as a positive regulator in the presence of urea, activating transcription of urease structural and accessory genes via sequences upstream of ureD. To address the role of the nonstructural regulatory and accessory genes, we constructed deletion, cassette, and linker insertion mutations throughout the ure gene cluster and determined the effect of these mutations on production and regulation of urease activity in Escherichia coli. Mutations were obtained, with locations determine by DNA sequencing, in all genes except ureA and ureE. In each case, the mutation resulted in a urease-negative phenotype.

Mutational analysis of the herpes simplex virus virion host shutoff protein: evidence that vhs functions in the absence of other viral proteins

Herpes simplex virus (HSV) virions contain one or more factors that trigger rapid shutoff of host protein synthesis and accelerated decay of cellular and viral mRNAs in infected cells. HSV isolates bearing mutations at the virion host shutoff (vhs) locus (gene UL41) are defective for both processes, indicating that the vhs protein is required; however, it is not clear whether the role of vhs in shutoff is direct or indirect and if other virion components are also necessary. We therefore used a transient-cotransfection assay to determine if the vhs protein displays activity in the absence of other viral gene products. We found that a vhs expression vector strongly suppressed expression of a cotransfected lacZ reporter gene and that this effect was eliminated by the vhs1 point mutation that abolishes virion-induced host shutoff during HSV infection. Further evidence for the biological relevance of the transfection assay came from the demonstration that five vhs in-frame linker insertion mutations yielded concordant results when assayed in cotransfected cells and following transfer into the viral genome: three mutations eliminated activity in both assays, while two had no effect. On the basis of these results, we conclude that the vhs protein can trigger host shutoff in the absence of other HSV proteins. The cotransfection assay was used to rapidly assess the activities of a panel of linker insertion mutants spanning the vhs polypeptide. All mutations that mapped to regions conserved among the vhs homologs of alphaherpesvirus inactivated function; in contrast, four of five mutations that mapped to regions that are absent from several vhs homologs had no effect. These results further support the biological relevance of the transfection assay and begin to delineate functional domains of the vhs polypeptide.

Bipartite DNA-binding region of the Epstein-Barr virus BMRF1 product essential for DNA polymerase accessory function

The Epstein-Barr virus (EBV) BMRF1 gene product is necessary for DNA polymerase catalytic subunit (BALF5) activity in 100 mM ammonium sulfate. To map regions of BMRF1 necessary for polymerase accessory function, linker insertion and deletion mutant BMRF1 polypeptides were expressed by in vitro transcription-translation and assayed for DNA polymerase elongation activity and binding to double-stranded DNA (dsDNA)-cellulose. Amino-terminal deletions up to residue 303 were defective for stimulation of elongation. Deletions between residues 44 and 194 and residues 238 and 303 abolished binding to dsDNA-cellulose. The region from residues 194 to 238, therefore, is necessary for stimulation of BALF5 elongation but dispensable for dsDNA-cellulose binding. Deletion analysis also localized reactive epitopes of two neutralizing monoclonal antibodies to BMRF1 to a carboxy-terminal region which is dispensable for activity. These data suggest that a bipartite DNA-binding region is an essential component of the DNA polymerase accessory function and that the two noncontiguous regions are separated by a region (residues 194 to 217) which is essential for stimulation; therefore, it may interact with the BALF5 catalytic subunit of EBV DNA polymerase. Both EBV BMRF1 and herpes simplex virus UL42 gene products are DNA polymerase accessory proteins which bind dsDNA and increase the processivity of their corresponding catalytic components. Outstanding similarities between their primary amino acid sequences are not evident. However, it appears that their structural organizations are similar.