Human herpesvirus 7 open reading frame U12 encodes a functional beta-chemokine receptor

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.     

The equine herpesvirus 2 E1 open reading frame encodes a functional chemokine receptor

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.     

E5 open reading frame of bovine papillomavirus type 1 encodes a transforming gene.

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.     

Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) open reading frame 4 protein (kaposica) is a functional homolog of complement control proteins

The genome analysis of Kaposi's sarcoma-associated herpesvirus (KSHV) has revealed the presence of an open reading frame (ORF 4) with sequence homology to complement control proteins. To assign a function to this protein, we have now expressed this ORF using the Pichia expression system and shown that the purified protein inhibited human complement-mediated lysis of erythrocytes, blocked cell surface deposition of C3b (the proteolytically activated form of C3), and served as a cofactor for factor I-mediated inactivation of complement proteins C3b and C4b (the subunits of C3 convertases). Thus, our data indicate that this KSHV inhibitor of complement activation (kaposica) provides a mechanism by which KSHV can subvert complement attack by the host.     

Yeast open reading frame YCR14C encodes a DNA beta-polymerase-like enzyme.

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.     

Norwalk virus open reading frame 3 encodes a minor structural protein

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.     

Bovine herpesvirus 1 U(L)3.5 interacts with bovine herpesvirus 1 alpha-transinducing factor

The bovine herpesvirus 1 (BHV-1) U(L)3.5 gene encodes a 126-amino-acid tegument protein. Homologs of U(L)3.5 are present in some alphaherpesviruses and have 20 to 30% overall amino acid homology that is concentrated in the N-terminal 50 amino acids. Mutant pseudorabies virus lacking U(L)3.5 is deficient in viral egress but can be complemented by BHV-1 U(L)3.5 (W. Fuchs, H. Granzow, and T. C. Mettenleiter, J. Virol. 71:8886-8892, 1997). The function of BHV-1 U(L)3.5 in BHV-1 replication is not known. To get a better understanding of its function, we sought to identify the proteins that interact with the BHV-1 U(L)3.5 protein. By using an in vitro pull-down assay and matrix-assisted laser desorption ionization mass spectrometry analysis, we identified BHV-1 alpha-transinducing factor (alphaBTIF) as a BHV-1 U(L)3. 5-interacting protein. The interaction was verified by coimmunoprecipitation from virus-infected cells using an antibody to either protein, by indirect immunofluorescence colocalization in both virus-infected and transfected cells, and by the binding of in vitro-translated proteins. In virus-infected cells, U(L)3.5 and alphaBTIF colocalized in a Golgi-like subcellular compartment late in infection. In transfected cells, they colocalized in the nucleus. Deletion of 20 amino acids from the N terminus of U(L)3.5, but not 40 amino acids from the C terminus, abolished the U(L)3.5-alphaBTIF interaction both in vitro and in vivo. The interaction between U(L)3. 5 and alphaBTIF may be important for BHV-1 maturation and regulation of alphaBTIF transactivation activity.     

The plant mitochondrial open reading frame orf221 encodes a membrane-bound protein

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.     

Murine gammaherpesvirus 68 open reading frame 11 encodes a nonessential virion component

Open reading frame 11 (ORF11) is a conserved gammaherpesvirus gene that remains undefined. We identified the product of murine gammaherpesvirus 68 (MHV-68) ORF11, p43, as a virion component with a predominantly perinuclear distribution in infected cells. MHV-68 lacking p43 grew normally in vitro but showed reduced lytic replication in vivo and a delay in seeding to the spleen. Subsequent latency amplification was normal. Thus, MHV-68 ORF11 encoded a virion component that was important for in vivo lytic replication but dispensable for the establishment of latency.     

Murine cytomegalovirus m41 open reading frame encodes a Golgi-localized antiapoptotic protein

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.     

Expression in a recombinant murid herpesvirus 4 reveals the in vivo transforming potential of the K1 open reading frame of Kaposi's sarcoma-associated herpesvirus

