Sequence determination of the extreme 5′ end of equine arteritis virus leader region

The extreme 5 end of the leader sequence of four equine arteritis virus (EAV) strains was obtained by using rapid amplification of cDNA end method (5 RACE), and sequenced. Seventeen more nucleotides were added upstream of the 5 end of the EAV published genomic sequence. A common feature among the analyzed EAV isolates was the presence of an AUG start codon within the added sequence and the appearance of an intraleader open reading frame (ORF) of 111 nucleotides which was predicted to encode a peptide of 37 amino acids. The role of this putative intraleader ORF has yet to be determined.     

RNA binding properties of core protein of the flavivirus Kunjin

Summary Kunjin virus (KUN) C is a typical flavivirus core protein which is truncated in vivo to a mature form of 105 residues enriched in lysine and arginine. In order to study the possible association of KUN C with RNA in vitro, we prepared several recombinant C proteins with specific deletions, each fused at the amino-terminus to glutathione-S-transferase (GST) and expressed inE. coli. They were reacted with KUN RNA probes transcribed in vitro from cDNA representing the 5 untranslated region (5 UTR, 93 of 96 nucleotides), the 3 UTR (624 nucleotides), and the 5 UTR plus most of the C coding region (t core, 440 nucleotides). Fusion protein C107 (incorporating mature C) bound strongly to all KUN RNA probes with apparent specificity, being completely resistant to inhibition by 800 mM NaCl, and to competition by a large excess of tRNA. In reactions with labelled KUN RNA probes putative binding sites were identified in the isolated amino-terminal (32 residues) and carboxyterminal (26 residues) basic amino acid domains; this binding was strongly competed by unlabelled KUN UTR probes but weakly or not at all by tRNA. These small domains probably acted co-operatively in binding of mature C to KUN RNA probes. The KUN RNA-core protein binding reactions are similar to those reported with other viral coat or capsid proteins and viral RNAs.     

Comparison of the 5′ Leader Sequences of North American Isolates of Reference and Field Strains of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

The 5 leader is documented to be an important regulatory element in many (+) ssRNA virus genome. To understand the significance of the 5 leader RNA of PRRSV, we determined the complete leader sequences of fifteen different North American strains of PRRSV and predicted their secondary structures. Viruses analysed included three reference strains and nine field strains originating from different geographic locations. To further examine the leader region, one of the field strains was adapted to grow in tissue culture, and three clones were isolated. We also predicted the secondary structures of two European strains based on their published sequences. The predicted RNA secondary structures of the leader sequences suggested the existence of three conserved domains formed by the 5 region of the leader among the North American strains, two of which were conserved in the European strains. A variable structural domain was predicted from the 3 region of the leader sequences of the North American strains, where all tissue culture-adapted isolates were characterized by a stem-loop while field isolates were characterized by an internal bulge within the stem-loop.     

Proteins specifically binding to the 3′ untranslated region of hepatitis A virus RNA in persistently infected cells

Summary Establishment of persistency is the common result of hepatitis A virus (HAV) infection in most HAV/cell culture systems. Previous studies provided evidence that shortly before or concomitantly with establishment of persistent infections synthesis of viral RNA is down-regulated. This may be an effect of regulating factors. Using RNA/protein binding assays it was shown that, at the critical time during virus replication, proteins accumulate which interact specifically with a distinct nucleotide sequence (HPE) within the 3 non-coding region of the HAV genome and/or (HME) within the 5 terminal region of the HAV antigenome. The sequences consist of 23 nucleotides (HPE: 5-AAAUUUUCUUAAAAUUUCUGAGG-3; HME: 5-CCUCAGAAAUUUUAAGAAAAUUU-3). A sequence with 79% similarity was found in the corresponding 3 non-coding region of poliovirus type I (Sabin) RNA. The latter sequence was shown to bind proteins from HAV infected cells but comparable proteins were absent in cells infected poliovirus.     

