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1.
The E4 region of human adenovirus type 9 (Ad9) transforms established rat embryo fibroblasts and encodes an essential determinant for the production of estrogen-dependent mammary tumors in rats. Testing of the seven Ad9 E4 open reading frames (ORFs) individually for transformation of the established rat embryo fibroblast cell line CREF indicated that only Ad9 E4 ORF1 possessed a significant ability to generate transformed foci on these cells. In contrast, the E4 ORF1 sequences from human Ad5 and Ad12 lacked the transforming potential exhibited by Ad9 E4 ORF1. Cell lines derived from Ad9 E4 ORF1-transformed foci expressed the 14-kDa Ad9 E4 ORF1 protein and formed colonies in soft agar. In addition, the Ad9 E4 ORF1 protein was required for initiation of mammary oncogenesis in vivo, as E4 ORF1 mutant viruses failed while E4 ORF2 and ORF3 mutant viruses succeeded in eliciting mammary tumors in animals. A role for Ad9 E4 ORF1 in tumor maintenance was suggested by the fact that 100% of virus-induced mammary tumors expressed the E4 ORF1 protein. Taken together, the facts that the Ad9 E4 ORF1 protein exhibits transforming potential in culture and is required by Ad9 to produce mammary tumors in animals suggest that Ad9 E4 ORF1 is a new viral oncoprotein.  相似文献   

2.
Human papillomavirus type 16 (HPV 16) DNA is capable of morphologically transforming rat 3Y1 cells. The expression plasmids, constructed from the simian virus 40-based expression vector pSV2-0 and specific DNA fragments from the putative early region of the HPV 16 genome, were tested for their transforming capacity. Among the various pSV2 plasmids, only those containing the intact E7 coding region were found to produce foci of the transformed rat cells which could grow in a soft-agar medium. The data indicate that expression of the HPV 16 E7 open reading frame is sufficient to induce focal transformation of rat cells.  相似文献   

3.
We have previously shown that the early region of the bovine papillomavirus type 1 genome contains two nonoverlapping segments that can independently induce the morphological transformation of cultured cells. The transforming gene from the 5' end of the early region is encoded by the E6 open reading frame. The second transforming segment was previously localized to a 2.3-kilobase fragment (2.3T) from the 3' end of the early region. To determine which of the four open reading frames (E2, E3, E4, and E5) located within 2.3T encodes a transforming gene, we have now introduced a series of insertion and deletion mutations into a clone (pHLB1) in which 2.3T is activated by the Harvey viral long terminal repeat, and we tested the mutants for their ability to induce focal transformation. Our results indicate that the E5 open reading frame, which could encode a low-molecular-weight hydrophobic peptide, is required for pHLB1-induced transformation of NIH 3T3 cells, but that the E2, E3, and E4 open reading frames are not.  相似文献   

4.
Human adenovirus type 9 (Ad9) elicits exclusively estrogen-dependent mammary tumors in rats, and an essential oncogenic determinant for this virus is Ad9 E4 open reading frame 1 (9ORF1), which encodes a 125-residue cytoplasmic protein with cellular growth-transforming activity in vitro. In this study, we engineered 48 different mutant 9ORF1 genes in an attempt to identify regions of this viral protein essential for transformation of the established rat embryo fibroblast cell line CREF. In initial assays with CREF cells, 17 of the 48 mutant 9ORF1 genes proved to be severely defective for generating transformed foci but only 7 of these defective genes expressed detectable amounts of protein. To further examine the defects of the seven mutant proteins, we selected individual cell pools of stable CREF transformants for the wild-type and mutant 9ORF1 genes. Compared to cell pools expressing the wild-type 9ORF1 protein, most cell pools expressing mutant proteins displayed decreased growth in soft agar, and all generated significantly smaller tumors in syngeneic animals. The altered amino acid residues of the seven mutant 9ORF1 polypeptides clustered within three separate regions referred to as region I (residues 34 to 41), region II (residues 89 to 91), and C-terminal region III (residues 122 to 125). By using indirect immunofluorescence, we also assessed whether the mutant proteins localized properly to the cytoplasm of cells. The region I and region II mutants displayed approximately wild-type subcellular localizations, whereas most region III mutants aberrantly accumulated within the nucleus of cells. In summary, we have identified three 9ORF1 protein regions necessary for cellular transformation and have demonstrated that C-terminal region III sequences significantly influence the proper localization of the 9ORF1 polypeptide in cells.  相似文献   

