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1.
Identification of an essential domain in the herpes simplex virus 1 UL34 protein that is necessary and sufficient to interact with UL31 protein 总被引:4,自引:0,他引:4
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Previous results have indicated that the herpes simplex virus 1 UL31 and UL34 proteins interact and form a complex at the inner nuclear membranes of infected cells, where both play important roles in the envelopment of nucleocapsids at the inner nuclear membrane. In the work described here, mapping studies using glutathione S-transferase pull-down assays indicated that amino acids 137 to 181 of the UL34 protein are sufficient to mediate an interaction with the UL31 protein. A recombinant virus (v3480) lacking UL34 codons 138 to 181 was constructed. Similar to a UL34 null virus, v3480 failed to replicate on Vero cells and grew to a limited extent on rabbit skin cells. A UL34-expressing cell line restored v3480 growth and plaque formation. Similar to the localization of UL31 protein in cells infected with a UL34 null virus, the UL31 protein was present in the nuclei of Hep2 cells infected with v3480. Hep2 cells infected with v3480 contained the UL34 protein in the cytoplasm, the nucleus, and the nuclear membrane, and this was noted to be similar to the appearance of cells infected with a UL31 null virus. In transient expression assays, the interaction between UL34 amino acids 137 to 181 and the UL31 protein was sufficiently robust to target green fluorescent protein and emerin to intranuclear sites that contained the UL31 protein. These data indicate that amino acids 137 to 181 of the UL34 protein are (i) sufficient to mediate interactions with the UL31 protein in vitro and in vivo, (ii) necessary for the colocalization of UL31 and UL34 in infected cells, and (iii) essential for normal viral replication. 相似文献
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DNA polymerase alpha from the nuclear matrix of cells infected with simian virus 40. 总被引:7,自引:1,他引:6
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The nuclear matrix prepared from normal, simian virus 40 (SV40)-infected, and SV40-transformed cells contained DNA polymerase activities. Approximately 12% of the total DNA polymerase activities in isolated nuclei remained with the nuclear matrix. alpha-polymerase was the major matrix DNA polymerase activity as judged by sensitivity to various inhibitors: aphidicolin, dideoxy-TTP, and N-ethylmaleimide. Approximately 2-4 fold higher DNA polymerase activity was detected in matrices obtained from lytically infected and virus-transformed cells than that found in normal cells. In lytically infected cells, 30-50% of the matrix-bound DNA polymerase activity solubilized by sonication co-sedimented with majority of the matrix T-antigen, and was co-precipitated with anti-T sera. The results suggest that alpha-polymerase and viral T-antigen may form a functional complex in the matrix. 相似文献
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L R Trusal A Anthony J J Docherty 《The journal of histochemistry and cytochemistry》1975,23(4):283-288
Infection of human embryonic lung cells with herpes simplex virus type 1 (HSV-1) and herpes simplex type 1 (HSV-2) resulted in: (a) qualitative (nuclear cytopathologic) alterations and quantitative (nuclear area) differences in infected compared to control nuclei; (b) increased Feulgen-deoxyribonucleic acid (F-DNA) amounts in infected cells, probably due to viral DNA; (c) higher F-DNA levels in HSV-2 infected cells; and (d) increased rates of F-DNA hydrolysis in viral-infected as compared to uninfected nuclei. 相似文献
6.
Adenovirus proteins associated with mRNA and hnRNA in infected HeLa cells. 总被引:14,自引:10,他引:4
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The proteins that interact with cytoplasmic and nuclear polyadenylated RNA in adenovirus type 5 (Ad5) infection of HeLa cells were examined by UV-induced RNA-protein cross-linking in intact cells. The Ad5 100-kilodalton late nonvirion protein (100K protein) was cross-linked to both host and viral polyadenylated cytoplasmic RNA (mRNA). The cross-linking of the 100K protein to mRNA appears to correlate with productive infection, because the protein is not cross-linked to mRNA in abortive infection of wild-type Ad5 in monkey cells (CV-1) even though normal amounts of it are produced. However, when CV-1 cells are infected with Ad5 hr404, and Ad5 mutant which overcomes the host restriction to wild-type Ad5 infection in these cells, the 100K protein is cross-linked to mRNA. To identify and obtain antibodies to RNA-contacting proteins, a mouse was immunized with oligo(dT)-selected cross-linked RNA-protein complexes from Ad5-infected cells and the serum was used for immunoblotting experiments. It was found that in addition to the 100K protein, the Ad5 72K DNA-binding protein is also associated with RNA in the infected cells. The 72K DNA-binding protein is cross-linked to polyadenylated nuclear RNA sequences. These findings indicate that adenovirus proteins interact with RNAs in the infected cell and suggest possible mechanisms for the effects of the virus on mRNA metabolism. 相似文献
7.