Specific Interaction of a 25-kilodalton Cellular Protein,a 40S Ribosomal Subunit Protein,with the Internal Ribosome Entry Site of Hepatitis C Virus Genome

Translation initiation of hepatitis C virus (HCV) RNA is controlled by an internal ribosome entry site (IRES) contained in 5 noncoding region (NCR) and in several nucleotides of the coding region. The ability of a 25-kilodalton cellular protein (p25) to bind the HCV 5 NCR is correlated with the efficiency of translation initiation of HCV RNA, indicating that this protein plays a critical role in HCV translation (S. Fukushi, C. Kurihara, N. Ishiyama, F. B. Hoshino, A. Oya, and K. Katayama, J Virol 71, 1662–1666, 1997). We have extended the study for identification of the IRES region required for p25 binding. For this purpose, we have performed UV cross-linking competition analyses using 5- or 3- deleted mutants of the HCV 5 NCR as competitor RNAs for binding of p25 to wild-type HCV 5 NCR. Competitor RNAs lacking nucleotides (nt) 47–74 or nt 279–331 did not inhibit p25 binding to the HCV IRES, indicating that these regions are necessary for interaction of the p25 and HCV IRES. Since p25 binding was not observed in the IRES elements of encephalomyocarditis virus and poliovirus in UV cross-linking competition analyses, the p25 binding may be specific for the HCV IRES. p25 bound to the HCV IRES was detected when a purified 40S ribosomal subunit was used for UV cross-linking experiment, indicating that p25 is one of 40S ribosomal subunit proteins. These results reveal an unique interaction between the 40S ribosomal subunit and HCV IRES to contribute to translation initiation of the HCV genome.     

Specific binding of polypyrimidine tract binding protein and hnRNP A1 to HIV-1 CRS elements

The human immunodeficiency virus (HIV) Rev and human T-cell leukemia virus (HTLV) Rex proteins regulate viral RNA processing. Both proteins act to overcome the block to viral structural gene expression, at least in part, by reversing the inhibitory effect of intronic RNA sequences, termed cis-acting repressive (CRS) sequences. Using HTLV type II (HTLV-II) as a model, we recently showed that the function of a 5 long terminal repeat (LTR) CRS correlates with in vitro binding by both polypyrimidine tract binding (PTB) protein (also known as hnRNP I) and hnRNP A1 to CRS RNA (1,2). Using radioimmunoprecipitation of proteins ultraviolet (UV) crosslinked to each HIV CRS RNA with monoclonal anti-hnRNP antibodies, we now demonstrate that hnRNP I and hnRNP A1 bind to two different HIV-1 CRS RNAs. In addition, we show that hnRNP I and hnRNP A1 binding to HIV-1 CRS RNAs can be specifically competed by HTLV-II CRS RNAs using electrophoretic mobility shift assay (EMSA)/UV crosslinking assays. Binding by both hnRNP I and hnRNP A1 to HIV-1 and HTLV-II CRS RNAs suggests a role for these proteins in CRS function that may be influenced by the Rev and Rex proteins, respectively.     

Mitochondrial HSP70, HSP40, and HSP60 bind to the 3′ untranslated region of the <Emphasis Type="Italic">Murine hepatitis virus</Emphasis> genome

Summary. We have previously shown that mitochondrial-aconitase binds specifically to the 3 terminal 42 nucleotides of the Murine hepatitis virus (MHV) RNA along with three additional proteins of 70, 58 and 40kDa to form a stable RNA-protein complex. Supershift and western blot assays have identified these three proteins as mitochondrial HSP70 (mtHSP70), HSP60, and HSP40. A series of co-immunoprecipitation assays have established that these four MHV RNA binding proteins are associated, even in the absence of MHV RNA. However, the presence of a synthetic RNA containing the sequence bound by these four proteins does increase the amount of co-precipitated protein, in particular the amount of HSP60 which is brought down with antibodies directed against HSP40 and mtHSP70. We have provided evidence for the interaction of these four proteins with the 3 end region of MHV RNA in infected cells by a series of immunoprecipitation RT-PCR assays. We believe it is likely that MHV RNA interacts with m-aconitase prior to its import into mitochondria in cooperation with extra-mitochondrial mtHSP70, HSP60, and HSP40.These authors contributed equally to this work.     

