US3 Protein Kinase Research Articles

US3 protein kinase of herpes simplex virus type 2 plays a role in protecting corneal epithelial cells from apoptosis in infected mice.

To clarify the biological role of US3 protein kinase of herpes simplex virus type 2 (HSV-2) in vivo, the expression of the viral antigen, the appearance of apoptotic bodies and DNA fragmentation were examined immunohistologically after corneal infection of mice with three different kinds of HSV-2 strain 186: the wild-type virus, a US3-deficient mutant (L1BR1) and its revertant (L1B-11). In both wild-type 186- and L1B-11-infected mice, viral antigen was diffusely found in the corneal epithelium; no apoptotic changes were detected in the epithelial cells. Whereas, in L1BR1-infected mice, HSV-immunoreactivity was localized around the virus-inoculated sites, and a large number of apoptotic bodies were observed in the corneal epithelium with dual-positive reactions for both HSV-immunostaining and TUNEL staining. These results suggest that the US3 protein kinase plays an important role in protecting HSV-2-infected cells from apoptotic death in vivo.

The genetic organization and transcriptional analysis of the short unique region in the genome of nononcogenic Marek's disease virus serotype 2

Studies on the Marek's disease virus (MDV) serotype 2 (MDV2) genome may be important for understanding the naturally nononcogenic nature of the virus. To determine the complete DNA sequence of MDV2 unique short (U s) region, genomic BamHI fragments F, M1 and R were sequenced. The MDV2 U s region is 12 109 bp long and contains 12 potential open reading frames (ORFs) likely to encode for proteins. Seven of them exhibit homologies to herpes simplex virus type 1 (HSV-1) US1 (ICP22), US2, US3 (protein kinase), US6 (gD), US7 (gI), US8 (gE) and US10 genes. These ORFs are conserved in a similar arrangement with those of HSV-1, except for US10 which is transposed in the U s regions of all three MDV serotypes. The predicted amino acid sequence of MDV2 ORF6 is homologous to SORF3 of the other serotypes of MDV serotype 1 (MDV1) and herpesvirus of turkeys (HVT) and to infectious laryngotracheitis virus SR1. In addition, four ORFs, which have been identified around the U s and inverted repeat junction regions, have no apparent relation to any other known herpesvirus genes. The identified ORFs in the MDV2 U s region were more colinear with their previously reported locations of MDV1 than with those of HVT and other alphaherpesviruses. Ten of the 12 ORFs in the MDV2 U s region were expressed and transcribed with 3′-coterminal transcripts and/or a unique transcript in the virus-infected cells. Compared to other MDV serotypes, the MDV2 U s-encoded proteins showed 46–70% and 33–59% identities with equivalent of MDV1 and HVT at the amino acid level, respectively. Our present data will be useful to understand the different pathogenicity among serotypes of MDV and to allow precise manipulation of the genes for a possible use in genetically engineered vaccines.

Subcellular localization of the US3 protein kinase of herpes simplex virus type 2.

One supposes that herpes simplex virus US3 gene product possessing serine/threonine protein kinase activity is a cytoplasmic enzyme. To determine its subcellular localization during viral replication we prepared an antiserum to a synthetic oligopeptide corresponding to the N-terminal region of the US3 protein of HSV type 2 strain 186. The US3 protein first appeared in the cytoplasm of infected cell at 4 h postinfection but strong fluorescence was detected in the nuclei at 8 h postinfection. At 12 h postinfection fluorescence was mainly detected in the cytoplasm, again. Further, the US3 protein expressed alone was widely distributed throughout the cell, indicating that the US3 protein by itself can be localized in the nuclei even in the absence of any other viral proteins. These observations suggest that the HSV-2 US3 protein kinase may function not only in the cytoplasm but also in the nuclei.

The antiviral xanthate compound D609 inhibits herpes simplex virus type 1 replication and protein phosphorylation

