The PK domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is required for immediate-early gene expression and virus growth

The large subunit of herpes simplex virus (HSV) ribonucleotide reductase (RR), RR1, contains a unique amino-terminal domain which has serine/threonine protein kinase (PK) activity. To examine the role of the PK activity in virus replication, we studied an HSV type 2 (HSV-2) mutant with a deletion in the RR1 PK domain (ICP10DeltaPK). ICP10DeltaPK expressed a 95-kDa RR1 protein (p95) which was PK negative but retained the ability to complex with the small RR subunit, RR2. Its RR activity was similar to that of HSV-2. In dividing cells, onset of virus growth was delayed, with replication initiating at 10 to 15 h postinfection, depending on the multiplicity of infection. In addition to the delayed growth onset, virus replication was significantly impaired (1,000-fold lower titers) in nondividing cells, and plaque-forming ability was severely compromised. The RR1 protein expressed by a revertant virus [HSV-2(R)] was structurally and functionally similar to the wild-type protein, and the virus had wild-type growth and plaque-forming properties. The growth of the ICP10DeltaPK virus and its plaque-forming potential were restored to wild-type levels in cells that constitutively express ICP10. Immediate-early (IE) genes for ICP4, ICP27, and ICP22 were not expressed in Vero cells infected with ICP10DeltaPK early in infection or in the presence of cycloheximide, and the levels of ICP0 and p95 were significantly (three- to sevenfold) lower than those in HSV-2- or HSV-2(R)-infected cells. IE gene expression was similar to that of the wild-type virus in cells that constitutively express ICP10. The data indicate that ICP10 PK is required for early expression of the viral regulatory IE genes and, consequently, for timely initiation of the protein cascade and HSV-2 growth in cultured cells.     

In vitro characterization of a herpes simplex virus type 1 ICP22 deletion mutant

We report the construction of a deletion mutant (del22Z) that is unable to synthesize any detectable messenger RNA or protein products from the herpes simplex virus type 1 (HSV-1) immediate early ICP22 gene upon infection. The del22Z deletion mutant lacks all but 18 nucleotides of the ICP22 coding sequence and carries the bacterial lacZ gene at the site of the deletion. No other known open reading frames or flanking sequences were disrupted. Del22Z was able to infect Vero cells productively but was severely restricted in human and rodent cells that were permissive for the parental HSV-1(F). The yield of del22Z was not enhanced significantly, either by increasing the multiplicity of infection or by increasing the duration of the infection. There was a prolonged expression of some early gene products and a delayed appearance of some late gene products in both permissive and restrictive cells. This phenotype of cell-line restricted growth and alteration of the normal gene expression cascade maps specifically to the ICP22 coding region.     

Physical interaction between the herpes simplex virus type 1 immediate-early regulatory proteins ICP0 and ICP4

The herpes simplex virus type 1 immediate-early protein ICP0 enhances expression of a spectrum of viral genes alone and synergistically with ICP4. To test whether ICP0 and ICP4 interact physically, we performed far-Western blotting analysis of proteins from mock-, wild-type-, and ICP4 mutant virus-infected cells with in vitro-synthesized [35S]Met-labeled ICP0 and ICP4 as probes. The ICP4 and ICP0 polypeptides synthesized in vitro exhibited molecular weights similar to those of their counterparts in herpes simplex virus type 1-infected cells, and the in vitro-synthesized ICP4 was able to bind to a probe containing the ICP4 consensus binding site. Far-Western blotting experiments demonstrated that ICP0 interacts directly and specifically with ICP4 and with itself. To further define the interaction between ICP0 and ICP4, we generated a set of glutathione S-transferase (GST)-ICP0 fusion proteins that contain GST and either ICP0 N-terminal amino acids 1 to 244 or 1 to 394 or C-terminal amino acids 395 to 616 or 395 to 775. Using GST-ICP0 fusion protein affinity chromatography and in vitro-synthesized [35S]Met-labeled ICP0 and ICP4, ICP4 was shown to interact preferentially with the fusion protein containing ICP0 C-terminal amino acids 395 to 775, whereas ICP0 interacted efficiently with both the N-terminal GST-ICP0 fusion proteins and the C-terminal GST-ICP0 fusion proteins containing amino acids 395 to 775. Fusion protein affinity chromatography also demonstrated that the C-terminal 235 amino acid residues of ICP4 are important for efficient interaction with ICP0. Collectively, these results reveal a direct and specific physical interaction between ICP0 and ICP4.     

