The UL7 gene of pseudorabies virus encodes a nonessential structural protein which is involved in virion formation and egress

Homologues of the UL7 gene of herpes simplex virus type 1 are conserved in alpha-, beta-, and gammaherpesviruses. However, little is known about their functions. Using a monospecific rabbit antiserum raised against a bacterial fusion protein, we identified the UL7 gene product of the neurotropic alphaherpesvirus pseudorabies virus (PrV). In Western blot analyses of infected cells and purified PrV particles the serum specifically detected a 29-kDa protein, which matches the calculated mass of the 266-amino-acid translation product of PrV UL7. For functional analysis, UL7 was deleted by mutagenesis of an infectious full-length clone of the PrV genome in Escherichia coli. The obtained recombinant PrV-DeltaUL7F was replication competent in rabbit kidney cells, but maximum virus titers were decreased nearly 10-fold and plaque diameters were reduced by ca. 60% compared to wild-type PrV. Electron microscopy of infected cells revealed that in the absence of UL7, formation and nuclear egress of nucleocapsids were not affected, whereas secondary envelopment of cytoplasmic nucleocapsids appeared to be delayed and release of mature virions was less efficient. The observed replication defects were corrected by repair of the viral UL7 gene or by propagation of PrV-DeltaUL7F in UL7-expressing cells. PrV-DeltaUL7F was moderately attenuated in mice. Compared to wild-type virus, mean survival times were prolonged from 2 to 3 days after intranasal infection. However, neuroinvasion and transneuronal spread of PrV were not abolished in the absence of UL7. Thus, UL7 encodes a virion protein of PrV, which plays a role during virion maturation and egress both in vitro and in vivo.     

The Interacting UL31 and UL34 Gene Products of Pseudorabies Virus Are Involved in Egress from the Host-Cell Nucleus and Represent Components of Primary Enveloped but Not Mature Virions

A 2.6-kbp fragment of the pseudorabies virus (PrV) genome was sequenced and shown to contain the homologues of the highly conserved herpesvirus genes UL31 and UL32. By use of a monospecific antiserum, the UL31 gene product was identified as a nuclear protein with an apparent molecular mass of 29 kDa. For functional analysis, UL31 was deleted by mutagenesis in Escherichia coli of an infectious full-length clone of the PrV genome. The resulting virus mutants were deficient in plaque formation, and titers were reduced more than 100-fold from those of wild-type PrV. Ultrastructural analyses demonstrated that capsid maturation and DNA packaging were not affected. However, neither budding at the inner nuclear membrane nor cytoplasmic or extracellular virus particles were observed. These replication defects were similar to those of a UL34 deletion mutant (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 74:10063-10073, 2000) and could be completely repaired in a cell line which constitutively expresses the UL31 protein. Yeast two-hybrid studies revealed that a UL31 fusion protein specifically interacts with plasmids of a PrV genome library expressing the N-terminal part of UL34. Vice versa, UL34 selected UL31-encoding plasmids from the library. Immunofluorescence studies and immune electron microscopy demonstrated that in cells infected with wild-type PrV, both proteins accumulate at the nuclear membrane, whereas in the absence of UL34 the UL31 protein is dispersed throughout the nucleus. Like the UL34 protein, the UL31 gene product is a component of enveloped virus particles within the perinuclear space and absent from mature virions. Our findings suggest that physical interaction between these two virus proteins might be a prerequisite for primary envelopment of PrV at the inner nuclear membrane and that this envelope is removed by fusion with the outer nuclear membrane.     