Effect of cordycepin on the replication of western equine encephalitis virus

Summary Cordycepin (3-deoxyadenosine) inhibited viral RNA synthesis in the replication of western equine encephalitis virus, thereby causing a reduction of virus production. The rate of inhibition of viral RNA synthesis was dependent on drug concentration and the period of treatment with the drug. These results suggest that the virus RNA synthesizing system is sensitive to the drug.With 3 Figures     

Evolution of a Common Structural Core in the Internal Ribosome Entry Sites of Picornavirus

The translational control involving internal ribosome binding occurs in poliovirus (PV), human rhinoviruses (HRV), encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), and hepatitis A virus (HAV). Internal ribosome binding utilizes cis-acting genetic elements of approximately 450 nucleotides (nt) termed the internal ribosome entry sites (IRES) found in these picornaviral 5-untranslated region (5UTR). Although these IRES elements are quite different in their primary sequence, a similar folding structure with a conserved 3 structural core exists in the IRES. Phylogenetic analysis and RNA folding of the 5 UTR of picornaviruses, including PV types 1–3, coxsackievirus types A and B, swine vesicular disease virus, echoviruses, enteroviruses (human and bovine), HRV, HAV, EMCV, mengovirus, Theilers murine encephalomyelitis viruses, FMDV, and equine rhinoviruses, indicates that the predicted conserved structural core is indeed a general structural feature for all members of the picornavirus family. The evolution of a common structural core likely occurred by the gradual addition or deletion of structural domains and elements to preserve a similar tertiary structure that facilitates the utilization of the IRES in specific host-cell environments.     

Completion of the Four Large Gene Sequences of Porcine Group C Cowden Rotavirus

The terminal nucleotide sequences of group C Cowden rotavirus gene segments 1-4 were determined. When compared with the published sequences, we found 14 to 29 additional nt at the 5 ends of the four reported gene sequences. For the 3 ends, we observed an additional 16 nt in gene 2 and 14 fewer nt in gene 4.     

Complete nucleotide sequence and genome organization of <Emphasis Type="Italic">Pelargonium flower break virus</Emphasis>

Summary. The complete nucleotide sequence of Pelargonium flower break virus (PFBV) has been determined. The genomic RNA is 3923 nucleotides (nt) long and contains five open reading frames (ORFs). The 5-proximal ORF encodes a 27kDa protein (p27) and terminates with an amber codon which may be read-through into an in-frame p56 ORF to generate a 86kDa protein (p86) containing the viral RNA dependent-RNA polymerase motifs. Two small ORFs, located in the central part of the viral genome, encode polypeptides of 7 (p7) and 12kDa (p12), respectively, which are very likely involved in virus movement. Interestingly, p12 presents a leucine zipper motif that has not been previously reported in related proteins. The 3-proximal ORF encodes a 37kDa capsid protein (CP). The p12 ORF is in-frame with the p86 ORF and a double read-through protein of 99kDa (p99) may be produced. Amino acid sequence comparisons revealed that the proteins encoded by ORFs 2, 3 and 4 are more similar to the corresponding gene products of Carnation mottle virus than to those of other carmoviruses, whereas the p27 and the CP show higher identity with the equivalent proteins of Saguaro cactus virus. Phylogenetic analysis conducted with the different viral products confirmed the assignment of PFBV to the genus Carmovirus.     

