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1.
Adenovirus early region 4 is required for efficient virus particle assembly. 总被引:9,自引:6,他引:3
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H2dl807, a defective deletion mutant of human adenovirus type 2 lacking parts of early regions 3 and 4 and all of late region 5, was severely defective for virus particle assembly on HeLa cells, producing about 1% of the normal yield of particles. On Vero cells, H2dl807 produced only 5% as many particles as wild type, while on W162 cells, a Vero cell derivative which supports the growth of early region 4 mutants, H2dl807 produced nearly 40% of the wild-type level of particles. Two other defective deletion mutants, H2dl802 and H5dl1021, which lack parts of early region 3 and which are incapable of making fiber, the product of late region 5, were wild type for virus assembly. These data suggest that the cause of the assembly defect of H2dl807 is the lack of a diffusible early region 4 product. H2dl807-infected Vero cells accumulated nearly wild-type amounts of viral late proteins in the nucleus and cytoplasm. Thus, the defect of the mutant in assembly on Vero cells is not due to a general lack of late proteins. Finally, the fact that H2dl802 and H5dl1021 make wild-type amounts of virus particles suggests that fiber is not essential for adenovirus assembly. 相似文献
2.
Linker insertion mutations in the human immunodeficiency virus type 1 gag gene: effects on virion particle assembly, release, and infectivity. 总被引:16,自引:13,他引:3
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The phenotypes of a series of mutant human immunodeficiency virus type 1 proviruses with linker insertion and deletion mutations within the gag coding region were characterized. These mutants were tested for their ability to make and release viral particles in COS7 cells and for their viability in vivo. Of the 12 mutant proviruses, 4 did not make extracellular virion particles when transfected into COS7 cells. All four of these mutants had mutations in the C-terminal domain of CA. These mutants appeared to have defects both in the ability to accumulate high-molecular-weight intracellular structures containing Gag and Pol products and in the ability to release virion particles. Seven of the mutant proviruses retained the ability to make, release, and process virion particles from COS7 cells. These particles contained the Env glycoprotein, viral genomic RNA, and the mature products of the Gag and Gag-Pol polyproteins, yet they were noninfectious or poorly infectious. The defect in these mutants appears to be in one of the early steps of the viral life cycle. Thus, multiple regions throughout Gag appear to be important in mediating the early steps of the viral life cycle. 相似文献
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cyt gene of adenoviruses 2 and 5 is an oncogene for transforming function in early region E1B and encodes the E1B 19,000-molecular-weight polypeptide 总被引:22,自引:14,他引:8
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A total of 59 cytocidal (cyt) mutants were isolated from adenovirus 2 (Ad2) and Ad5. In contrast to the small plaques and adenovirus type of cytopathic effects produced by wild-type cyt+ viruses, the cyt mutants produced large plaques, and the cytopathic effect was characterized by marked cellular destruction. cyt mutants were transformation defective in established rat 3Y1 cells. cyt+ revertants and cyt+ intragenic recombinants recovered fully the transforming ability of wild-type viruses. Thus, the cyt gene is an oncogene responsible for the transforming function of Ad2 and Ad5. Genetic mapping in which we used three Ad5 deletion mutants (dl312, dl313, and dl314) as reference deletions located the cyt gene between the 3' ends of the dl314 deletion (nucleotide 1,679) and the dl313 deletion (nucleotide 3,625) in region E1B. Restriction endonuclease mapping of these recombinants suggested that the cyt gene encodes the region E1B 19,000-molecular-weight (175R) polypeptide (nucleotides 1,711 to 2,236). This was confirmed by DNA sequencing of eight different cyt mutants. One of these mutants has a single missense mutant, two mutants have double missense mutations, and five mutants have nonsense mutations. Except for one mutant, these point mutations are not located in any other known region E1B gene. We conclude that the cyt gene codes for the E1B 19,000-molecular-weight (175R) polypeptide, that this polypeptide is required for morphological transformation of rat 3Y1 cells, and that simple amino acid substitutions in the protein can be sufficient to produce the cyt phenotype. 相似文献
5.
