Evidence for a Dual Strategy in the Expression of Cauliflower Mosaic Virus Open Reading Frames I and IV

Studies have indicated that cauliflower mosaic virus (CaMV) gene expression is mediated by the translation of polycistronic 35S pregenomic RNA, but the involvement of some minor subgenomic RNA species is also suspected. We examined the involvement of the 35S promoter in the expression of CaMV open reading frames (ORFs) I and IV using both 35S RNA-driven and promoter-less ORF I- and ORF IV-β-glucuronidase (GUS) fusion constructs. In addition to the 35S promoter-dependent expression of both ORF I- and IV-GUS fusions, we detected the 35S promoter-independent expression of both fusion genes via subgenomic mRNAs, which were detected by Northern blotting in the protoplasts transfected with the 35S promoter-driven constructs as well as in those transfected with the promoter-less constructs. These results suggest the involvement of subgenomic RNAs in the expression of CaMV ORFs I and IV, and the operation of a dual strategy in the expression of two viral genes.     

Splicing of cauliflower mosaic virus 35S RNA is essential for viral infectivity.

A splicing event essential for the infectivity of a plant pararetrovirus has been characterized. Transient expression experiments using reporter constructs revealed a splice donor site in the leader sequence of the cauliflower mosaic virus (CaMV) 35S RNA and three additional splice donor sites within open reading frame (ORF) I. All four donors use the same splice acceptor within ORF II. Splicing between the leader and ORF II produces an mRNA from which ORF III and, in the presence of the CaMV translational transactivator, ORF IV can be translated efficiently. The other three splicing events produce RNAs encoding ORF I-II in-frame fusions. All four spliced CaMV RNAs were detected in CaMV-infected plants. Virus mutants in which the splice acceptor site in ORF II is inactivated are not infectious, indicating that splicing plays an essential role in the CaMV life cycle. The results presented here suggest a model for viral gene expression in which RNA splicing is required to provide appropriate substrate mRNAs for the specialized translation mechanisms of CaMV.     

Identification of the defective genes in three mutant groups of influenza virus.

Seven complementation-recombination groups of temperature-sensitive (ts) influenza WSN virus mutants have been previously isolated. Recently two of these groups (IV and VI) were shown to possess defects in the neuraminidase and the hemagglutinin gene, respectively, and two groups (I and III) were reported to have defects in the P3 and P1 proteins which are required for complementary RNA synthesis. In this communication we report on the defects in the remaining three mutant groups. Wild-type (ts+) recombinants derived from ts mutants and different non-ts influenza viruses were analyzed on RNA polyacrylamide gels. This technique permitted the identification of the P2 protein, the nucleoprotein, and the M protein as the defective gene products in mutant groups II, V, and VII, respectively. Based on the physiological behavior of mutants in groups II and V, it appears that P2 protein and nucleoprotein are required for virion RNA synthesis during influenza virus replication.     

The cauliflower mosaic virus virion-associated protein is dispensable for viral replication in single cells

Cauliflower mosaic virus (CaMV) open reading frame III (ORF III) codes for a virion-associated protein (Vap), which is one of two viral proteins essential for aphid transmission. However, unlike the aphid transmission factor encoded by CaMV ORF II, Vap is also essential for systemic infection, suggesting that it is a multifunctional protein. To elucidate the additional function or functions of Vap, we tested the replication of noninfectious ORF III-defective mutants in transfected turnip protoplasts. PCR and Western blot analyses revealed that CaMV replication had occurred with an efficiency similar to that of wild-type virus and without leading to reversions. Electron microscopic examination revealed that an ORF III frameshift mutant formed normally structured virions. These results demonstrate that Vap is dispensable for replication in single cells and is not essential for virion morphogenesis. Analysis of inoculated turnip leaves showed that the ORF III frameshift mutant does not cause any detectable local infection. These results are strongly indicative of a role for Vap in virus movement.     

Insertional mutagenesis of the cauliflower mosaic virus genome

A series of small insertions has been introduced into the various translational reading frames of the DNA of a "severe" strain of cauliflower mosaic virus (CaMV). A selectable gene (the kanamycin phosphotransferase gene of Tn903), flanked by a series of symmetrically arranged cloning sites taken from M13mp7, was used to prepare the site-specific mutants. In-phase insertions of 12 or 30 bp, which introduced unique SalI sites into reading regions I, III, IV, V and into the amino-proximal portion of region VI, destroyed infectivity. Insertions in the amino-distal portion of region VI, in the large intergenic region, and in region II retained infectivity. The amino-distal insertions in region VI reduced the severity of symptoms in plants. The insertion in region II destroyed aphid transmissibility. Longer DNA segments when inserted into region II or into the amino-distal portion of region VI destroyed infectivity, but similar insertions in the intergenic region were without effect on virus infection or development.     