5.
K Neary  B H Horwitz    D DiMaio 《Journal of virology》1987,61(4):1248-1252
Open reading frame (ORF) E4 is a 353-base-pair ORF of bovine papillomavirus type 1. To determine the biological activities of this ORF in mouse C127 cells, we analyzed the effects of two constructed mutations which are predicted to prevent synthesis of ORF E4 proteins while leaving the amino acid sequence encoded by the overlapping ORF E2 unchanged. Neither mutation interfered with the abilities of the mutants to efficiently induce focus formation, induce growth in soft agarose, or transactivate an inducible bovine papillomavirus type 1 enhancer. Also, neither mutation prevented establishment of the viral DNA as an extrachromosomal plasmid in transformed cells. These results suggest that ORF E4 proteins are not required for these biological activities, and they are consistent with the observation of others (J. Doorbar, D. Campbell, R. J. A. Grand, and P. H. Gallimore, EMBO J. 5:355-362, 1986) that the ORF E4 protein of a human papillomavirus is associated with late gene expression during papilloma formation.  相似文献   

6.
Several herpesviruses contain open reading frames (ORFs) that encode potential homologs of eucaryotic genes. Equine herpesvirus 2 (EHV-2) is a gammaherpesvirus related to other lymphotropic herpesviruses such as herpesvirus saimiri and Epstein-Barr virus. The E1 ORF of EHV-2, a G protein-coupled receptor homolog, shows 31 to 47% amino acid identity with known CC chemokine receptors. To investigate whether E1 may encode a functional receptor, we cloned the E1 ORF and expressed it in stably transfected cell lines. We report here the identification of the CC chemokine eotaxin as a functional ligand for the EHV-2 E1 receptor. Chemokines are likely to play a role in the regulation of immune functions in equine hosts during EHV-2 infection and, via interaction with E1, may affect viral replication and/or escape from immune responses.  相似文献   

7.
8.
Norwalk virus open reading frame 3 encodes a minor structural protein   总被引:12,自引:0,他引:12       下载免费PDF全文
Norwalk virus (NV) is a causative agent of acute epidemic nonbacterial gastroenteritis in humans. The inability to cultivate NV has required the use of molecular techniques to examine the genome organization and functions of the viral proteins. The function of the NV protein encoded by open reading frame 3 (ORF 3) has been unknown. In this paper, we report the characterization of the NV ORF 3 protein expressed in a cell-free translation system and in insect cells and show its association with recombinant virus-like particles (VLPs) and NV virions. Expression of the ORF 3 coding region in rabbit reticulocyte lysates resulted in the production of a single protein with an apparent molecular weight of 23,000 (23K protein), which is not modified by N-linked glycosylation. The ORF 3 protein was expressed in insect cells by using two different baculovirus recombinants; one recombinant contained the entire 3' end of the genome beginning with the ORF 2 coding sequences (ORFs 2+3), and the second recombinant contained ORF 3 alone. Expression from the construct containing both ORF 2 and ORF 3 resulted in the expression of a single protein (23K protein) detected by Western blot analysis with ORF 3-specific peptide antisera. However, expression from a construct containing only the ORF 3 coding sequences resulted in the production of multiple forms of the ORF 3 protein ranging in size from 23,000 to 35,000. Indirect-immunofluorescence studies using an ORF 3 peptide antiserum showed that the ORF 3 protein is localized to the cytoplasm of infected insect cells. The 23K ORF 3 protein was consistently associated with recombinant VLPs purified from the media of insect cells infected with a baculovirus recombinant containing the entire 3' end of the NV genome. Western blot analysis of NV purified from the stools of NV-infected volunteers revealed the presence of a 35K protein as well as multiple higher-molecular-weight bands specifically recognized by an ORF 3 peptide antiserum. These results indicate that the ORF 3 protein is a minor structural protein of the virion.  相似文献   

9.
The proteins encoded by the open reading frames of papillomavirus genomes and the minor polypeptides detected in purified virions are still poorly defined. We show here by its expression in Escherichia coli that the open reading frame L2 of human papillomavirus type 1a codes for a minor structural protein of Mr 76,000. Antisera raised against a truncated L2-beta-galactosidase fusion protein in which the conserved N-terminal region of L2 is missing are type specific for human papillomavirus type 1 virions and are reactive at high dilutions. Expression of the L2-encoded type-specific antigens thus provides a powerful new tool for the identification of papillomaviruses.  相似文献   