The mechanism of antiviral action of tricyclodecan-9-yl-xanthogenate (D609) was investigated in vitro. D609 inhibited herpes simplex virus type 1 (HSV-1) replication without apparent cytotoxicity. It reduced phosphorylation of virus-infected cell polypeptides and inhibited the HSV-1 encoded protein kinase (US3 PK) and, to a lesser extent, cellular protein kinase C in vitro. Virus production was reduced by D609 at concentrations greater than 3.8 μM, with complete inhibition at 75.2 μM at an MOI of 1 PFU/cell or less. Addition of D609 could be delayed until 7 h post-infection and still inhibit virus replication. Phosphorylation of infected cell viral polypeptides of 34 (similar molecular weight to the substrate of the viral US3 protein kinase) and 69 kDa was inhibited at 18.4 μM. Treatment of infected or uninfected cells with 37.6 μM D609 reduced protein phosphorylation to background levels. A concentration of 1.9 μM D609 in vitro inhibited the viral US3-encoded PK, which had been purified from infected cell lysates by affinity chromatography and identified by specific antibody. Purified cellular protein kinase C was inhibited at 75.2 μM D609 whereas other cellular kinases including casein kinase 1 and cAMP dependent kinase were not inhibited at concentrations as high as 188 μM D609. Collectively these data indicate that the mechanism of antiviral action of D609 is by inhibition of protein kinases and protein phosphorylation affecting a late step in HSV replication.

Purification and Characterization of the Protein Kinase Encoded by the UL13 Gene of Herpes Simplex Virus Type 2

The proteins encoded by the UL13 genes of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) have been predicted to be protein kinases. To identify the UL13 gene product, we have raised a rabbit polyclonal antiserum against a His · Tag-HSV-1 UL13 fusion protein. The antibody specifically reacted with the 60-kDa UL13 fusion protein expressed inEscherichia coliand also recognized 56- to 57-kDa late proteins in nuclear fractions of HSV-1- and HSV-2-infected cells. On the other hand, novel casein kinase activity was induced at the late stage of infection when Vero cells were infected with HSV-1 and HSV-2. The induction of the activity was most prominent in the nuclear fractions of HSV-2-infected cells and therefore we purified the protein kinase (PK) from the nuclear extracts by successive column chromatography (phosphocellulose, DEAE-cellulose, and hydroxyapatite) using casein as an exogenous substrate. The final preparation of the enzyme contained a single major protein with an apparent molecular weight of 56 kDa which was specifically reacted with the UL13 antiserum. The PK activity was optimal in the absence of NaCl and at relatively high pH. Acidic proteins such as casein and phosvitin were efficiently phosphorylated by the PK. A basic protein, protamine, which is the best substrate for the HSV-2 US3 PK, was not detectably phosphorylated but histone was a relatively good substrate for the UL13 PK. Phosphoamino acid analysis revealed that the PK phosphorylated serine and threonine but not tyrosine. Moreover the enzyme was found to be highly resistant to heparin, a potent inhibitor of casein kinase II (CK II) and also resistant to CK I-7, a synthetic inhibitor of CK I, but very sensitive to a bioflavonoid quercetin. These results indicate that the HSV-2 UL13 PK had unique catalytic properties different from those of cellular CK I, CK II, and the viral PK encoded by the US3 gene. We have also determined the complete nucleotide sequence of the HSV-2 UL13 gene. The overall amino acid homology between the HSV-2 and HSV-1 UL13 PKs was 85.9% and the homology was highly conserved in the C-terminal region.

The herpes simplex virus 1 protein kinase US3 is required for protection from apoptosis induced by the virus.

An earlier report showed that a disabled mutant lacking both copies of the major regulatory gene (alpha4) of herpes simplex virus 1 induced DNA degradation characteristic of apoptosis in infected cells, whereas the wild-type virus protected cells from apoptosis induced by thermal shock. More extensive analyses of the disabled mutant revealed a second mutation which disabled US3, a viral gene encoding a protein kinase known to phosphorylate serine/threonine within a specific arginine-rich consensus sequence. Analyses of cells infected with a viral mutant carrying a wild-type alpha4 gene but from which the US3 gene had been deleted showed that it induced fragmentation of cellular DNA, whereas a recombinant virus in which the deleted sequences of the US3 gene had been restored did not cause the cellular DNA to fragment. These results point to the protein kinase encoded by the US3 gene as the principal viral product required to block apoptosis.

The US3 protein kinase of herpes simplex virus type 2 is associated with phosphorylation of the UL12 alkaline nuclease in vitro.

Herpes simplex virus type 2 (HSV-2) gene US3 encodes a serine-threonine protein kinase. We previously described the isolation of a US3-inactivated mutant which is able to replicate in Vero cells but not in murine macrophages. To learn more about the biological role of the US3 protein kinase, we have sought to identify the target proteins of the enzyme. Studies of in vitro phosphorylation with extracts of infected cells demonstrate that the US3 protein kinase is involved in phosphorylation of the UL12 alkaline nuclease in vitro, suggesting that the nuclease is a possible target of the protein kinase.