A single species of pX mRNA of human T-cell leukemia virus type I encodes trans-activator p40x and two other phosphoproteins.

Human T-cell leukemia virus type I (HTLV-I) contains the pX sequence which codes for the trans-activator of the long terminal repeat (LTR) and is thus postulated to be associated with leukemogenesis in adult T-cell leukemia. Overlapping open reading frames (ORF) in the pX sequence were recently found to code for p27x-III and p21x-III by ORF III, in addition to p40x coded for by ORF IV. The mechanism of expression of these newly identified proteins and their possible association with trans-activation were studied. On transfection of an expression plasmid that contains a cDNA sequence of the pX mRNA, products from both ORFs III and IV were detected in the cells. The RNA was synthesized in vitro from the cDNA clone by SP6 RNA polymerase and translated in a rabbit reticulocyte lysate. As translation products, two proteins, p27x-III and p21x-III, were detected in addition to p40x. Elimination of the first and second ATG codons in ORF III resulted in loss of the ability to code for p27x-III and p21x-III, respectively, which indicated that the translations from these two ATG codons were independent. A mutant that lacked both ATG codons was fully active in trans-activation of chloramphenicol acetyltransferase gene expression directed by the LTR. These results indicate that a 2.1-kilobase pX mRNA of HTLV-I independently encodes three proteins, p40x, p27x-III, and p21x-III, by different ORFs and that the last two proteins are not involved in trans-activation of the unintegrated LTR.     

Biochemical and functional analysis of the Borna disease virus G protein.

The Borna disease virus (BDV) antigenome is comprised of five major open reading frames (ORFs). Products have been reported only for ORFs I, II, and III, encoding N (p40), P (p24/p23), and M (gp18), respectively. ORF IV predicts a 57-kDa protein with several potential glycosylation sites. Analysis of radiolabeled extracts from BDV-infected C6 cells and BHK-21 cells transfected with a Semliki Forest virus vector that contains ORF IV demonstrated the presence of a 94-kDa protein (G protein) which was sensitive to tunicamycin, endoglycosidase F/N-glycosidase, and endoglycosidase H but not to O-glycosidase. Sera from BDV-infected rats detected the G protein and had neutralization activity that was reduced following immunoadsorption with the G protein. Preincubation of cells with the G protein interfered with BDV infectivity. This effect was enhanced by treatment of the G protein with the exoglycosidase alpha-mannosidase and reduced after subsequent treatment with N-acetyl-beta-D-glucosaminidase. In concert these findings indicate that ORF IV encodes a 94-kDa N-linked glycoprotein with extensive high mannose- and/or hybrid-type oligosaccharide modifications. The presence of neutralization epitopes on the G protein and its capacity to interfere with infectivity suggest that the G protein is important for viral entry.     

Cauliflower mosaic virus ORF III product forms a tetramer in planta: its implication in viral DNA folding during encapsidation.

Cauliflower mosaic virus (CaMV) open reading frame (ORF) III encodes a 15 kDa protein; the function of which is as yet unknown. This protein has non-sequence-specific DNA binding activity and is associated with viral particles, suggesting that the ORF III product (P3) is involved in the folding of CaMV DNA during encapsidation. In this study, we demonstrated that P3 forms a tetramer in CaMV-infected plants. A P3-related protein with an apparent molecular weight of 60 kDa was detected by Western blotting analysis using anti-P3 antiserum under non-reducing conditions, while only 15 kDa P3 was detected under reducing conditions. Analysis of P3 using viable mutants with a 27-bp insertion in either ORF III or IV revealed that the 60 kDa protein was a tetramer of P3. The P3 tetramer co-sedimented with viral coat protein in multiple fractions on sucrose gradient centrifugation, suggesting that P3 tetramer binds to mature and immature virions. These results strongly suggested that CaMV P3 forms a tetramer in planta and that disulfide bonds are involved in its formation and/or stabilization. The finding of P3 tetramer in planta suggested that viral DNA would be folded compactly by the interaction with multiple P3 molecules, which would form tetramers, while being packaged into the capsid shell.     

The biosynthesis of highly branched N-glycans: studies on the sequential pathway and functional role of N-acetylglucosaminyltransferases I, II, III, IV, V and VI