10.
The E3 regions of adenovirus types 2 and 5, respectively, are known to synthesize proteins of 19,000 Mr (19K) and 11.6K, but information regarding the identity and characterization of other potential E3 proteins encoded by the six remaining open reading frames (ORFs) is lacking. In this study, we show that the last ORF of region E3, which encodes a 14.7K protein, is expressed in adenovirus-infected cells. This information was largely derived from analysis of an E3 deletion mutant (H2dl801) in which an extensive deletion (1,939 base pairs) was found to eliminate all ORFs except for two proteins of 12.5K and 14.7K. The 14.7K protein was translated from RNA isolated from H2dl801-infected cells that had been hybridization selected to E3 DNA; hybridization-selected RNA from wild-type adenovirus type 5-infected cells translated both the 19K and the 14.7K proteins. Moreover, an antiserum directed against a bacterial 14.7K fusion protein (A. E. Tollefson and W. S. M. Wold, J. Virol. 62:33-39, 1988) immunoprecipitated the 14.7K translation product synthesized by wild-type and mutant H2dl801 adenovirus mRNAs.  相似文献   

11.
We have shown that the open reading frame orf221 is an active mitochondrial gene which encodes a novel mitochondrial polypeptide. The orf221 sequence is common to higher plants but absent in animal and fungal mitochondria. A mitochondrial polypeptide with an apparent molecular weight of 21 000 was detected with a polyclonal antibody raised against an ORF221 fusion protein. In organello translation followed by immunoprecipitation with the anti-ORF221 antibody demonstrated that this polypeptide is encoded by the orf221 gene in plant mitochondria. The ORF221 was found to be a mitochondrial membrane protein in normal (N), cms-T, and cms-C cytoplasms of several inbred lines of maize (Zea mays L.) and in other plant species.  相似文献   

12.
We have previously observed that bovine papillomavirus type 1 (BPV-1) induces the appearance of five cellular proteins in C127 mouse fibroblasts, four of which appear to arise by altered processing of resident endoplasmic reticulum proteins. Studies of various cell lines revealed that expression of the 3' end of the BPV early region was sufficient for induction of these changes. To identify the BPV gene responsible, we have utilized the simian virus 40 (SV40)/BPV-1 recombinant virus Pava-1, which expresses the 3' end of the BPV early region behind an SV40 early promoter. C127 cells infected with Pava-1 for 48 h show the expected BPV-associated alterations, as do cells infected with Pava constructs mutated in the E5 or E2 genes. However, a mutation in the start codon of a previously ignored open reading frame extending from nucleotides 4013 to 4170 (E5B) eliminated the BPV-associated changes. Similar results were obtained with COS cells infected with the Pava mutants and C127 cells transformed by full-length mutated BPV. Despite its influence on the processing of cellular endoplasmic reticulum proteins, this mutation in E5B did not alter BPV-transforming efficiency or the ability of transformants to form colonies in soft agar. The E5B open reading frame encodes a hydrophobic 52-amino-acid polypeptide that shares structural similarities with HPV6 E5A and HPV16 E5. Speculations on a role for E5B in the viral life cycle are discussed.  相似文献   

13.
Brune W  Nevels M  Shenk T 《Journal of virology》2003,77(21):11633-11643
Viruses have evolved various strategies to prevent premature apoptosis of infected host cells. Some of the viral genes mediating antiapoptotic functions have been identified by their homology to cellular genes, but others are structurally unrelated to genes of known function. In this study, we used a random, unbiased approach to identify such genes in the murine cytomegalovirus genome. From a library of random transposon insertion mutants, a mutant virus that caused premature cell death was isolated. The transposon was inserted within open reading frame m41. An independently constructed m41 deletion mutant showed the same phenotype, whereas deletion mutants lacking the adjacent genes m40 and M42 did not. Apoptosis occurred in different cell types, could be blocked by caspase inhibitors, and did not require p53. Within the murine cytomegalovirus genome, m41, m40, and m39 form a small cluster of genes of unknown function. They are homologous to r41, r40, and r39 of rat cytomegalovirus, but lack sequence homology to UL41, UL40, and UL37 exon 1 (UL37x1) which are located at the corresponding positions of the human cytomegalovirus genome. Unlike UL37x1 of human cytomegalovirus, which encodes a mitochondrion-localized inhibitor of apoptosis that is essential for virus replication, m41 encodes a protein that localizes to the Golgi apparatus. The murine cytomegalovirus m41 product is the first example of a Golgi-localized protein that prevents premature apoptosis and thus extends the life span of infected cells.  相似文献   