Cloning of the gene for the capsid protein of potato leafroll virus

Summary DNA complementary to the RNA of purified potato leafroll virus (PLRV) was synthesized and cloned into the insertion vector NM1149. Over-lapping PLRV-specific cDNA clones were isolated that represent some 80% of the viral genome. Sequences coding for the capsid protein were identified by subcloning size-selected cDNAs into the expression vector gt11 and screening with PLRV-specific antisera. The gene for the viral capsid protein was shown to reside in the 3 terminal half of the genomic RNA. Sequence comparisons with the recently published genomes of the beet western yellows virus (BWYV) and the barley yellow dwarf virus (BYDV) reveal some 65% protein sequence homology between the capsid proteins of BWYV and PLRV and some 45% homology between BYDV and PLRV. Furthermore, it is evident that the structural organization of the PLRV genome in the CP gene region is similar to that of BWYV and BYDV.     

The complete nucleotide sequence and genome organization of barley mild mosaic virus (Na1 strain)

Summary The 5-terminal half of RNA 1 and whole of RNA 2 of barley mild mosaic bymovirus Na1 strain (BaMMV-Na1) were sequenced to give together with the published data its complete genomic sequence. BaMMV-Na1 RNA 1 and RNA 2 consist of 7 263 and 3 516 nucleotides, excluding the 3 poly A tails, respectively. RNA 1 encodes a single large polyprotein of 2 258 amino acids (M_r 256 K), containing eight putative functional proteins, and RNA 2 also encodes one polyprotein of 891 amino acids (M_r 98 K), containing two functional proteins. These functional proteins are arranged in the same manner as in RNA 1 and RNA 2 of barley yellow mosaic bymovirus (BaYMV), and significant amino acid sequence homology (25–58%) exists between the proteins of the two viruses. The BaMMV-Na1 proteins show less amino acid sequence homology (18–32%) with the corresponding proteins of potyviruses or rymoviruses than with those of Ba YMV. Comparisons of the BaMMV-Na1 proteins with the corresponding proteins of other partially sequenced BaMMV isolates show 87–98% amino acid sequence identity. There is 91–94% nucleotide sequence identity between the 3 non-coding regions (NCRs) in RNA 1 or RNA 2 of BaMMV-Na1 and other BaMMV isolates, but only 68–72% identity between the 5 NCRs in RNA 2 of BaMMV-Na1 and other BaMMV isolates.The nucleotide sequence data described in this paper will appear in the DDBJ, EMBL and GenBank nucleotide databases under the accession numbers D83408 (RNA 1) and D83409 (RNA 2).     

Nucleotide sequence of dengue type 3 virus genomic RNA encoding viral structural proteins

Complementary DNAs to the 5 proximal region of the dengue virus type 3 RNA were cloned into bacterial plasmids and the nucleotide sequence of 3,000 bases from the 5 terminus of the genome were determined by DNA and RNA sequencing methods using dideoxy chain-termination reactions. Comparison of the nucleotide sequence thus obtained with those of other flavivirus genomes revealed significant homology existing in nucleotide sequence of the flavivirus genomes. When we compared amino acid sequence deduced from the nucleotide sequence with those of other flaviviruses, this genome region was found to include sequences encoding three viral structural proteins C, M, and E and a part of the viral nonstructural protein NS1 in this order in addition to the 5-noncoding sequence. The characteristics and functions of these proteins were discussed based on the deduced amino acid sequences and their hydrophobic profiles. The genetic relationship of flaviviruses was also discussed based on the genetic variation observed in their genomes.     

A Defective RNA Associated with Bamboo Mosaic Virus and the Possible Common Mechanisms for RNA Recombination in Potexviruses