Pseudorabies virus UL3 gene codes for a nuclear protein which is dispensable for viral replication

Many of the products of the ca. 80 genes encoded by alphaherpesviruses have already been identified and, at least tentatively, functionally characterized. Among the least characterized proteins are the products of the genes homologous to herpes simplex virus UL3, which are present only in the subfamily Alphaherpesvirinae: To identify the UL3 protein of the porcine alphaherpesvirus pseudorabies virus (PrV), the complete PrV UL3 open reading frame was cloned, expressed in Escherichia coli as a glutathione S-transferase fusion protein, and used for immunization of a rabbit. In Western blots, the generated antiserum specifically detected a 34-kDa protein in PrV-infected cells, which was absent from purified virus preparations, indicating that PrV UL3 encodes a nonstructural protein. In indirect immunofluorescence analysis, the anti-UL3 serum produced predominantly nuclear staining in transfected as well as in infected cells, which was not altered in the absence of other virus-encoded nuclear proteins such as the UL31 and UL34 gene products. To investigate UL3 function, a deletion mutant, PrV-DeltaUL3F2, was constructed and characterized. This mutant replicated and formed plaques on noncomplementing cells indistinguishable from wild-type PrV, demonstrating that PrV UL3 is not required for virus propagation in cultured cells. Moreover, ultrastructural examinations revealed no impairment of capsid formation in the nucleus, nuclear egress of capsids, virion maturation in the cytoplasm, or virus release. Thus, the overall properties of PrV UL3 are similar to those described for the homologous herpes simplex virus proteins which may be indicative of a common, yet hitherto unknown, function in alphaherpesvirus replication. However, based on our studies, an involvement of the UL3 homologs in virion formation appears unlikely.     

Identification and characterization of the pseudorabies virus tegument proteins UL46 and UL47: role for UL47 in virion morphogenesis in the cytoplasm

Proteins encoded by the UL46 and UL47 genes of herpes simplex virus type 1 (HSV-1) constitute major components of the viral tegument. However, their functions have so far not been elucidated in detail. By use of monospecific antisera directed against bacterially expressed glutathione-S-transferase fusion proteins, the homologous UL46 and UL47 proteins of the alphaherpesvirus pseudorabies virus (PrV) were identified in virus-infected cells and in virions. The PrV UL46 gene product of 693 amino acids (aa) exhibits an apparent molecular mass of 95 kDa, whereas the UL47 product of 750 aa was identified as a 97-kDa protein. Both are present in purified virions, correlating with their role as tegument proteins. Immunofluorescence analysis by confocal laser scan microscopy showed that late in infection the UL46 product is detectable in the cytoplasm, whereas the UL47 product was observed to be diffuse in the cytoplasm and speckled in the nucleus. Virus mutants lacking either the UL46 or the UL47 gene or both were isolated on noncomplementing cells, demonstrating that these genes either singly or in combination are not required for productive viral replication. However, plaque sizes were decreased. Interestingly, in one-step growth analysis, UL47 deletion mutants exhibited an approximately 10-fold decrease in final titers, whereas the UL46 deletion mutant was not affected. This finding correlated with ultrastructural observations which showed unimpaired virion morphogenesis in the absence of the UL46 protein, whereas in the absence of the UL47 protein intracytoplasmic aggregates of partially tegumented capsids were observed. In summary, we identified the PrV UL46 and UL47 proteins and show that the UL47 protein plays an important role in virion assembly in the cytoplasm.     

Composition of pseudorabies virus particles lacking tegument protein US3, UL47, or UL49 or envelope glycoprotein E

Proteins located in the tegument layer of herpesvirus particles play important roles in the replicative cycle at both early and late times after infection. As major constituents of the virion, they execute important functions in particular during formation of progeny virions. These functions have mostly been elucidated by construction and analysis of mutant viruses deleted in single or multiple tegument protein-encoding genes (reviewed in the work of T. C. Mettenleiter, Virus Res. 106:167-180, 2004). However, since tegument proteins have been shown to be involved in numerous protein-protein interactions, the impact of single protein deletions on the composition of the virus particle is unknown, but they could impair correct interpretation of the results. To analyze how the absence of single virion constituents influences virion composition, we established a procedure to assay relative amounts of virion structural proteins in deletion mutants of the alphaherpesvirus Pseudorabies virus (PrV) in comparison to wild-type particles. The assay is based on the mass spectrometric quantitation of virion protein-derived peptides carrying stable isotope mass tags. After deletion of the US3, UL47, UL49, or glycoprotein E gene, relative amounts of a capsid protein (UL38), a capsid-associated protein (UL25), several tegument proteins (UL36 and UL47, if present), and glycoprotein H were unaffected, whereas the content of other tegument proteins (UL46, UL48, and UL49, if present) varied significantly. In the case of the UL48 gene product, a specific increase in incorporation of a smaller isoform was observed after deletion of the UL47 or UL49 gene, whereas a larger isoform remained unaffected. The cellular protein actin was enriched in virions of mutants deficient in any of the tegument proteins UL47, UL49, or US3. By two-dimensional gel electrophoresis multiple isoforms of host cell-derived heat shock protein 70 and annexins A1 and A2 were also identified as structural components of PrV virions.     