Murid herpesvirus 4 (commonly called MHV-68) is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV) and provides an excellent model system for investigating gammaherpesvirus-associated pathogenesis. MHV-76 is a naturally occurring deletion mutant of MHV-68 that lacks 9,538 bp of the left end of the unique portion of the genome encoding nonessential pathogenesis-related genes. The KSHV K1 protein has been shown to transform rodent fibroblasts in vitro and common marmoset T lymphocytes in vivo. Using homologous recombination techniques, we successfully generated recombinants of MHV-76 that encode green fluorescent protein (MHV76-GFP) and KSHV K1 (MHV76-K1). The replication of MHV76-GFP and MHV76-K1 in cell culture was identical to that of MHV-76. However, infection of BALB/c mice via the intranasal route revealed that MHV76-K1 replicated to a 10-fold higher titer than MHV76-GFP in the lungs at day 5 postinfection (p.i.). We observed type 2 pneumocyte proliferation in areas of consolidation and interstitial inflammation of mice infected with MHV76-K1 at day 10 p.i. MHV76-K1 established a 2- to 3-fold higher latent viral load than MHV76-GFP in the spleens of infected mice on days 10 and 14 p.i., although this was 10-fold lower than that established by wild-type MHV-76. A salivary gland tumor was present in one of four mice infected with MHV76-K1, as well as an increased inflammatory response in the lungs at day 120 p.i. compared with that of mice infected with MHV-76 and MHV76-GFP.     

Adenovirus type 9 E4 open reading frame 1 encodes a transforming protein required for the production of mammary tumors in rats.

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.     

Evidence that the intron open reading frame of the phage T4 td gene encodes a specific endonuclease

The phage T4 thymidylate synthase (td) gene contains an intron open reading frame that encodes a 245-amino acid-long basic protein (Chu, F. K., Maley, G. F., West, D. K., Belfort, M., and Maley, F. (1986) Cell 45, 157-166). The open reading frame (Irf) has been cloned as a fusion protein behind a phage T7 promoter and overexpressed in Escherichia coli. The amplified Irf protein is associated with insoluble inclusion bodies and migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis about 7 kDa smaller than expected. Data obtained from DNA sequencing, amino acid sequencing of the fusion protein, and carboxypeptidase Y digestion suggest that although the cloned gene is not altered and the protein is made from the expected start codon, it appears to terminate about 90 amino acids before the encoded stop codon. Proteolytic cleavage during or soon after synthesis appears to be responsible for the truncated Irf. The expressed protein is solubilized in guanidine HCl and renatured by dialysis against high salt. This partially purified preparation has been found to contain a DNA endonuclease activity specific for the td delta I gene, which contains a precise deletion of the intron.     

Human papillomavirus type 16 open reading frame E7 encodes a transforming gene for rat 3Y1 cells.

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.     

Human adenovirus type 9 E4 open reading frame 1 encodes a cytoplasmic transforming protein capable of increasing the oncogenicity of CREF cells.

The induction of estrogen-dependent rat mammary tumors by human adenovirus type 9 (Ad9) requires the Ad9 E4 open reading frame 1 (9ORF1) protein, which alone can transform that rat embryo fibroblast cell line CREF in vitro. In the present study, independent pools of both 9ORF1-expressing and control CREF cells were generated by selection with G418 and compared with respect to transformed properties. Indirect immunofluorescence analyses revealed that more than 99% of the cells that made up the 9ORF1-transfected pools expressed 9ORF1 protein and, together with confocal laser scanning microscopy, indicated that this E4 protein was located predominantly within the cytoplasm of cells. With regard to transformation, cells of the 9ORF1-expressing pools differed from those of control pools by forming foci, displaying morphological alterations, growing more efficiently in soft agar, and reaching higher saturation densities. Following injection into immunocompetent syngeneic rats, the 9ORF1-expressing pool cells exhibited greatly enhanced oncogenicity compared with control pool cells. These results show that 9ORF1 protein (i) localizes predominantly within the cytoplasm, (ii) confers multiple general transformed characteristics to CREF cells in vitro, and (iii) increases the tumorigenic properties of these cells in vivo.     

Deletion mutants of herpesvirus saimiri define an open reading frame necessary for transformation.

Analysis of a 5,549-base-pair sequence at the left end of herpesvirus saimiri unique (L-) DNA revealed two open reading frames and genes for five small nuclear U RNAs (herpesvirus saimiri U RNAs). Replication-competent deletion mutants were constructed in order to assess the importance of these genetic features for transformation by this oncogenic herpesvirus. Although not required for replication, one of the open reading frames was found to be required for immortalization of marmoset T lymphocytes into continuously growing cell lines. The protein predicted by this reading frame (STP; saimiri transformation-associated protein) has a highly hydrophobic stretch of 26 amino acids sufficient for a membrane-spanning domain near its carboxy terminus; this domain is immediately preceded by a sequence appropriate for formation of a metal-binding domain (His X2 His X6 Cys X2 Cys, where Xs are other amino acids). One of two poly(A)+ RNAs that could encode STP is bicistronic, while the other has a long 5' untranslated region (approximately 1.5 kilobases). Although some analogies can be drawn between STP and LMP (lymphocyte membrane protein) of Epstein-Barr virus, STP is not related in sequence to LMP.     

The L2 open reading frame of human papillomavirus type 1a encodes a minor structural protein carrying type-specific antigens.

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.