14.
We report the discovery of a novel gene in the varicella-zoster virus (VZV) genome, designated open reading frame (ORF) S/L. This gene, located at the left end of the prototype VZV genome isomer, expresses a polyadenylated mRNA containing a splice within the 3' untranslated region in virus-infected cells. Sequence analysis reveals significant differences between the ORF S/Ls of wild-type and attenuated strains of VZV. Antisera raised to a bacterially expressed portion of ORF S/L reacted specifically with a 21-kDa protein synthesized in cells infected with a VZV clinical isolate and with the original vaccine strain of VZV (Oka-ATCC). Cells infected with other VZV strains, including a wild-type strain that has been extensively passaged in tissue culture and commercially produced vaccine strains of Oka, synthesize a family of proteins ranging in size from 21 to 30 kDa that react with the anti-ORF S/L antiserum. MeWO cells infected with recombinant VZV harboring mutations in the C-terminal region of the ORF S/L gene lost adherence to the stratum and adjacent cells, resulting in an altered plaque morphology. Immunohistochemical analysis of VZV-infected cells demonstrated that ORF S/L protein localizes to the cytoplasm. ORF S/L protein was present in skin lesions of individuals with primary or reactivated infection and in the neurons of a dorsal root ganglion during virus reactivation.  相似文献   

15.
Human herpesvirus 7 (HHV-7), which belongs to the betaherpesvirus subfamily, infects mainly CD4+ T cells in vitro and infects children during infancy. After the primary infection, HHV-7 becomes latent. HHV-7 contains two genes (U12 and U51) that encode putative homologs of cellular G-protein-coupled receptors. To analyze the biological function of the U12 gene, we cloned the gene and expressed the U12 protein in cells. The U12 gene encoded a calcium-mobilizing receptor for the EBI1 ligand chemokine-macrophage inflammatory protein 3beta (ELC/MIP-3beta) but not for other chemokines, suggesting that the chemokine selectivity of the U12 gene product is distinct from that of the known mammalian chemokine receptors. These studies revealed that U12 activates distinct transmembrane signaling pathways that may mediate biological functions by binding with a beta-chemokine, ELC/MIP-3beta.  相似文献   

16.
17.
Several herpesviruses encode Fc receptors that may play a role in preventing antibody-mediated clearance of the virus in vivo. Human cytomegalovirus (HCMV) induces an Fc-binding activity in cells upon infection, but the gene that encodes this Fc-binding protein has not been identified. Here, we demonstrate that the HCMV AD169 open reading frame TRL11 and its identical copy, IRL11, encode a type I membrane glycoprotein that possesses IgG Fc-binding capabilities.  相似文献   

18.
Sequencing of the E5 open reading frame (ORF) of human papillomavirus type 16 revealed an additional nucleotide, a thymidine residue, at position 3903 compared with the original sequence (Seedorf et al., Virology 145:181-185, 1985). The additional T had two effects; first, in reading frame 2, in which the original E5 ORF was predicted, the additional T changed the reading frame downstream of position 3903 to create an ORF, which we designated E5, that terminated at position 4018 and potentially encoded a 52-amino-acid polypeptide. Secondly, in reading frame 3, a new ORF was created (positions 3807 to 4097), which we propose is the authentic papillomavirus type 16 E5 ORF. It contained a methionine residue and encoded an additional 82 amino acids. Both ORFs have been cloned into bacterial expression vectors (pATH), and the fusion proteins have been used to generate polyclonal antibodies in rabbits.  相似文献   

19.
20.
We have shown by activity gel that overexpression in E. coli of a yeast chromosome 3 open reading frame (ORF) designated YCR14C and bearing homology to mammalian DNA polymerases beta results in a new DNA polymerase in the host cells. The molecular mass of this enzyme corresponded to the YCR14C-predicted 67 kDa protein, and NH2-terminal amino acid sequencing confirmed that the expressed protein was encoded by the yeast ORF. This new yeast DNA polymerase was purified to homogeneity from E.coli. In a fashion similar to that of mammalian beta-polymerases, the purified yeast enzyme exhibited distributive DNA synthesis on DNA substrate with a single-stranded template and processive gap-filling synthesis on a short-gapped DNA substrate. Activity of this yeast beta-polymerase-like enzyme was sensitive to the beta-polymerase inhibitor ddNTP and resistant to both 1 mM NEM and neutralizing antibody to E. coli DNA polymerase I. These results, therefore, indicate that YCR14C encodes a DNA beta-polymerase-like enzyme in yeast, and we name it DNA polymerase IV. Yeast strains harboring a deletion mutation of the pol IV gene are viable, they exhibit no increase in sensitivity to ultraviolet light, ionizing radiation or alkylating agents, and sporulation and spore viability are not affected in the mutant.  相似文献   

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