The UL48 tegument protein of pseudorabies virus is critical for intracytoplasmic assembly of infectious virions

The pseudorabies virus (PrV) homolog of the tegument protein encoded by the UL48 gene of herpes simplex virus type 1 (HSV-1) was identified by using a monospecific rabbit antiserum against a bacterial fusion protein. UL48-related polypeptides of 53, 55, and 57 kDa were detected in Western blots of infected cells and purified virions. Immunofluorescence studies demonstrated that the PrV UL48 protein is predominantly localized in the cytoplasm but is also found in the nuclei of infected cells. Moreover, it is a constituent of extracellular virus particles but is absent from primary enveloped perinuclear virions. In noncomplementing cells, a UL48-negative PrV mutant (PrV-DeltaUL48) exhibited delayed growth and significantly reduced plaque sizes and virus titers, deficiencies which were corrected in UL48-expressing cells. RNA analyses indicated that, like its HSV-1 homolog, the PrV UL48 protein is involved in regulation of immediate-early gene expression. However, the most salient effect of the UL48 gene deletion was a severe defect in virion morphogenesis. Late after infection, electron microscopy of cells infected with PrV-DeltaUL48 revealed retention of newly formed nucleocapsids in the cytoplasm, whereas enveloped intracytoplasmic or extracellular complete virions were only rarely observed. In contrast, capsidless particles were produced and released in great amounts. Remarkably, the intracytoplasmic capsids were labeled with antibodies against the UL36 and UL37 tegument proteins, whereas the capsidless particles were labeled with antisera directed against the UL46, UL47, and UL49 tegument proteins. These findings suggested that the UL48 protein is involved in linking capsid and future envelope-associated tegument proteins during virion formation. Thus, like its HSV-1 homolog, the UL48 protein of PrV functions in at least two different steps of the viral life cycle. The drastic inhibition of virion formation in the absence of the PrV UL48 protein indicates that it plays an important role in virion morphogenesis prior to secondary envelopment of intracytoplasmic nucleocapsids. However, the UL48 gene of PrV is not absolutely essential, and concomitant deletion of the adjacent tegument protein gene UL49 also did not abolish virus replication in cell culture.     

Identification of cell surface molecules that interact with pseudorabies virus.

The alphaherpesvirus pseudorabies virus (PrV) has been shown to attach to cells by interaction between the viral glycoprotein gC and cell membrane proteoglycans carrying heparan sulfate chains (HSPGs). A secondary binding step requires gD and presumably another, hitherto unidentified cellular receptor. By use of a virus overlay protein binding assay (VOPBA), cosedimentation analyses, and affinity chromatography, we identified three species of cell membrane constituents that bind PrV. By treatment with EDTA, peripheral HSPGs of very high apparent molecular mass (>200 kDa) could be extracted from Madin-Darby bovine kidney cells. Binding of PrV to these HSPGs in the VOPBA was sensitive to enzymatic digestion with heparinase or papain. Cosedimentation analyses indicated that binding between PrV and high-molecular-weight HSPG depended on the presence of gC in the virion. In addition, adsorption of radiolabeled PrV virions to cells could be inhibited by the addition of purified high-molecular-weight HSPG. By using urea extraction buffer, a second species of HSPG of approximately 140 kDa could be solubilized. Binding of PrV to this HSPG in the VOPBA was also dependent on the presence of heparan sulfate, since reactivity was abolished after suppression of glycosaminoglycan biosynthesis with NaClO3 and after heparinase treatment. In addition to HSPG, in cellular membrane extracts obtained by treatment with mild detergent, a 85-kDa membrane protein was demonstrated to bind PrV in the VOPBA and affinity chromatography. In summary, we identified three species of cell membrane constituents that bind PrV: a peripheral HSPG of high molecular weight, an integral HSPG of approximately 140 kDa, and an integral membrane protein of 85 kDa. It is tempting to speculate that interaction between PrV and the two species of HSPG mediates primary attachment of PrV and that the 85-kDa protein is involved in a subsequent attachment step.     