Burckhardt CJ Suomalainen M Schoenenberger P Boucke K Hemmi S Greber UF 《Cell host & microbe》2011,10(2):105-117
Viral particle binding to plasma membrane receptors elicits virus motions, recruits signaling proteins, and triggers membrane bending and fission, finally resulting in endocytic virus uptake. Here we analyze how human adenovirus engages its receptor coxsackievirus adenovirus receptor (CAR) and coreceptor αv integrin to move on the plasma membrane. Virus binding to CAR through fiber knobs gave rise to diffusive motions and actomyosin-2-dependent drifts, while integrin-targeted viruses were spatially more confined. Diffusions, drifts, and confined motions were specifically observed with viral particles that were subsequently internalized. CAR-mediated drifts together with integrin binding supported fiber shedding from adenovirus particles, leading to?exposure of the membrane-lytic internal virion protein VI and enhanced viral escape from endosomes. Our results show that adenovirus uncoating is initiated at the plasma membrane by CAR drifting motion and binding to immobile integrins. 相似文献
6.
Deletion of the gene encoding the adenovirus 5 early region 1b 21,000-molecular-weight polypeptide leads to degradation of viral and host cell DNA. 总被引:38,自引:27,他引:11
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The adenovirus 5 mutant H5dl337 lacks 146 base pairs within early region 1B. The deletion removes a portion of the region encoding the E1B 21,000-molecular-weight (21K) polypeptide, but does not disturb the E1B-55K/17K coding region. The virus is slightly defective for growth in cultured HeLa cells, in which its final yield is reduced ca. 10-fold compared with wild-type virus. The mutant displays a striking phenotype in HeLa cells. The onset of cytopathic effect is dramatically accelerated, and both host cell and viral DNAs are extensively degraded late after infection. This defect has been described previously for a variety of adenovirus mutants and has been termed a cytocidal (cyt) phenotype. H5dl337 serves to map this defect to the loss of E1B-21K polypeptide function. In addition to its defect in the productive growth cycle, H5dl337 is unable to transform rat cells at normal efficiency. 相似文献
7.
The simian virus 40 sequences between 0.169 and 0.423 map units are not essential to immortalize early-passage rat embryo cells. 总被引:6,自引:2,他引:4
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F8dl is a simian virus 40 early-region deletion mutant that lacks the sequences between 0.169 and 0.423 map units. We show that cloned F8dl DNA immortalized early-passage Fisher rat embryo cells with an efficiency that was about 20% of that of cloned wild-type simian virus 40 DNA. In contrast, we detected no immortalized colonies when we transfected the cells with DNA of five other early-region deletion mutants that do not make stable truncated forms of T antigen. Since all five of these mutants have intact early- and late-region control sequences, we conclude that these control sequences are not sufficient for immortalization. Three of the mutants that did not immortalize did make a normal small t antigen, suggesting that the expression of this protein alone is not sufficient for immortalization of early-passage Fisher rat embryo cells. 相似文献
8.
Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: mapping and functional analysis of the head fiber gene.
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A set of mutants of Bacillus subtilis bacteriophage phi29 unable to synthesize the head fiber protein has been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Infectious phage are produced during restrictive infection. We have focused on mutant sus 8.5(900) because the mutation is suppressible by both the su(+3) and su(+44) hosts, and it can be mapped by three- and four-factor crosses. After restrictive infection with mutant sus 8.5(900), a fragment about 70% of the size of the normal fiber is produced as well as particles that are fast-sedimenting in sucrose gradients relative to phi29(+). These particles have the buoyant density of particles with the fibers removed and have the absolute plating efficiency of phi29(+). Fiber protein is absent from prohead as well as virion. A second set of mutants produces fiber protein with a slightly altered electrophoretic mobility. This type of fiber protein is either present or absent on both prohead and virion. A third class of mutants, typified by 914, produces a "normal" fiber, but a major head protein of altered electrophoretic mobility. After infection by this mutant, the fiber is absent from both prohead and virion, and the biological and physical properties of the 914(-) particle are similar to those of particles produced after infection of the su(-) host by sus8.5(900). These observations suggest that the head fiber is not an essential component of the prohead or virion and that the assembly process is efficient in the absence of fiber protein. Three- and four-factor genetic crosses have established the order sus8(769)-8(914)-sus8.5(900)-sus9(756) and indicate that cistrons 8 and 8.5 code for the major head protein and head fiber protein, respectively. 相似文献
9.