Effects of lamin A/C, lamin B1, and viral US3 kinase activity on viral infectivity, virion egress, and the targeting of herpes simplex virus U(L)34-encoded protein to the inner nuclear membrane
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Previous results indicated that the U(L)34 protein (pU(L)34) of herpes simplex virus 1 (HSV-1) is targeted to the nuclear membrane and is essential for nuclear egress of nucleocapsids. The normal localization of pU(L)34 and virions requires the U(S)3-encoded kinase that phosphorylates U(L)34 and lamin A/C. Moreover, pU(L)34 was shown to interact with lamin A in vitro. In the present study, glutathione S-transferase/pU(L)34 was shown to specifically pull down lamin A and lamin B1 from cellular lysates. To determine the role of these interactions on viral infectivity and pU(L)34 targeting to the inner nuclear membrane (INM), the localization of pU(L)34 was determined in LmnA(-/-) and LmnB1(-/-) mouse embryonic fibroblasts (MEFs) by indirect immunofluorescence and immunogold electron microscopy in the presence or absence of U(S)3 kinase activity. While pU(L)34 INM targeting was not affected by the absence of lamin B1 in MEFs infected with wild-type HSV as viewed by indirect immunofluorescence, it localized in densely staining scalloped-shaped distortions of the nuclear membrane in lamin B1 knockout cells infected with a U(S)3 kinase-dead virus. Lamin B1 knockout cells were relatively less permissive for viral replication than wild-type MEFs, with viral titers decreased at least 10-fold. The absence of lamin A (i) caused clustering of pU(L)34 in the nuclear rim of cells infected with wild-type virus, (ii) produced extensions of the INM bearing pU(L)34 protein in cells infected with a U(S)3 kinase-dead mutant, (iii) precluded accumulation of virions in the perinuclear space of cells infected with this mutant, and (iv) partially restored replication of this virus. The latter observation suggests that lamin A normally impedes viral infectivity and that U(S)3 kinase activity partially alleviates this impediment. On the other hand, lamin B1 is necessary for optimal viral replication, probably through its well-documented effects on many cellular pathways. Finally, neither lamin A nor B1 was absolutely required for targeting pU(L)34 to the INM, suggesting that this targeting is mediated by redundant functions or can be mediated by other proteins. 相似文献
8.
The immunohistochemical localization of the heat shock proteins (Hsp70 and Hsp90) and histone protein in healthy and Paenibacillus larvae infected honeybee (Apis mellifera L.) larvae has been studied. Hsp70 was found in the nuclei and the cytoplasm of infected midgut, salivary gland cells and haemocytes, but not in uninfected larvae. Hsp90 was localized in both infected and uninfected cells. Exposed histone proteins were localized in the nuclei of dying uninfected cells undergoing programmed cell death. The distribution of histone protein in uninfected cells of midgut, salivary gland, and other tissues was nuclear and indicative of normal programmed cell death at levels between 1 and 5%.After applying histone protein antibodies to P. larvae infected honeybee larvae, the DAB based reaction product was located in the nuclei or immediate surroundings of all larval cells. The Hsp70, Hsp90 and histone protein distribution patterns are discussed in relation to the morphological, cytochemical and immunocytochemical characteristics of programmed cell death and pathological necrosis. Results produced by methyl green-pyronin staining confirm an elevation of RNA levels in normal programmed cell death and a reduced staining for RNA in necrotic infected cells. 相似文献
9.