A proteomic study of the arabidopsis nuclear matrix

The eukaryotic nucleus has been proposed to be organized by two interdependent nucleoprotein structures, the DNA-based chromatin and the RNA-dependent nuclear matrix. The functional composition and molecular organization of the second component have not yet been resolved. Here, we describe the isolation of the nuclear matrix from the model plant Arabidopsis, its initial characterization by confocal and electron microscopy, and the identification of 36 proteins by mass spectrometry. Electron microscopy of resinless samples confirmed a structure very similar to that described for the animal nuclear matrix. Two-dimensional gel electrophoresis resolved approximately 300 protein spots. Proteins were identified in batches by ESI tandem mass spectrometry after resolution by 1D SDS-PAGE. Among the identified proteins were a number of demonstrated or predicted Arabidopsis homologs of nucleolar proteins such as IMP4, Nop56, Nop58, fibrillarins, nucleolin, as well as ribosomal components and a putative histone deacetylase. Others included homologs of eEF-1, HSP/HSC70, and DnaJ, which have also been identified in the nucleolus or nuclear matrix of human cells, as well as a number of novel proteins with unknown function. This study is the first proteomic approach towards the characterization of a higher plant nuclear matrix. It demonstrates the striking similarities both in structure and protein composition of the operationally defined nuclear matrix across kingdoms whose unicellular ancestors have separated more than one billion years ago.     

The UL49.5 gene of pseudorabies virus codes for an O-glycosylated structural protein of the viral envelope.

Sequence analysis of BamHI fragment 1 of the pseudorabies virus (PrV) genome identified a novel PrV gene located upstream of the UL50 gene encoding PrV dUTPase. The deduced protein product displayed homology to the product of the herpes simplex virus type 1 UL49.5 protein. The predicted PrV UL49.5 protein consists of 98 amino acids with a calculated molecular mass of 10,155 Da. It contains putative signal peptide and transmembrane domains but lacks a consensus sequence for N glycosylation. PrV UL49.5 was expressed as a fusion protein with glutathione S-transferase in Escherichia coli, and a rabbit antiserum was generated. In Western blots (immunoblots) of purified virions, the antiserum detected a protein with an apparent molecular mass of 14 kDa. After fractionation of the virions, the 14-kDa protein was detected in the envelope fraction. Localization of the UL49.5 protein in the viral envelope was confirmed by immunoelectron microscopy. The treatment of purified virions with glycosidases led to a reduction of the apparent molecular mass in Western blots by approximately 2 kDa following digestion with neuraminidase and O-glycosidase. Our results demonstrate that the PrV UL49.5 protein is an O-glycosylated structural component of the viral envelope. It represents the 10th PrV glycoprotein described. According to the unified nomenclature for alphaherpesvirus glycoproteins, we propose to designate it glycoprotein N (gN).     

Infectivity of a Pseudorabies Virus Mutant Lacking Attachment Glycoproteins C and D

