Capsid coding sequence is required for efficient replication of human rhinovirus 14 RNA.

Mechanisms by which the plus-sense RNA genomes of picornaviruses are replicated remain poorly defined, but existing models do not suggest a role for sequences encoding the capsid proteins. However, candidate RNA replicons (delta P1 beta gal and delta P1Luc), representing the sequence of human rhinovirus 14 virus (HRV-14) with reporter protein sequences (beta-galactosidase or luciferase, respectively) replacing most of the P1 capsid-coding region, failed to replicate in transfected H1-HeLa cells despite efficient primary cleavage of the polyprotein. To determine which P1 sequences might be required for RNA replication, HRV-14 mutants in which segments of the P1 region were removed to frame from the genome were constructed. Mutants with deletions involving the 5'proximal 1,489 nucleotides of the P1 region replicated efficiently, while those with deletions involving the 3' 1,079 nucleotides did not. Reintroduction of the 3' P1 sequence into the nonreplicating delta P1Luc construct resulted in a new candidate replicon, delta P1Luc/VP3, which replicated well and expressed luciferase efficiently. Capsid proteins provided in trans by helper virus failed to rescue the nonreplicating delta P1Luc genome but were able to package the larger-than-genome-length delta P1Luc/VP3 replicon. Thus, a 3'-distal P1 capsid-coding sequence has a previously unrecognized cis-active function related to replication of HRV-14 RNA.     

Partial nucleotide sequence of the Murray Valley encephalitis virus genome. Comparison of the encoded polypeptides with yellow fever virus structural and non-structural proteins

The sequence of 5400 bases corresponding to the 5'-terminal half of the Murray Valley encephalitis virus genome has been determined. The genome contains a 5' non-coding region of about 97 nucleotides, followed by a single continuous open reading frame that encodes the structural proteins followed by the non-structural proteins. Amino acid sequence homology between the Murray Valley encephalitis and yellow fever (Rice et al., 1985) polyproteins is 42% over the region sequenced. The start points of the various Murray Valley encephalitis virus-coded proteins have been assigned on the basis of this homology and a consistent set of potential proteolytic cleavage sites identified, the sequences of which are similar in Murray Valley encephalitis and yellow fever. The deduced Murray Valley encephalitis gene order is 5'-C-prM (M)-E-NS1-ns2a-ns2b-NS3-3'. The genome organization of Murray Valley encephalitis and yellow fever appears to be identical and the sizes of the predicted virus-coded proteins similar between the two viruses. Both viruses encode a basic capsid protein followed by three glycoproteins; the glycoproteins appear to have the conventional topology of N terminus outside with a C-terminal membrane-spanning domain. There are conserved glycosylation sites in prM, the precursor to the M protein of the virion, and in NS1, a non-structural protein of uncertain function. The glycosylation sites in E, the major envelope protein of the virion, are not conserved as to position. We predict the existence, in flavivirus-infected cells, of two small, hydrophobic peptides, ns2a and ns2b, which show only limited amino acid sequence homology. Finally, about half of the amino acid sequence of NS3 has been obtained; NS3 is a hydrophilic non-structural protein that shows 55% amino acid sequence similarity between Murray Valley encephalitis and yellow fever over the region sequenced and is probably involved in RNA replication.     

The Nucleotide Sequence of the 3'-Terminal Region of Clover Yellow Vein Potyvirus Isolated from Calanthe sp.

The sequence of 1399 nucleotides from the 3'region of the RNA of clover yellow vein potyvirus (CIYVV-C) isolated from Calanthe sp. was determined. The capsid protein cistron was composed of 915 nucleotides, and corresponded to a region encoding 305 amino acids with a calculated Mr of 34900: the adjacent 3'non-coding region was 74 nucleotides long. The length of the capsid protein gene of CIYVV-C is longer, but the 3'non-coding region is shorter than those of other CIYVV strains (CIYVV-30.CIYVV-NZ and CIYVV-B). The nucleotide sequence of the capsid protein gene and the 3'non-coding region of CIYVV-C RNA showed significantly higher homology with those of other CIYVV strains. These data suggest that the CIYVV-C capsid gene arose from a frame-shift mutation of the capsid gene of another CIYVV strain. or vice versa. CIYVV-C has a closer affinity to other CIYVV strains and to bean yellow mosaic virus than to the other potyviruses.     

The rhinovirus type 14 genome contains an internally located RNA structure that is required for viral replication.