Inhibition of nuclear import and alteration of nuclear pore complex composition by rhinovirus
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Nucleocytoplasmic trafficking pathways and the status of nuclear pore complex (NPC) components were examined in cells infected with rhinovirus type 14. A variety of shuttling and nonshuttling nuclear proteins, using multiple nuclear import pathways, accumulated in the cytoplasm of cells infected with rhinovirus. An in vitro nuclear import assay with semipermeabilized infected cells confirmed that nuclear import was inhibited and that docking of nuclear import receptor-cargo complexes at the cytoplasmic face of the NPC was prevented in rhinovirus-infected cells. The relocation of cellular proteins and inhibition of nuclear import correlated with the degradation of two NPC components, Nup153 and p62. The degradation of Nup153 and p62 was not due to induction of apoptosis, because p62 was not proteolyzed in apoptotic HeLa cells, and Nup153 was cleaved to produce a 130-kDa cleavage product that was not observed in cells infected with poliovirus or rhinovirus. The finding that both poliovirus and rhinovirus cause inhibition of nuclear import and degradation of NPC components suggests that this may be a common feature of the replicative cycle of picornaviruses. Inhibition of nuclear import is predicted to result in the cytoplasmic accumulation of a large number of nuclear proteins that could have functions in viral translation, RNA synthesis, packaging, or assembly. Additionally, inhibition of nuclear import also presents a novel strategy whereby cytoplasmic RNA viruses can evade host immune defenses by preventing signal transduction into the nucleus. 相似文献
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Emerin is hyperphosphorylated and redistributed in herpes simplex virus type 1-infected cells in a manner dependent on both UL34 and US3 总被引:7,自引:4,他引:3
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Leach N Bjerke SL Christensen DK Bouchard JM Mou F Park R Baines J Haraguchi T Roller RJ 《Journal of virology》2007,81(19):10792-10803
Cells infected with wild-type herpes simplex virus type 1 (HSV-1) show disruption of the organization of the nuclear lamina that underlies the nuclear envelope. This disruption is reflected in changes in the localization and phosphorylation of lamin proteins. Here, we show that HSV-1 infection causes relocalization of the LEM domain protein emerin. In cells infected with wild-type virus, emerin becomes more mobile in the nuclear membrane, and in cells infected with viruses that fail to express UL34 protein (pUL34) and US3 protein (pUS3), emerin no longer colocalizes with lamins, suggesting that infection causes a loss of connection between emerin and the lamina. Infection causes hyperphosphorylation of emerin in a manner dependent upon both pUL34 and pUS3. Some emerin hyperphosphorylation can be inhibited by the protein kinase Cdelta (PKCdelta) inhibitor rottlerin. Emerin and pUL34 interact physically, as shown by pull-down and coimmunoprecipitation assays. Emerin expression is not, however, necessary for infection, since virus growth is not impaired in cells derived from emerin-null transgenic mice. The results suggest a model in which pUS3 and PKCdelta that has been recruited by pUL34 hyperphosphorylate emerin, leading to disruption of its connections with lamin proteins and contributing to the disruption of the nuclear lamina. Changes in emerin localization, nuclear shape, and lamin organization characteristic of cells infected with wild-type HSV-1 also occur in cells infected with recombinant virus that does not make viral capsids, suggesting that these changes occur independently of capsid envelopment. 相似文献
12.
Simian virus 40 (SV40)-specific proteins associated with the nuclear matrix isolated from adenovirus type 2-SV40 hybrid virus-infected HeLa cells carry SV40 U-antigen determinants. 总被引:6,自引:3,他引:3
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W Deppert 《Journal of virology》1978,26(1):165-178
The distribution of simian virus 40 (SV40)-specific proteins in nuclear subfractions of pulse-chase-labeled HeLa cells infected with nondefective adenovirus type 2 (Ad2)-SV40 hybrid viruses was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The SV40-specific proteins of Ad2+ND1, Ad2+ND2, and Ad2+ND5 specifically associate with the nuclear matrix and are virtually absent from the high-salt nuclear extract. In Ad2+ND4-infected HeLa cells, the SV40-specific proteins with molecular weights of 64,000 (64K) and lower also specifically associate with the nuclear matrix. The SV40-specific 72K, 74K, and 95K proteins were found both in the nuclear matrix and in the high-salt nuclear extract. Analyses of the nuclear matrices isolated from hybrid virus-infected cells by immunofluorescence microscopy showed that SV40 U-antigen-positive sera from SV40 tumor-bearing hamsters react with SV40-specific proteins integrated into nuclear matrices of HeLa cells infected by Ad2+ND1, Ad2+ND2, and Ad2+ND4, but not with nuclear matrices of HeLa cells infected by Ad2+ND5. This suggests that SV40-specific proteins of Ad2+ND1, Ad2+ND2, and Ad2+ND4 integrated into the nuclear matrix carry SV40 U-antigen determinants. The apparent discrepancy in the subcellular localization of SV40-specific proteins in hybrid virus-infected cells when analyzed by biochemical cell fractionation procedures and when analyzed by immunofluorescence staining is discussed. 相似文献
13.