Changing nature of physician satisfaction with health maintenance organization and fee-for-service practices

We have identified a new, slightly unstable alpha chain hemoglobin variant, present in a Mexican-American family. Amino acid sequencing and mass spectral analysis of the aberrant peptide (alpha T-9) of the variant revealed that the aspartic acid is deleted either at position 74 or 75 of one of the alpha-globin chains. Sequencing of the amplified alpha 2- or alpha 1-globin genes revealed a trinucleotide deletion (GAC) at codon 74 or 74 of the alpha 2 gene. Although the aspartic acid residues of 74 and 75 of the alpha chain are neither a heme nor an inter chain contact, the slight instability of Hb Watts may be due to disturbance of the central cavity of hemoglobin by the deletion of an aspartic acid residue in the EF helix. Hb Watts is the first example of a trinucleotide deletion in the alpha 2-globin gene.     

Repression of gene expression upon infection of cells with herpes simplex virus type 1 mutants impaired for immediate-early protein synthesis

Herpes simplex virus type 1 (HSV-1) mutants defective in immediate-early (IE) gene expression do not readily enter productive replication after infection of tissue culture cells. Instead, their genomes are retained in a quiescent, nonreplicating state in which the production of viral gene products cannot be detected. To investigate the block to virus replication, we used the HSV-1 triple mutant in1820K, which, under appropriate conditions, is effectively devoid of the transactivators VP16 (a virion protein), ICP0, and ICP4 (both IE proteins). Promoters for the HSV-1 IE ICP0 gene or the human cytomegalovirus (HCMV) major IE gene, cloned upstream of the Escherichia coli lacZ coding sequences, were introduced into the in1820K genome. The regulation of these promoters and of the endogenous HSV-1 IE promoters was investigated upon conversion of the virus to a quiescent state. Within 24 h of infection, the ICP0 promoter became much less sensitive to transactivation by VP16 whereas the same element, when used to transform Vero cells, retained its responsiveness. The HCMV IE promoter, which is not activated by VP16, also became less sensitive to the HCMV functional homolog of VP16. Both elements remained available for transactivation by HSV-1 IE proteins at 24 h postinfection, showing that the in1820K genome was not irreversibly inactivated. The promoters controlling the HSV-1 ICP4, ICP22, and ICP27 genes also became essentially unresponsive to transactivation by VP16. The ICP0 promoter was induced when hexamethylene bisacetamide was added to cultures at the time of infection, but the response to this agent was also lost by 24 h after infection. Therefore, promoter elements within the HSV-1 genome are actively repressed in the absence of IE gene expression, and repression is not restricted specifically to HSV-1 IE promoters.     

Self-association of herpes simplex virus type 1 ICP35 is via coiled-coil interactions and promotes stable interaction with the major capsid protein

The ordered copolymerization of viral proteins to form the herpes simplex virus (HSV) capsid occurs within the nucleus of the infected cell and is a complex process involving the products of at least six viral genes. In common with capsid assembly in double-stranded DNA bacteriophages, HSV capsid assembly proceeds via the assembly of an outer capsid shell around an interior scaffold. This capsid intermediate matures through loss of the scaffold and packaging of the viral genomic DNA. The interior of the HSV capsid intermediate contains the viral protease and assembly protein which compose the scaffold. Proteolytic processing of these proteins is essential for and accompanies capsid maturation. The assembly protein (ICP35) is the primary component of the scaffold, and previous studies have demonstrated it to be capable of intermolecular association with itself and with the major capsid protein, VP5. We have defined structural elements within ICP35 which are responsible for intermolecular self-association and for interaction with VP5. Yeast (Saccharomyces cerevisiae) two-hybrid assays and far-Western studies with purified recombinant ICP35 mapped a core self-association domain between Ser165 and His219. Site-directed mutations in this domain implicate a putative coiled coil in ICP35 self-association. This coiled-coil motif is highly conserved within the assembly proteins of other alpha herpesviruses. In the two-hybrid assay the core self-association domain was sufficient to mediate stable self-association only in the presence of additional structural elements in either N- or C-terminal flanking regions. These regions also contain conserved sequences which exhibit a high propensity for alpha helicity and may contribute to self-association by forming additional short coiled coils. Our data supports a model in which ICP35 molecules have an extended conformation and associate in parallel orientation through homomeric coiled-coil interactions. In additional two-hybrid experiments we evaluated ICP35 mutants for association with VP5. We discovered that in addition to the C-terminal 25 amino acids of ICP35, previously shown to be required for VP5 binding, an additional upstream region was required. This region is between Ser165 and His234 and contains the core self-association domain. Site-directed mutations and construction of chimeric molecules in which the self-association domain of ICP35 was replaced by the GCN4 leucine zipper indicated that this region contributes to VP5 binding through mediating self-association of ICP35 and not through direct binding interactions. Our results suggest that self-association of ICP35 strongly promotes stable association with VP5 in vivo and are consistent with capsid formation proceeding via formation of stable subassemblies of ICP35 and VP5 which subsequently assemble into capsid intermediates in the nucleus.     