Initiation of herpesvirus infection requires attachment of virions to the host cell followed by fusion of virion envelope and cellular cytoplasmic membrane during penetration. In several alphaherpesviruses, glycoprotein C (gC) is the primary attachment protein, interacting with cell-surface heparan sulfate proteoglycans. Secondary binding is mediated by gD, which, normally, is also required for penetration. Recently, we described the isolation of a gD-negative infectious pseudorabies virus (PrV) mutant, PrV gD⁻ Pass (J. Schmidt, B. G. Klupp, A. Karger, and T. C. Mettenleiter, J. Virol. 71:17–24, 1997). In PrV gD⁻ Pass, attachment and penetration occur in the absence of gD. To assess the importance of specific attachment for infectivity of PrV gD⁻ Pass, the gene encoding gC was deleted, resulting in mutant PrV gCD⁻ Pass. Deletion of both known attachment proteins reduced specific infectivity compared to wild-type PrV by more than 10,000-fold. Surprisingly, the virus mutant still retained significant infectivity and could be propagated on normal noncomplementing cells, indicating the presence of another receptor-binding virion protein. Selection of bovine kidney (MDBK) cells resistant to infection by PrV gCD⁻ Pass resulted in the isolation of a cell clone, designated NB, which was susceptible to infection by wild-type PrV but refractory to infection by either PrV gCD⁻ Pass or PrV gD⁻ Pass, a defect which could partially be overcome by polyethylene glycol (PEG)-induced membrane fusion. However, even after PEG-induced infection plaque formation of PrV gCD⁻ Pass or PrV gD⁻ Pass did not ensue in NB cells. Also, phenotypic gD complementation of PrV gCD⁻ Pass or PrV gD⁻ Pass rescued the defect in infection of NB cells but did not restore plaque formation. Glycosaminoglycan analyses of MDBK and NB cells yielded identical results, and NB cells were normally susceptible to infection by other alphaherpesviruses as well as vesicular stomatitis virus. Infectious center assays after PEG-induced infection of NB cells with PrV gD⁻ Pass on MDBK cells indicated efficient exit of virions from infected NB cells. Together, our data suggest the presence of another receptor and receptor-binding virion protein which can mediate PrV entry and cell-to-cell spread in MDBK cells.     

Nuclear heat shock response and novel nuclear domain 10 reorganization in respiratory syncytial virus-infected a549 cells identified by high-resolution two-dimensional gel electrophoresis

The pneumovirus respiratory syncytial virus (RSV) is a leading cause of epidemic respiratory tract infection. Upon entry, RSV replicates in the epithelial cytoplasm, initiating compensatory changes in cellular gene expression. In this study, we have investigated RSV-induced changes in the nuclear proteome of A549 alveolar type II-like epithelial cells by high-resolution two-dimensional gel electrophoresis (2DE). Replicate 2D gels from uninfected and RSV-infected nuclei were compared for changes in protein expression. We identified 24 different proteins by peptide mass fingerprinting after matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS), whose average normalized spot intensity was statistically significant and differed by +/-2-fold. Notable among the proteins identified were the cytoskeletal cytokeratins, RNA helicases, oxidant-antioxidant enzymes, the TAR DNA binding protein (a protein that associates with nuclear domain 10 [ND10] structures), and heat shock protein 70- and 60-kDa isoforms (Hsp70 and Hsp60, respectively). The identification of Hsp70 was also validated by liquid chromatography quadropole-TOF tandem MS (LC-MS/MS). Separate experiments using immunofluorescence microscopy revealed that RSV induced cytoplasmic Hsp70 aggregation and nuclear accumulation. Data mining of a genomic database showed that RSV replication induced coordinate changes in Hsp family proteins, including the 70, 70-2, 90, 40, and 40-3 isoforms. Because the TAR DNA binding protein associates with ND10s, we examined the effect of RSV infection on ND10 organization. RSV induced a striking dissolution of ND10 structures with redistribution of the component promyelocytic leukemia (PML) and speckled 100-kDa (Sp100) proteins into the cytoplasm, as well as inducing their synthesis. Our findings suggest that cytoplasmic RSV replication induces a nuclear heat shock response, causes ND10 disruption, and redistributes PML and Sp100 to the cytoplasm. Thus, a high-resolution proteomics approach, combined with immunofluorescence localization and coupled with genomic response data, yielded unexpected novel insights into compensatory nuclear responses to RSV infection.     

The pseudorabies virus UL51 gene product is a 30-kilodalton virion component.