Identification of a target protein of US3 protein kinase of herpes simplex virus type 2

Herpes simplex virus type 2 (HSV-2) gene US3 has been shown to encode a serine-threonine protein kinase. In this study, we have tried to identify target proteins of the US3 protein kinase using a US3 lacZ insertion mutant of HSV-2. When permeabilized cells were labelled with [gamma-32P]ATP under the optimum conditions for the US3 enzyme, the most striking difference between wild-type HSV-2 strain 186- and mutant-infected cells was observed in the phosphorylation of proteins ranging in M(r) values from 14K to 22K. Studies of in vitro phosphorylation with purified virions and with cells infected with a US9-defective HSV-1 mutant suggested that a tegment phosphoprotein encoded by the US9 gene may be a target of HSV-2 US3 protein kinase.

Purification and Biochemical Characterization of the Protein Kinase Encoded by the US3 Gene of Herpes Simplex Virus Type 2

Post-ribosomal cytoplasmic fractions from Vero cells mock-infected or infected with wild-type herpes simplex virus type 2 (HSV-2) or a US3 gene-disrupted mutant of HSV-2 were fractionated with DEAE-cellulose chromatography, and the peak fraction of the protein kinase which was detectable only in the extract of wild-type virus-infected cells was subjected to succesive chromatography. The enzyme was purified more than 1000-fold from the post-ribosomal supernatant, and the final preparation contained one major protein of apparent molecular weight 66 kilodalton (K), which was phosphorylated in the autophosphorylation reaction. Western blotting analysis showed that antibodies to an synthetic peptide corresponding to the 15 amino acids of the predicted HSV-2 US3 protein sequence strongly reacted with a 66 K protein in the enzyme fractions. On Superose 12 HR chromatography, the protein kinase activity was eluted as a single major peak at a position corresponding to an apparent molecular mass of approximately 60 K. These results suggest that the 66 K protein is the protein kinase encoded by the US3 gene of HSV-2 and that it acts as a monomer. The HSV-2 protein kinase was relatively resistant to high concentrations of salt, but KCl above 400 mM exerted a significant inhibitory effect. When the substrate specificity was investigated using synthetic oligopeptides, the peptides containing arginyl residues on the amino-terminal side of the target seryl residue were found to be the best substrates for the protein kinase. However, the replacement of the seryl residue to threonine markedly reduced the rate of phosphorylation by this enzyme, suggesting that threonine is a poor phosphate acceptor of the protein kinase. The enzyme was resistant to heparin, a potent inhibitor of casein kinase II, but was moderately sensitive to H-9 (N-(2-aminoethyl)-5-isoquinolinesulfonamide dihydrochloride), a potent inhibitor of cyclic nucleotide-dependent protein kinases and protein kinase C. Quercetin, a bioflavonoid, also inhibited the protein kinase and the inhibitory effect was competitive towards ATP (Ki = 10 μM). The results indicate that the biochemical properties of the HSV-2 US3 protein kinase are very similar to those of the HSV-1 counterpart and pseudorabies virus-encoded 38-kDa protein kinase, but are different from those in several respects.

The pathogenicity of a US3 protein kinase-deficient mutant of herpes simplex virus type 2 in mice.

We have investigated the pathogenicity of a US3 protein kinase-deficient mutant (L1 BR1) of herpes simplex virus type 2 (HSV-2) for 4-week-old ICR mice to define the role of the viral protein kinase in virus-host interaction. When mice were intraperitoneally infected with 10(5)PFU of L1 BR1, the virus disappeared from the peritoneal cavity by 2 days postinfection and failed to induce any significant histopathological changes in the liver and spleen although viral antigens were occasionally detected in the epithelial cells of small bile ducts and small vascular wall. The parental virus (HSV-2 186) and a revertant of the mutant (L1 B-11) both caused severe hepatitis, and viral antigens were clearly detected in the hepatocytes and Kupffer cells in the focal necrotic areas. Both of the virulent viruses, unlike L1 BR1, could produce infectious progeny and cytopathic effects in freshly harvested peritoneal macrophages. The growth of L1 BR1 in peritoneal macrophages was restricted at a stage of or prior to viral DNA synthesis but after the induction of viral DNA polymerase. In addition, the production and/or the spread of mutant in mouse embryo fibroblasts (MEF) was found to be much more effectively suppressed by cocultivation of peritoneal macrophages than that of 186. An almost complete inhibition of L1 BR1-plaque formation was observed at a macrophage-to-MEF ratio of 4:1. These results suggest that the attenuation of L1 BR1 following intraperitoneal infection is primarily due to its high sensitivity to intrinsic and extrinsic inhibition of peritoneal macrophages and that the US3 protein kinase may play a role in viral DNA replication in peritoneal macrophages.