Complementary change in cis determinants and trans factors in the evolution of an mRNP stability complex

RNA-protein (RNP) complexes play significant roles in the fate and expression of mRNAs. The prolonged half-life of human alpha-globin mRNA, a major determinant of normal erythroid differentiation, is dependent on the assembly of a sequence-specific 3'-untranslated region (3'UTR) RNP (alpha-complex). We demonstrate that the stability of murine alpha-globin mRNA is controlled by a parallel mechanism. Unexpectedly, however, the respective 3'UTR RNP complexes that stabilize the h(alpha)- and m(alpha)-globin mRNAs differ in structure. While the cis determinants in both species are encoded in polypyrimidine tracks, the human determinant is C-rich (CCUCC motif) while the mouse alpha-3'UTR consists of an equal distribution of Cs and Us (CCUUCU motif). The protein components of the corresponding human and murine alpha-complexes differ in a complementary manner: the previously described 39 kDa poly(C) binding protein (PCBP) present in the human alpha-complex is replaced in the mouse alpha-complex by a 48 kDa cytoplasmic poly(CU) binding protein (CUBP). These results reveal that drift in the primary sequences of the alpha-globin mRNA 3'UTR polypyrimidine tracks in a comparison between mouse and human is paralleled by an alteration in the composition of the corresponding trans-acting components. Surprisingly, these structurally distinct complexes appear to perform the identical function of stabilizing the corresponding alpha-globin mRNAs.     

Mutational analysis of ICP0R, a transrepressor protein created by alternative splicing of the ICP0 gene of herpes simplex virus type 1

The immediate-early protein ICP0 (infected-cell polypeptide 0) of herpes simplex virus type 1 (HSV-1) is a promiscuous transactivator of both viral and nonviral promoters in transient expression assays. Failure to splice the second of two introns in the ICP0 gene results in the utilization of an alternate stop codon that generates a truncated form of ICP0 called ICP0R. This protein exists in low levels in HSV-1-infected cells and functions as a dominant negative repressor of ICP0-mediated transactivation in transient expression assays. To conduct a detailed structure-function analysis of ICP0R, a series of insertion and deletion mutants of this protein were generated and analyzed in transfection assays. These studies indicated that segments of ICP0R that were rich in acidic amino acid residues (amino acids 9 to 76 and 233 to 241) or glycine residues (amino acids 242 to 262) were dispensable for the dominant negative phenotype. In contrast, the RING finger domain (amino acids 116 to 156) and surprisingly the sequences carboxy terminal to it (amino acids 157 to 232) were absolutely essential for transdominant repression. Consistent with these findings, the amino acid sequences of these two regions were conserved among other alphaherpesvirus ICP0 homologs. A construct containing only amino acids 76 to 232 inhibited ICP0-mediated transactivation almost as efficiently as wild-type ICP0R and represented the minimal sequences necessary for the dominant negative phenotype. These results demonstrated that the critical functional domain shared by both ICP0R and ICP0 is much more complex than a simple RING finger motif. Western blot (immunoblot) analyses of transfected cell lysates revealed that nearly all of the mutant constructs directed the expression of stable ICP0R proteins of the predicted molecular weight. However, there was a striking inverse correlation between the ability of a mutant construct to mediate transrepression and the amount of protein that it synthesized, indicating that dominant negative inhibition is achieved through the action of very little ICP0R protein.     