Positional homologs to the UL51 open reading frame of herpes simplex virus type 1 have been identified throughout the herpesvirus family. However, no respective protein has so far been described for any of the herpesviruses. With rabbit antisera directed against oligopeptides predicted to comprise antigenic regions of the deduced pseudorabies virus (PrV) UL51 protein, a polypeptide with a size of 30 kDa was identified in PrV-infected cell lysates and in purified virions. This molecular mass correlates reasonably well with the predicted mass of 25 kDa of the 236-amino-acid deduced UL51 protein. Antisera raised against peptides derived from different predicted antigenic regions all detected the 30-kDa protein in Western blot (immunoblot) analyses. Specificity was ascertained by peptide competition. Subcellular fractionation showed the presence of the UL51 protein mainly in the nucleus of infected cells. After separation of purified virion preparations into envelope and capsid, the PrV UL51 protein was detected in the capsid fraction. In summary, we identified the first herpesvirus UL51 protein and demonstrate that it represents a structural component of PrV virions.     

The pseudorabies virus US3 protein is a component of primary and of mature virions

Herpesviruses acquire a primary envelope by budding of capsids at the inner leaflet of the nuclear membrane. They then traverse into the cytoplasm after fusion of the primary envelope with the outer leaflet of the nuclear membrane. In the alphaherpesvirus pseudorabies virus (PrV), the latter process is impaired when the US3 protein is absent. Acquisition of final tegument and envelope occurs in the cytoplasm. Besides the capsid components, only the UL31 and UL34 gene products of PrV have unequivocally been shown to be part of primary enveloped virions, whereas they lack several tegument proteins present in mature virions (reviewed by T. C. Mettenleiter, J. Virol. 76:1537-1547, 2002). Using immunoelectron microscopy, we show that the US3 protein is present in primary enveloped as well as in mature virions. It is also detectable in intracytoplasmic inclusions produced in the absence of other viral tegument components or envelope-associated glycoproteins. In particular, inclusions formed in the absence of the inner tegument protein UL37 contained the US3 protein. Thus, the US3 protein is a tegument component of both forms of enveloped alphaherpes virions. We hypothesize that US3 protein in primary virions modulates deenvelopment at the outer leaflet of the nuclear membrane and is either lost from primary virions during nuclear egress and subsequently reacquired early during tegumentation or is retained during transit of the nucleocapsid through the nuclear membrane.     

The nuclear matrix from cells of different origin. Evidence for a common set of matrix proteins

We compared the protein composition of the nuclear matrix isolated from several murine embryonal carcinoma cells and mature tissues by two-dimensional gel electrophoresis. Two nuclear matrix fractions were investigated: the "peripheral" nuclear matrix (matrix proteins that remain insoluble after reduction), and the "internal" nuclear matrix (matrix proteins released by reduction). The two subfractions have completely different protein compositions. Although numerous differences in nuclear matrix protein composition among different cell types were observed, a limited set of polypeptides common to all mouse cell types was identified. A majority of these common proteins was also present in cells from other mammalian species (i.e. rat and human). For this set of proteins, we coin the term "minimal matrix." As expected, lamin B, known to be expressed throughout differentiation, is part of the common set of peripheral nuclear matrix proteins. Lamins A and C are not because these proteins were absent from undifferentiated embryonal carcinoma cells. Since these common nuclear matrix proteins occur in all mammalian nuclear matrices analyzed so far, it is likely that they have a basic role in nuclear organization and function.     

The UL25 protein of pseudorabies virus associates with capsids and localizes to the nucleus and to microtubules

The UL25 gene of pseudorabies virus (PrV) can encode a protein of about 57 kDa which is well conserved among herpesviruses. The UL25 protein of herpes simplex virus type 1 is a capsid constituent involved in virus penetration and capsid maturation. To identify and characterize the UL25 gene product of PrV, polyclonal mouse anti-UL25 antibodies were raised to a bacterially expressed fusion protein. In immunoblotting and immunoprecipitation assays of PrV-infected cell lysates, these anti-UL25 antisera specifically recognized a protein of the expected size with late expression kinetics. This 57-kDa product was also present in purified virions and was found to be associated with all types of capsids. Synthesis of a protein migrating at the same size point was directed from the eukaryotic expression plasmid pCG-UL25. To determine the subcellular localization of UL25, immunofluorescence studies with anti-UL25 antisera were performed on Nonidet P-40-extracted COS-7 cells infected with PrV or transfected with pCG-UL25. In PrV-infected cells, newly synthesized UL25 is directed mainly to distinct nuclear compartments, whereas UL25 expressed in the absence of other viral proteins is distributed more uniformly in the nucleus and colocalizes also with microtubules. To study the fate of UL25 at very early stages of infection, immunofluorescence experiments were performed on invading PrV particles in the presence or absence of drugs that specifically depolymerize components of the cytoskeleton. We found that the incoming nucleocapsids colocalize with microtubules during their transport to the nucleus and that UL25 remains associated with nucleocapsids during this transport.     