Effects of poliovirus infection on nucleo-cytoplasmic trafficking and nuclear pore complex composition
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Infection of eukaryotic cells with lytic RNA viruses results in extensive interactions of viral gene products with macromolecular pathways of the host, ultimately leading to death of the infected cells. We show here that infection of cells with poliovirus results in the cytoplasmic accumulation of a variety of shuttling and non-shuttling nuclear proteins that use multiple nuclear import pathways. In vitro nuclear import assays using semi-permeabilized infected cells confirmed that nuclear import was blocked and demonstrated that docking of nuclear import receptor-cargo complexes at the cytoplasmic face of the nuclear pore complex (NPC) was prevented. Analysis of components of the NPC revealed that two proteins, Nup153 and p62, were proteolyzed during poliovirus infection. These results suggest that the cytoplasmic relocalization of numerous cellular proteins is caused by the inhibition of multiple nuclear import pathways via alterations in NPC composition in poliovirus-infected cells. Blocking of nuclear import points to a novel strategy by which cytoplasmic RNA viruses can evade host immune defenses, by preventing signal transduction to the nucleus. 相似文献
14.
To define the roles of subnuclear structure in SV40 infection, the relative distribution of T-antigen (T-ag) in various subnuclear fractions obtained from both lytically infected and transformed African green monkey kidney cells was determined. Depending on the differential sensitivity of nuclear T-ag to extraction by salt and detergent, nuclear T-ag could be separated into nucleoplasmic T-ag, salt-sensitive T-ag and matrix-bound T-ag subclasses. At least fivefold less matrix-bound T-ag was found in transformed cells than in lytically infected cells. While a cAMP-independent protein kinase was detected in the nuclear matrix, the matrix-bound T-ag (94K) could not be phosphorylated in vitro. The removal of cellular chromosomes by DNase caused changes in the interaction of T-ag with nuclear components. The results suggest that the compartmentalization of nuclear T-ag may be determined by its interaction with host chromosomes. 相似文献
15.
Nakajima-Shimada J Zou C Takagi M Umeda M Nara T Aoki T 《Biochimica et biophysica acta》2000,1475(2):175-183
Trypanosoma cruzi-infected and normal control mammalian cells were subjected to analysis of Fas-mediated apoptosis stimulated by an agonistic anti-Fas monoclonal antibody. The infected cells showed markedly hampered apoptotic changes in nuclear morphology, phosphatidylethanolamine translocation from the inside to the outside of the plasma membrane, and DNA fragmentation into multiples of 180 bp, relative to normal control cells. Upstream of these morphological and biochemical consequences, the caspase-3 activity was elevated by the Fas stimulation in a significantly greater proportion of intact control cells, but at a highly reduced rate of infected cells. The rapid elevation of caspase-8 activity in control, apoptotic cells was completely inhibited in infected cells. In an examination of the specificity of other stimulants, X-ray radiation or chemicals such as hydrogen peroxide, colchicine or etoposide did not cause significant differences in apoptotic rates between control and infected cells; tumor necrosis factor-alpha, however, induced a high rate of apoptosis in control cells, with an extremely lowered rate in infected cells. This study demonstrates, for the first time, that T. cruzi infection inhibits one of the earliest steps of death receptor-mediated apoptosis, an effect that most probably involves the inhibition of caspase-8. Differential apoptotic responses in cells infected with T. cruzi and other intracellular parasites are discussed. 相似文献
16.
Fate of the inner nuclear membrane protein lamin B receptor and nuclear lamins in herpes simplex virus type 1 infection
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During herpesvirus egress, capsids bud through the inner nuclear membrane. Underlying this membrane is the nuclear lamina, a meshwork of intermediate filaments with which it is tightly associated. Details of alterations to the lamina and the inner nuclear membrane during infection and the mechanisms involved in capsid transport across these structures remain unclear. Here we describe the fate of key protein components of the nuclear envelope and lamina during herpes simplex virus type 1 (HSV-1) infection. We followed the distribution of the inner nuclear membrane protein lamin B receptor (LBR) and lamins A and B(2) tagged with green fluorescent protein (GFP) in live infected cells. Together with additional results from indirect immunofluorescence, our studies reveal major morphologic distortion of nuclear-rim LBR and lamins A/C, B(1), and B(2). By 8 h p.i., we also observed a significant redistribution of LBR-GFP to the endoplasmic reticulum, where it colocalized with a subpopulation of cytoplasmic glycoprotein B by immunofluorescence. In addition, analysis by fluorescence recovery after photobleaching reveals that LBR-GFP exhibited increased diffusional mobility within the nuclear membrane of infected cells. This is consistent with the disruption of interactions between LBR and the underlying lamina. In addition to studying stably expressed GFP-lamins by fluorescence microscopy, we studied endogenous A- and B-type lamins in infected cells by Western blotting. Both approaches reveal a loss of lamins associated with virus infection. These data indicate major disruption of the nuclear envelope and lamina of HSV-1-infected cells and are consistent with a virus-induced dismantling of the nuclear lamina, possibly in order to gain access to the inner nuclear membrane. 相似文献
17.