Cis-acting RNA signals are required for replication of positive-strand viruses such as the picornaviruses. Although these generally have been mapped to the 5' and/or 3' termini of the viral genome, RNAs derived from human rhinovirus type 14 are unable to replicate unless they contain an internal cis-acting replication element (cre) located within the genome segment encoding the capsid proteins. Here, we show that the essential cre sequence is 83-96 nt in length and located between nt 2318-2413 of the genome. Using dicistronic RNAs in which translation of the P1 and P2-P3 segments of the polyprotein were functionally dissociated, we further demonstrate that translation of the cre sequence is not required for RNA replication. Thus, although it is located within a protein-coding segment of the genome, the cre functions as an RNA entity. Computer folds suggested that cre sequences could form a stable structure in either positive- or minus-strand RNA. However, an analysis of mutant RNAs containing multiple covariant and non-covariant nucleotide substitutions within these putative structures demonstrated that only the predicted positive-strand structure is essential for efficient RNA replication. The absence of detectable minus-strand synthesis from RNAs that lack the cre suggests that the cre is required for initiation of minus-strand RNA synthesis. Since a lethal 3' noncoding region mutation could be partially rescued by a compensating mutation within the cre, the cre appears to participate in a long-range RNA-RNA interaction required for this process. These data provide novel insight into the mechanisms of replication of a positive-strand RNA virus, as they define the involvement of an internally located RNA structure in the recognition of viral RNA by the viral replicase complex. Since internally located RNA replication signals have been shown to exist in several other positive-strand RNA virus families, these observations are potentially relevant to a wide array of related viruses.     

Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication

Sequences in the 5' and 3' termini of plus-strand RNA viruses harbor cis-acting elements important for efficient translation and replication. In case of the hepatitis C virus (HCV), a plus-strand RNA virus of the family Flaviviridae, a 341-nucleotide-long nontranslated region (NTR) is located at the 5' end of the genome. This sequence contains an internal ribosome entry site (IRES) that is located downstream of an about 40-nucleotide-long sequence of unknown function. By using our recently developed HCV replicon system, we mapped and characterized the sequences in the 5' NTR required for RNA replication. We show that deletions introduced into the 5' terminal 40 nucleotides abolished RNA replication but only moderately affected translation. By generating a series of replicons with HCV-poliovirus (PV) chimeric 5' NTRs, we could show that the first 125 nucleotides of the HCV genome are essential and sufficient for RNA replication. However, the efficiency could be tremendously increased upon the addition of the complete HCV 5' NTR. These data show that (i) sequences upstream of the HCV IRES are essential for RNA replication, (ii) the first 125 nucleotides of the HCV 5' NTR are sufficient for RNA replication, but such replicon molecules are severely impaired for multiplication, and (iii) high-level HCV replication requires sequences located within the IRES. These data provide the first identification of signals in the 5' NTR of HCV RNA essential for replication of this virus.     

甜菜坏死黄脉病毒RNA4 cDNA克隆、序列分析及其编码蛋白基因在大肠杆菌中的表达

以甜菜坏死黄脉病毒(Beet Necrotic Yellow Vein Virus,简称BNYVV)内蒙分离物(NM)RNA为模板,通过反转录和PCR扩增得到了BNYVV RNA4基因组的cDNA克隆pGBF6。序列分析结果表明,pGBF6含有全长RNA4 cDNA插入片段,大小为1465个核苷酸,含有一个849个核苷酸的开放阅读框架,编码产生由282个氨基酸组成的分子量为31kDa的蛋白。与法国F2分离物RNA4相比,其核苷酸序列和由此推导的氨基酸序列同源性分别为97.1％和96.4％,并在5'端非编码区比F2分离物缺失了3个核苷酸。将RNA4编码区cDNA克隆到原核表达载体pFLAG·MAC上,获得融合蛋白表达质粒pFMBF87。所构建的融合蛋白由载体序列编码的14个氨基酸和31kDa蛋白C端的233个氨基酸组成。经IPTG诱导,Westem blotting分析表明,该融合蛋白在大肠杆菌中得到高效表达。本文还对内蒙分离物的株系划分进行了讨论。     

Requirements at the 3' end of the sindbis virus genome for efficient synthesis of minus-strand RNA