Grit Kupgan Danielle C. Hentges Nathan J. Muschinske William D. Picking Wendy L. Picking Joshua D. Ramsey 《Molecular biotechnology》2014,56(11):979-991
While fiberless adenovirus has the potential for use as a vaccine or gene delivery vector, some groups have observed instability issues associated with the modified virus. To investigate the effect of fiber modification on adenovirus stability, we produced mutant adenovirus particles that contained the tail and a portion of the shaft domain without the knob. The shaft domain was either completely removed (i.e., fiberless) or truncated to 7-, 14-, or 21-repeats. The mutants were evaluated by biophysical characterization techniques to determine their relative stabilities based on temperature-induced changes to the secondary, tertiary, and quaternary structures of the virus and its constituent proteins. Data acquired using circular dichroism, intrinsic/extrinsic fluorescence, and static/dynamic light scattering were compiled into a comprehensive empirical phase diagram, which showed that native adenovirus was the most stable followed by fiberless adenovirus and then the mutants with truncated fiber protein. In summary, the individual biophysical measurements and the empirical phase diagram showed that providing several repeats of shaft protein negatively impacted the structural stability of the virus more so than completely removing the fiber protein. 相似文献
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The molecular mechanism for packaging of the adenovirus (Ad) genome into the capsid is likely similar to that of DNA bacteriophages and herpesviruses-the insertion of viral DNA through a portal structure into a preformed prohead driven by an ATP-hydrolyzing molecular machine. It is speculated that the IVa2 protein of adenovirus is the ATPase providing the power stroke of the packaging machinery. Purified IVa2 binds ATP in vitro and, along with a second Ad protein, the L4 22-kilodalton protein (L4-22K), binds specifically to sequences in the Ad genome that are essential for packaging. The efficiency of binding of these proteins in vitro was correlated with the efficiency of packaging in vivo. By utilizing a virus unable to express IVa2, pm8002, it was reported that IVa2 plays a role in assembly of the empty virion. We wanted to address the question of whether the ATP binding, and hence the putative ATPase activity, of IVa2 was required for its role in virus assembly. Our results show that ATPase activity was not required for the assembly of empty virus particles. In addition, we present evidence that particles were assembled in the absence of IVa2 by using two viruses null for IVa2-a deletion mutant virus, ΔIVa2, and the previously described mutant virus, pm8002. Empty virus particles produced by these IVa2 mutant viruses did not contain detectable viral DNA. We conclude that the major role of IVa2 is in viral DNA packaging. A characterization of the empty particles obtained from the IVa2 mutant viruses compared to wild-type empty particles is presented. 相似文献
12.
Composition of pseudorabies virus particles lacking tegument protein US3, UL47, or UL49 or envelope glycoprotein E
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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. 相似文献
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Evaluation of the roles of specific regions of the Cucumber necrosis virus coat protein arm in particle accumulation and fungus transmission
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The Cucumber necrosis virus (CNV) particle is a T=3 icosahedron composed of 180 identical coat protein (CP) subunits. Each CP subunit includes a 34-amino-acid (aa) arm which connects the RNA binding and shell domains. The arm is comprised of an 18-aa "beta" region and a 16-aa "epsilon" region, with the former contributing to a beta-annular structure involved in particle stability and the latter contributing to quasiequivalence and virion RNA binding. Previous work has shown that specific regions of the CNV capsid play important roles in transmission by zoospores of the fungal vector Olpidium bornovanus and that particle expansion is essential for this process. To assess the importance of the two arm regions in particle accumulation, stability, and virus transmission, five CP arm deletion mutants were constructed. Our findings indicate that beta(-) mutants are capable of producing particles in plants; however, the arm(-) and epsilon(-) mutants are not. In addition, beta(-) particles bind zoospores less efficiently than wild-type CNV and are not fungally transmissible. Beta(-) particles are also less thermally stable and disassemble under swelling conditions. Our finding that beta(-) mutants can accumulate in plants suggests that other features of the virion, such as RNA/CP interactions, may also be important for particle stability. 相似文献
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Mutations in the E1a gene of type 5 adenovirus result in oncogenic transformation of Fischer rat embryo cells 总被引:1,自引:0,他引:1
G J Duigou L E Babiss W S Liaw S G Zimmer H S Ginsberg P B Fisher 《Journal of cellular biochemistry》1987,33(2):117-126