A naturally occurring 1.1 kb RNA was isolated from purified virions of bamboo mosaic potexvirus isolate S (BaMV-S). This RNA is a defective RNA (D RNA) derived from a single internal deletion of the BaMV genome. A cDNA clone representing the complete nucleotide sequence of the BaMV-S D RNA was generated and its nucleotide sequence was determined. The BaMV D cDNA is 1015 nts in length [excluding the poly(A) tail] and consists of two regions corresponding to 867 nts of the 5 terminus and 148 nts of the 3 terminus of the BaMV genomic RNA. BaMV D cDNA contains a single open reading frame (ORF) encoding a putative 29.7 kDa protein comprised of a fusion of the first 258 amino acids of BaMV ORF 1 and the last 2 amino acids of coat protein. The coding capacity of D RNA was verified by in vitro translation of native BaMV-S D RNA and of 1.1 kb RNA transcribed in vitro from the full-length D cDNA. BaMV D RNA can be reproducibly generated by serial passages of BaMV-S in Nicotiana benthamiana and is the first D RNA in the potexvirus group shown to be generated de novo. Alignments of sequences surrounding the 5 and 3 junction borders of reported potexvirus D RNAs reveal a 65.2–84.6% sequence identity, suggesting that common mechanisms for viral RNA recombination are involved in the generation of potexvirus D RNAs.     

Nucleotide Sequences of Double-Stranded RNA Segments from a Hypovirulent Strain of the White Root Rot Fungus Rosellinia necatrix: Possibility of the First Member of the Reoviridae from Fungus

Twelve double-stranded (ds) RNA segments were detected from a hypovirulent strain W370 of the white root rot fungus Rosellinia necatrix. The estimated molecular weights ranged from 0.41×10⁶ to 2.95×10⁶. Full length cDNA clones for eight segments were obtained. Northern blot analysis suggested that each segment was genetically unique. The nucleotide sequences of eight full length dsRNA segments were determined. One long open reading frame was found in each segment. Conserved sequences at the 5-end (5-ACAAUUU-3) and at the 3-end (5-UGCAGAC-3) were identified in all eight segments. Segment-specific panhandle structures, formed by inverted terminal repeats, were also found in all segments. Comparative analyses of the predicted translational products of eight dsRNA segments showed that the deduced amino acid sequence partially matched those of the Reoviridae family members: Colorado tick fever virus, Nilaparvata lugens reovirus, and rice black streaked dwarf virus. The results suggested that W370 dsRNA is derived from a new member of the family Reoviridae detected in fungus.     

The Conserved, 5′ Termini of RNAs 1 and 2 of <Emphasis Type="Italic">Tomato aspermy virus</Emphasis> are Dispensable for Infection but Affect Virulence

The 5 terminus of each of the three genomic RNAs (RNAs 1, 2 and 3) of Tomato aspermy virus (TAV) begins with the sequence 5-GUUU, which is also shared by a number of other viruses. Mutagenic analyses showed that the 5-GUUU sequence of RNAs 1 and 2 of TAV was dispensable for viral infection and did not prevent symptom induction. On the other hand, substitution of U at position 5 for G in RNA 1, but not RNA 2, induced veinal necrosis symptoms in Nicotiana glutinosa. The mutants constructed included insertion of UUU into the 5-GUUU sequence of TAV RNAs 1 and/or 2. All RNA 2 mutants induced more severe symptoms than viral RNAs containing either mutated RNA 1 or most combinations of mutated RNAs 1 and 2. Some combinations of mutated RNAs 1 and 2 also induced veinal necrosis in N. glutinosa. Virulence was unrelated to the levels of viral RNA accumulation. Sequence analysis of progeny viral RNAs showed that only the mutant viral RNAs with a G to U substitution in RNA 1 and the deletion of the 5-GUUU in both RNAs 1 and 2 were able to maintain the same sequence as the inoculum. The other mutants either reverted to the wildtype sequence or underwent further deletion or insertion. None of the constructed mutants were able to compete for accumulation with the wildtype virus after co-inoculation to the plant species tested.     