At least 6 N-acetylglucosaminyltransferases (GlcNAc-T I, II, III, IV, V and VI) are involved in initiating the synthesis of the various branches found in complex asparagine-linked oligosaccharides (N-glycans), as indicated below: GlcNAc beta 1-6 GlcNAc-T V GlcNAc beta 1-4 GlcNAc-T VI GlcNAc beta 1-2Man alpha 1-6 GlcNAc-T II GlcNAc beta 1-4Man beta 1-4-R GlcNAc T III GlcNAc beta 1-4Man alpha 1-3 GlcNAc-T IV GlcNAc beta 1-2 GlcNAc-T I where R is GlcNAc beta 1-4(+/- Fuc alpha 1-6)GlcNAcAsn-X. HPLC was used to study the substrate specificities of these GlcNAc-T and the sequential pathways involved in the biosynthesis of highly branched N-glycans in hen oviduct (I. Brockhausen, J.P. Carver and H. Schachter (1988) Biochem. Cell Biol. 66, 1134-1151). The following sequential rules have been established: GlcNAc-T I must act before GlcNAc-T II, III and IV; GlcNAc-T II, IV and V cannot act after GlcNAc-T III, i.e., on bisected substrates; GlcNAc-T VI can act on both bisected and non-bisected substrates; both Glc-NAc-T I and II must act before GlcNAc-T V and VI; GlcNAc-T V cannot act after GlcNAc-T VI. GlcNAc-T V is the only enzyme among the 6 transferases cited above which can be essayed in the absence of Mn2+. In studies on the possible functional role of N-glycan branching, we have measured GlcNAc-T III in pre-neoplastic rat liver nodules (S. Narasimhan, H. Schachter and S. Rajalakshmi (1988) J. Biol. Chem. 263, 1273-1281). The nodules were initiated by administration of a single dose of carcinogen 1,2-dimethyl-hydrazine.2 HCl 18 h after partial hepatectomy and promoted by feeding a diet supplemented with 1% orotic acid for 32-40 weeks. The nodules had significant GlcNAc-T III activity (1.2-2.2 nmol/h/mg), whereas the surrounding liver, regenerating liver 24 h after partial hepatectomy and control liver from normal rats had negligible activity (0.02-0.03 nmol/h/mg). These results suggest that GlcNAc-T III is induced at the pre-neoplastic stage in liver carcinogenesis and are consistent with the reported presence of bisecting GlcNAc residues in N-glycans from rat and human hepatoma gamma-glutamyl transpeptidase and their absence in enzyme from normal liver of rats and humans (A. Kobata and K. Yamashita (1984) Pure Appl. Chem. 56, 821-832).     

Mutant adenovirus type 9 E4 ORF1 genes define three protein regions required for transformation of CREF cells.

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.     

Amino acids essential for RNase H activity of hepadnaviruses are also required for efficient elongation of minus-strand viral DNA.

The hepadnavirus P gene contains amino acid sequences which share homology with all known RNases H. In this study, we made four mutants in which single amino acids of the duck hepatitis B virus (DHBV) RNase H region were altered. In two of them, amino acids at locations comprising the putative catalytic site were changed, while the remaining mutants had alterations at amino acids conserved among hepadnaviruses. Transfection of these mutant genomes into permissive cells resulted in synthesis of several discrete viral nucleic acid species, ranging in apparent sizes from approximately 500 to 3,000 bp, numbered I, II, III, IV, and V. While the locations of the species were similar in all mutants, the proportions of the species varied among the mutants. Analysis of the nucleic acid species revealed that they were hybrid molecules of RNA and minus-strand DNA, indicating that the RNase H activity was missing or greatly reduced in these mutants. Primer extension experiments showed that the mutant viruses initiated minus-strand viral DNA synthesis normally. The 3' termini of minus-strand DNA in species II, III, and IV were mapped just downstream of nucleotides 1659, 1220, and 721, respectively. Species V contained essentially full-length minus-strand viral DNA. A parallel amino acid change in the putative catalytic site of the HBV RNase H domain resulted in accumulation of low-molecular-weight hybrid molecules consisting of RNA and minus-strand DNA and similar in size and pattern to those seen with DHBV. These studies demonstrate experimentally the involvement of the C-terminal portion of the P gene in RNase H activity in both DHBV and human hepatitis B virus and indicate that the amino acids essential for RNase H activity of hepadnavirus P protein are also important for the efficient elongation of minus-strand viral DNA.     

Complementation of adenovirus E4 mutants by transient expression of E4 cDNA and deletion plasmids.

Human adenovirus mutants that carry a large deletion in early region 4 (E4) are severely defective in the synthesis of viral late proteins. Plasmids that carry intact E4 sequences can complement the late protein synthetic defect of such mutants when introduced into infected cells by transfection, presumably due to the transient expression of E4 products. Cells transfected with cDNA clones capable of expressing E4 open reading frame (ORF) 6, or deletion mutant clones expected to express either E4 ORF 6 or E4 ORF 3, also complement the mutants' defects. Thus, these E4 ORFs can individually satisfy the requirement for E4 products in viral late gene expression, and function effectively in the absence of other E4 products. Some E4 deletion mutants also exhibit a defect in the production of viral DNA. All of the clones that stimulate late gene expression also enhance one such mutant's ability to accumulate viral DNA. Thus, the ORF 3 and ORF 6 products are also individually sufficient to provide an E4 function necessary for normal viral DNA replication in the absence of other E4 products.