Transformation of a specific clone of Fischer rat embryo (CREF) cells with wild-type 5 adenovirus (Ad5) or the E1a plus E1b transforming gene regions of Ad5 results in epithelioid transformants that grow efficiently in agar but that do not induce tumors when inoculated into nude mice or syngeneic Fischer rats. In contrast, CREF cells transformed by a host-range Ad5 mutant, H5hrl, which contains a single base-pair deletion of nucleotide 1055 in E1a resulting in a 28-kd protein (calculated) in place of the wild-type 51-kd acidic protein, display a cold-sensitive transformation phenotype and an incomplete fibroblastic morphology but surprisingly do induce tumors in nude mice and syngeneic rats. Tumors develop in both types of animals following injection of CREF cells transformed by other cold-sensitive Ad5 E1a mutants (H5dl101 and H5in106), which contain alterations in their 13S mRNA and consequently truncated 289AA proteins. CREF cells transformed with only the E1a gene (0-4.5 m.u.) from H5hrl or H5dl101 also produce tumors in these animals. To directly determine the role of the 13S E1a encoded 289AA protein and the 12S E1a encoded 243AA protein in initiating an oncogenic phenotype in adenovirus-transformed CREF cells, we generated transformed cell lines following infection with the Ad2 mutant pm975, which synthesizes the 289AA E1a protein but not the 243AA protein, and the Ad5 mutant H5dl520 and the Ad2 mutant H2dl1500, which do not produce the 289AA E1a protein but synthesize the normal 243AA E1a protein. All three types of mutant adenovirus-transformed CREF cells induced tumors in nude mice and syngeneic rats. Tumor formation by these mutant adenovirus-transformed CREF cells was not associated with changes in the arrangement of integrated adenovirus DNA or in the expression of adenovirus early genes. These results indicate, therefore, that oncogenic transformation of CREF cells can occur in the presence of a wild-type 13S E1a protein or a wild-type 12S E1a protein when either protein is present alone, but does not occur when both wild-type E1a proteins are present. 相似文献
18.
Morphogenesis of human adenovirus type 2 studied with fiber- and fiber and penton base-defective temperature-sensitive mutants. 总被引:10,自引:8,他引:2
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The nature, polypeptide composition, and antigenic composition of the particles formed by six human adenovirus type 2 temperature-sensitive (ts) mutants were studied. ts115, ts116, and ts125 were phenotypically fiber-defective mutants, and ts103, ts104, and ts136 failed to synthesize detectable amounts of fiber plus penton base at 39.5 degrees C. The mutants belonged to five complementation groups, one group including ts116 and ts125. Except for ts103 and ts136, the other mutants were capable of producing particles at 39.5 degrees C. ts116 and ts125 accumulated light assembly intermediate particles (or top components) at nonpermissive temperatures, with few virus particles. The sodium dodecyl sulfate polypeptide pattern of ts116- or ts125-infected cells, intermediate particles, and virus particles showed that polypeptide IV (fiber) was smaller by a molecular weight of 2,000 than that in the wild-type virion and was glycosylated. In fiber plus penton base-defective ts104-infected cells, equivalent quantities of top components and viruses with a buoyant density (rho) of 1.345 g/ml (rho = 1.345 particles) were produced at 39.5 degrees C. These rho = 1.345 particles corresponded to young virions, as evidenced by the presence of uncleaved precursors to proteins VI, VIII, and VII. These young virions matured upon a shift down. Virus capsid vertex antigenic components underwent a phase of eclipse during their incorporation into mature virus particles. No antigenic penton base or IIa was detected in intermediate particles of all the ts mutants tested. Only hexon and traces of fiber antigens were found in ts104 young virions. Penton base and IIIa appeared as fully antigenically expressed capsid subunits in mature wild-type virions or ts104 virions after a shift down. The ts104 lesion is postulated to affect a regulatory function related in some way to penton base and fiber overproduction and the maturation processing of precursors PVI, PVII, and PVII. 相似文献
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Identification of adenovirus 2 early genes required for induction of cellular DNA synthesis in resting hamster cells. 总被引:6,自引:2,他引:4
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tsAF8 cells are temperature-sensitive (ts) mutants of BHK-21 cells that arrest at the nonpermissive temperature in the G1 phase of the cell cycle. When made quiescent by serum restriction, they can be stimulated to enter the S phase by 10% serum at 34 degrees C, but not at 40.6 degrees C. Infection by adenovirus type 2 or type 5 stimulates cellular DNA synthesis in tsAF8 cells at both 34 and 40.6 degrees C. Infection of these cells with deletion Ad5dl312, Ad5dl313, Ad2 delta p305, and Ad2+D1) and temperature-sensitive (H5ts125, H5ts36) mutants of adenovirus indicates that the expression of both early regions 1A and 2 is needed to induce quiescent tsAF8 cells to enter the S phase at the permissive temperature. This finding has been confirmed by microinjection of selected adenovirus DNA fragments into the nucleus of tsAF8 cells. In addition, we have shown that additional viral functions encoded by early regions 1B and 5 are required for the induction of cellular DNA synthesis at the nonpermissive temperature. 相似文献