Persistence and expression of the herpes simplex virus genome in the absence of immediate-early proteins

The immediate-early (IE) proteins of herpes simplex virus (HSV) function on input genomes and affect many aspects of host cell metabolism to ensure the efficient expression and regulation of the remainder of the genome and, subsequently, the production of progeny virions. Due to the many and varied effects of IE proteins on host cell metabolism, their expression is not conducive to normal cell function and viability. This presents a major impediment to the use of HSV as a vector system. In this study, we describe a series of ICP4 mutants that are defective in different subsets of the remaining IE genes. One mutant, d109, does not express any of the IE proteins and carries a green fluorescent protein (GFP) transgene under the control of the human cytomegalovirus IE promoter (HCMVIEp). d109 was nontoxic to Vero and human embryonic lung (HEL) cells at all multiplicities of infection tested and was capable of establishing persistent infections in both of these cell types. Paradoxically, the genetic manipulations that were required to eliminate toxicity and allow the genome to persist in cells for long periods of time also dramatically lowered the level of transgene expression. Efficient expression of the HCMVIEp-GFP transgene in the absence of ICP4 was dependent on the ICP0 protein. In d109-infected cells, the level of transgene expression was very low in most cells but abundant in a small subpopulation of cells. However, expression of the transgene could be induced in cells containing quiescent d109 genomes weeks after the initial infection, demonstrating the functionality of the persisting genomes.     

Accumulation of alpha- and beta-globin messenger RNAs in mouse erythroleukemia cells

The accumulation of alpha- and beta-globin mRNA sequences in murine erythroleukemia cells (MELC) treated with various inducers has been studied using specific alpha- and beta-globin complementary DNAs (cDNAs). In cells cultured with dimethylsulfoxide (Me2SO), hexamethylene bisacetamide (HMBA) or butyric acid, accumulation of alpha-globin mRNA is detectable after 16, 12 and 8 hr of culture, respectively. An increase in beta-globin mRNA sequences is not detected until 20-24 hr after culture. In cells exposed to hemin, both alpha- and beta-globin mRNAs are detectable by 6 hr of culture, and a constant ratio of alpha/beta-mRNA is maintained during induction. In maximally induced cells, the alpha/beta-globin mRNA ratios are approximately 1 in cells induced by Me2SO and HMBA, and 0.66 and 0.3-0.50 in cells induced by butyric acid and hemin, respectively. Thus different inducers of erythroid differentiation in MELC lead to different times of onset of the expression of alpha- and beta-like genes. In addition, the relative accumulation of alpha- and beta-globulin mRNAs in induced cells differs with various types of inducers.     

Reduction of the major swine xenoantigen, the alpha-galactosyl epitope by transfection of the alpha2,3-sialyltransferase gene

alpha2,3-Sialyltransferase represents a putative enzyme that reduces the Galalpha1-3Gal beta1-4GlcNAc-R (the alpha-galactosyl epitope) by intracellular competition with alpha1,3-galactosyltransferase for a common acceptor substrate. This study demonstrates that the overexpression of the alpha2,3-sialyltransferase gene suppresses the antigenicity of swine endothelial cells to human natural antibodies by 77% relative to control cells and by 30% relative to cells transfected with alpha1,2-fucosyltransferase, and in addition, it reduces the complement-mediated cell lysis by 75% compared with control cells and by 22% compared with cells transfected with alpha1, 2-fucosyltransferase. The mechanism by which the alpha-galactosyl epitope was reduced was also studied. Suppression of alpha1, 3-galactosyltransferase activity by 30-63% was observed in the transfectants with alpha2,3-sialyltransferase, and mRNA expression of the alpha1,3-galactosyltransferase gene was reduced as well. The data suggest that the alpha2,3-sialyltransferase effectively reduced the alpha-galactosyl epitope as well as or better than the alpha1, 2-fucosyltransferase did and that the reduction of the alpha-galactosyl epitope is due not only to substrate competition but also to an overall reduction of endogenous alpha1, 3-galactosyltransferase enzyme activity.