The oligodendroglial reaction to brain stab wounds: An immunohistochemical study

Summary Myelin/oligodendrocyte specific protein was compared to glial fibrillary acidic protein and 23-cyclic nucleotide 3-phosphodiesterase expression in normal rat brains and following stab wounds to the cerebral cortex, corpus callosum and hippocampus. Animals with stab wounds were allowed to recover for 5,15,28, 45 and 70 days post-operation before fixation by perfusion. Sections were reacted with antibodies against myelin/oligodendrocyte specific protein, glial fibrillary acidic protein and 23-cyclic nucleotide 3-phosphodiesterase, and observed by light and electron microscopy. Normal cerebral cortex had very few myelin/oligodendrocyte specific protein-positive and 23-cyclic nucleotide 3-phosphodiesterasepositive cells, but some glial fibrillary acidic protein-positive cells. The myelinated fibres of the corpus callosum were heavily stained for myelin/oligodendrocyte specific protein but unstained by glial fibrillary acidic protein or 23-cyclic nucleotide 3-phosphodiesterase antibodies. Some immunopositive cells were present in the corpus callosum and hippocampus with all three antibodies. After stab wound myelin/oligodendrocyte specific protein-positive reactive cells had more and longer processes and stained more intensely than equivalent cells in normal brain. These cells were distributed along the wound track, including within the cerebral cortex. The numbers of these cells increased until 28 days post-operation and then decreased so that very few were found at 70 days post-operation except in the corpus callosum. Where demyelination occurred myelin/oligodendrocyte specific protein-staining was lost. Staining for 23-cyclic nucleotide 3-phosphodiesterase revealed a similar pattern. Glial fibrillary acidic protein-positive reactive cells, which were also more robust than the normal cells, were more widely distributed. They increased in number throughout the time periods studied and gliosis was evident on the contralateral side. The glial fibrillary acidic protein-positive astrocytes were also different from the myelin/oligodendrocyte specific protein-positive and 23-cyclic nucleotide 3-phosphodiesterase-positive oligodendrocytes in terms of cell shape. With electron microscopy myelin/oligodendrocyte specific protein-positive cells showed features typical of immature oligodendrocytes. We conclude that the injury caused a numerical increase in oligodendrocytes and that myelin/ oligodendrocyte specific protein is a good marker for the oligodendroglial response and demyelination in pathological conditions.     

Cloning,sequencing, and expression inEscherichia coli of the coat protein gene of a new potyvirus infecting South AfricanPassiflora

Summary Complementary DNA representing 1418 nucleotides (nt) of the 3-poly(A)-proximal tract of the genomic RNA of a potyvirus causing woodiness disease in South African passionfruit, was cloned and sequenced. The sequence contained a single long open reading frame (ORF) of 1188 nt with no initiation codon, and a 3-non-coding region (3-NCR) of 230 nt followed by a poly-adenylate tract. Comparison of the ORF with other potyviral coat protein (CP) sequences led to the prediction that a 279 residue CP of MW 31722 is encoded by 836 nt at the 3-terminus of the ORF. This virus is not merely a South African strain of passionfruit woodiness virus (PWV): the deduced CP sequence is only distantly related to CPs of other sequenced strains of PWV, although it is part of a distinct subgroup of potyviruses related to PWV. The virus was therefore designated as South African passiflora virus (SAPV). The DNA containing the putative CP was cloned into the pUEX 2 bacterial expression vector and expressed inEscherichia coli as a -gal-CP fusion protein. The fusion protein reacted with polyclonal antisera raised against the native virus, and antisera raised against partially purified fusion protein reacted with viral CP in Western blots.     

Investigation of the 5′ terminal structures of genomic and subgenomic RNAs of potato virus S

Hybrid arrest translation involving an antisense RNA, generated from a cloned cDNA close to the 5 region of potato virus S genomic RNA, blocked the synthesis of genomic encoded products but had little effect on subgenomic RNA encoded products. Similarly, the synthesis of PVS genomic RNA-directed peptides was inhibited by the cap analogue m⁷G⁵ ppp⁵G, suggesting the presence of a cap structure at the 5 terminus whilst subgenomic RNA encoded products remained unaffected, suggesting an uncapped structure. This was confirmed by artificially produced uncapped subgenomic RNAs translating as efficiently in in vitro translation systems as authentic viral subgenomic RNAs.