Frances Kallman John M. Adams Robley C. Williams David T. Imagawa 《The Journal of cell biology》1959,6(3):379-382
Cells which are infected with measles virus have been known for some time to contain inclusion material that is distinguishable from normal cellular components by application of traditional staining methods and observation in the light microscope. The fine structure of the inclusion material contained in HeLa cells infected with Edmonston strain of measles virus has been examined in the electron microscope. Two steps have been found necessary in this study: (1) the recognition by phase-contrast microscopy of the living cell of bodies that are defined as inclusion material when the cells are classically stained; and (2) the recognition in the electron microscope of inclusion-body material that had previously been identified in the living cell. The fine structure of the nuclear and cytoplasmic inclusion material in osmium-treated cells was found to consist mainly of randomly arrayed filaments of low electron density. Dense, highly ordered arrays of filaments were found near the center of the nuclear inclusions, sometimes as a two-dimensional, nearly orthogonal arrangement. If the size of the measles virus is taken to be around 100 mµ in diameter, the strands seen in the inclusions cannot be fully formed virus. 相似文献
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E. D. Ammar 《Journal of Phytopathology》1994,141(2):153-158
Samples of maize leaves naturally infected with maize streak virus (MSV) from Malawi and South Africa, as well as sugarcane leaves naturally infected with sugarcane streak virus (SSV) from Egypt, were examined by light (LM) and transmission electron, microscopy (TEM). Intranuclear inclusions, detectable by both methods, were found mainly in mesophyll and bundle sheath cells, and less frequently in vascular parenchyma and immature phloem cells. At higher TEM magnifications, these inclusions consisted, of crystalline or noncrystalline aggregates of isometric geminivirus–like particles (VLP) that occurred either singly or in geminate arrays. Cytopathological changes in these cells were confined to the nuclei, which were usually larger than normal, with peripheral chromatin and nucleoli. The nuclear envelope of some inclusion–containing nuclei was ruptured, and occasionally a crystal of VLP was found in the cytoplasm of cells in which no intact nuclei were detected. No differences in cytopathology were found between MSV and SSV, or between the two MSV isolates examined. 相似文献
20.
Similarities and Differences in the Development of Laboratory Strains and Freshly Isolated Strains of Herpes Simplex Virus in HEp-2 Cells: Electron Microscopy 总被引:30,自引:22,他引:8
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HEp-2 cells infected with two laboratory strains (mP and MP) and two freshly isolated strains (F and G) of herpes simplex virus were fixed at intervals between 4 and 50 hr postinfection and sectioned, and were then examined with the electron microscope. These studies revealed the following. (i) All four strains caused identical segregation of nucleoli and aggregation of host chromosomes at the nuclear membrane. (ii) The development of MP virus could not be differentiated from that of its parent mP strain. (iii) There were quantitative differences between laboratory (mP) and freshly isolated (F) type 1 strains. Thus, cells infected with F contained numerous nuclear crystals of nucleocapsids and relatively few cytoplasmic structures containing enveloped nucleocapsids. Conversely, cells infected with mP or with MP virus contained numerous cytoplasmic structures with enveloped nucleocapsids and relatively few nuclear crystals of nucleocapsids. (iv) There were qualitative differences between type 2 strain (G) isolated from genital lesions and type 1 strains. Thus, cells infected with the G strain contain numerous filaments in nuclei and unenveloped and partially enveloped nucleocapsids in the cytoplasm. Of particular interest is the finding that cytoplasmic membranes in apposition to nucleocapsids were thickened and bent as if they were enveloping the particle. The significance of the qualitative differences in the development of the four strains is discussed. 相似文献