Sequence determination and genetic content of an 8.9-kb restriction fragment in the short unique region and the internal inverted repeat of Marek's disease virus type 1 DNA.

The DNA sequence (8.9 kb) covering about 70% of the short unique region (Us) and part of the short inverted repeat of the Marek's disease virus type 1 GA strain was determined. Computer analysis of the sequence showed the presence of nine potential open reading frames (ORFs), consisting of more than 300 nucleotides in the Us region. Of these ORFs, four were found to be homologous to US10 (minor virion protein), US3 (protein kinase), US2, and US6 (gD) in the Us region of alpha-herpesvirus herpes simplex virus type 1. The protein kinase homologue is especially well conserved in alpha-herpesviruses. No counterpart of the nine MDV1 ORFs was found in the beta-herpes virus human cytomegalovirus and gamma-herpesvirus Epstein-Barr virus, suggesting that MDV1 is more similar to the alpha-herpesviruses. The junction of the Us region and the short inverted repeat was also determined by comparison between the sequences of the DNA fragments, including the terminal and internal repeats. Northern blot analysis showed that the Us region within the 8.9 kb sequence was transcriptionally active in MDV1-infected cells.

Construction of a US3 lacZ insertion mutant of herpes simplex virus type 2 and characterization of its phenotype in vitro and in vivo

We have constructed and characterized a mutant of herpes simplex virus type 2 (HSV-2) which was inserted a modified lacZ gene, placed under the control of HSV-1 β8 promotor, into the US3 protein kinase gene. The mutant, L1BR1, could not induce the virus-encoded protein kinase activity, but could replicate in Vero cells as efficiently as the parental virus. When the biological properties of L1BR1 were examined in mice by using four routes (footpad, intraperitoneal, corneal, and intracerebral) of infection, the mutant displayed the route-dependent reduction of virulence; after inoculation by footpad and intraperitoneal routes, the mutant was more than 10,000-fold less virulent than the parental virus, but it exhibited only about a 10-fold decrease in virulence following the corneal and intracerebral infection. In the intraperitoneal inoculation into adult mice, the replication of L1BR1 in the liver and spleen was severely restricted, but in newborn mice the mutant could grow as well as the parental virus in these organs. The adoptive transfer of peritoneal macrophages from adult mice resulted in a marked inhibition in the replication of L1BR1 in the liver and spleen of newborn mice, while the transfer exhibited little or no effect on the production of the wild-type virus in these organs. We also found that the mutant, unlike the parental virus, could not replicate in precultured peritoneal macrophages from adult mice. Taking these observations together, it seems likely that L1 BR1 lost the ability to overcome the mononuclear-phagocytic defense system and thereby lost its pathogenicity by intraperitoneal and footpad routes. Furthermore, the mutant was shown to be rescued by a 4.8-kb HindIII/ XbaI fragment containing the entire US3 open reading frame. However, we could not rule out the possibility that some of the phenotypes of L1BR1 are due to mutations in the US3-neighboring genes, US2 and US4.

The UL13 gene of herpes simplex virus 1 encodes the functions for posttranslational processing associated with phosphorylation of the regulatory protein alpha 22.

The herpes simplex virus 1 genome was shown to encode two genes, US3 and UL13, exhibiting amino acid sequence motifs common to protein kinases. Elsewhere this laboratory reported that the prominent substrate of the US3 protein kinase is the product of the UL34 gene, an essential nonglycosylated membrane protein. In the absence of the US3 kinase, the UL34 protein remains unphosphorylated but forms a complex with four proteins that become phosphorylated uniquely when UL34 is not. To investigate the role of UL13 protein in this process, recombinant viruses lacking UL13 or both UL13 and US3 were constructed. We report that UL13 is dispensable for viral replication in cell culture and is not involved in the processing of UL34 or of associated phosphoproteins. UL13 is, however, responsible for the posttranslational processing associated with phosphorylation of infected-cell protein 22, the product of the alpha 22 gene. This gene was previously reported to play a regulatory role in selected cell lines. UL13 appears to be either a protein kinase or a phosphotransferase and its major substrate is the alpha 22 protein.