人成骨肉瘤MG-63细胞在HMBA诱导分化后核基质蛋白表达的变化

HMBA诱导处理人成骨肉瘤MG-63细胞分化后,选择性抽提核基质蛋白,经双向凝胶电泳技术分析．共有12个蛋白点表达发生变化,经肽指纹图谱分析鉴定了9个蛋白质,其中,MHCⅡ类抗原、IFN刺激的基因因子3α蛋白、DKFZp434M2221．1蛋白、8-羟基-鸟嘌呤糖基化酶同功酶oggl和波形蛋白表达上调,hnRNP A2/B1和肌动蛋白表达下调,60S核糖体蛋白L21和ST2蛋白仅在分化的MG-63细胞中表达。研究结果表明肿瘤细胞增殖分化过程中伴随核基质蛋白表达的变化,并为深入揭示核基质蛋白与细胞癌变和逆转的关系以及阐明细胞增殖分化的基因表达调控原理提供了依据。     

The pseudorabies virus UL11 protein is a virion component involved in secondary envelopment in the cytoplasm

Homologs of the small tegument protein encoded by the UL11 gene of herpes simplex virus type 1 are conserved throughout all herpesvirus subfamilies. However, their function during viral replication has not yet been conclusively shown. Using a monospecific antiserum and an appropriate viral deletion and rescue mutant, we identified and functionally characterized the UL11 protein of the alphaherpesvirus pseudorabies virus (PrV). PrV UL11 encodes a protein with an apparent molecular mass of 10 to 13 kDa that is primarily detected at cytoplasmic membranes during viral replication. In the absence of the UL11 protein, viral titers were decreased approximately 10-fold and plaque sizes were reduced by 60% compared to wild-type virus. Intranuclear capsid maturation and nuclear egress resulting in translocation of DNA-containing capsids into the cytoplasm were not detectably affected. However, in the absence of the UL11 protein, intracytoplasmic membranes were distorted. Moreover, in PrV-DeltaUL11-infected cells, capsids accumulated in the cytoplasm and were often found associated with tegument in aggregated structures such as had previously been demonstrated in cells infected with a PrV triple-mutant virus lacking glycoproteins E, I, and M (A. R. Brack, J. M. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). Thus, the PrV UL11 protein, like glycoproteins E, I, and M, appears to be involved in secondary envelopment.     

Effect of bacteriophage M13 infection on phosphorylation of dnaK protein and other Escherichia coli proteins

1. The effects of infection with the filamentous phage M13 on the phosphorylation of Escherichia coli proteins were studied. Phosphorylated proteins were labeled with [32P]orthophosphate and analyzed by the O'Farrell two-dimensional gel technique and autoradiography. 2. Phage infection was shown to induce significant changes in the pattern of protein phosphorylation. At least eight different proteins were found to be phosphorylated to a larger extent while seven others were, by contrast, much less labeled than in uninfected bacteria. 3. Labeling experiments with [35S]methionine demonstrated that these quantitative changes in protein phosphorylation were not connected, in any case, with changes in the amount of protein synthesized. They rather seemed to result from a variation of the phosphorylating capacity of the relevant protein kinase(s). 4. The individual proteins, whose phosphorylation was affected by phage infection, were characterized by both their molecular mass and isoelectric point. One of them, whose phosphorylation was increased by a factor of 7, was identified as the dnaK protein which is necessary for both cellular and phage DNA replication. 5. The chemical analysis of the phosphorylated moiety of dnaK protein showed that it was modified exclusively at serine residues during normal growth of cells, and mostly at threonine residues after phage infection. These results were discussed in terms of stimulation of the protein activity by phosphorylation.