The 3'-untranslated region of the Sindbis virus genome is 0.3 kb in length with a 19-nucleotide conserved sequence element (3' CSE) immediately preceding the 3'-poly(A) tail. The 3' CSE and poly(A) tail have been assumed to constitute the core promoter for minus-strand RNA synthesis during genome replication; however, their involvement in this process has not been formally demonstrated. Utilizing both in vitro and in vivo analyses, we have examined the role of these elements in the initiation of minus-strand RNA synthesis. The major findings of this study with regard to efficient minus-strand RNA synthesis are the following: (i) the wild-type 3' CSE and the poly(A) tail are required, (ii) the poly(A) tail must be a minimum of 11 to 12 residues in length and immediately follow the 3' CSE, (iii) deletion or substitution of the 3' 13 nucleotides of the 3' CSE severely inhibits minus-strand RNA synthesis, (iv) templates possessing non-wild-type 3' sequences previously demonstrated to support virus replication do not program efficient RNA synthesis, and (v) insertion of uridylate residues between the poly(A) tail and a non-wild-type 3' sequence can restore promoter function to a limited extent. This study shows that the optimal structure of the 3' component of the minus-strand promoter is the wild-type 3' CSE followed a poly(A) tail of at least 11 residues. Our findings also show that insertion of nontemplated bases can restore function to an inactive promoter.     

Two regions of an avian hepadnavirus RNA pregenome are required in cis for encapsidation.

We have constructed a series of deletion mutants spanning the genome of duck hepatitis B virus in order to determine which regions of the viral genome are required in cis for packaging of the pregenome into capsid particles. Deletion of sequences within either of two nonadjacent regions prevented replication of the mutant viral genomes expressed in a permissive avian hepatoma cell line in the presence of functionally active viral core and P proteins. Extraction of RNA from cells transfected with these replication-defective mutants showed that the mutants retained the capacity to be transcribed into a pregenomic-size viral RNA, but that these RNA species were not packaged into viral capsids. The two regions defined by these deletions are located 36 to 126 (region I) and 1046 to 1214 (region II) nucleotides downstream of the 5' end of the pregenome and contain sequences which are required in cis for encapsidation of the duck hepatitis B virus pregenome.     

The complete nucleotide sequence of the RNA coding for the primary translation product of foot and mouth disease virus.

The complete nucleotide sequence of the coding region of foot and mouth disease virus RNA (strain A1061) is presented. The sequence extends from the primary initiation site, approximately 1200 nucleotide from the 5' end of the genome, in an open translational reading frame of 6,999 nucleotides to a termination codon 93 nucleotides from the 3' terminal poly (A). Available amino acid sequence data correlates with that predicted from the nucleotide sequence. The amino acid sequence around cleavage sites in the polyprotein shows no consistency, although a number of the virus-coded protease cleavage sites are between glutamate and glycine residues.     

Molecular characterization of the 3' terminus of the simian hemorrhagic fever virus genome.

The 3' end of the simian hemorrhagic fever virus (SHFV) single-stranded RNA genome was cloned and sequenced. Adjacent to the 3' poly(A) tract, we identified a 76-nucleotide noncoding region preceded by two overlapping reading frames (ORFs). The ultimate 3' ORF of the viral genome encodes the capsid protein, and the penultimate ORF encodes the smallest SHFV envelope protein. These two ORFs overlap each other by 26 nucleotides. Northern (RNA) blot hybridization analyses of cytoplasmic RNA extracts from SHFV-infected MA-104 cells with gene-specific probes revealed the presence of full-length genomic RNA as well as six subgenomic SHFV-specific mRNA species. The subgenomic mRNAs are 3' coterminal. In its virion morphology and size, genome structure and length, and replication strategy, SHFV is most similar to lactate dehydrogenase-elevating virus, equine arteritis virus, and porcine reproductive and respiratory syndrome virus.     

The 5'-terminal region of the Aichi virus genome encodes cis-acting replication elements required for positive- and negative-strand RNA synthesis

Aichi virus is a member of the family Picornaviridae. It has already been shown that three stem-loop structures (SL-A, SL-B, and SL-C, from the 5' end) formed at the 5' end of the genome are critical elements for viral RNA replication. In this study, we further characterized the 5'-terminal cis-acting replication elements. We found that an additional structural element, a pseudoknot structure, is formed through base-pairing interaction between the loop segment of SL-B (nucleotides [nt] 57 to 60) and a sequence downstream of SL-C (nt 112 to 115) and showed that the formation of this pseudoknot is critical for viral RNA replication. Mapping of the 5'-terminal sequence of the Aichi virus genome required for RNA replication using a series of Aichi virus-encephalomyocarditis virus chimera replicons indicated that the 5'-end 115 nucleotides including the pseudoknot structure are the minimum requirement for RNA replication. Using the cell-free translation-replication system, we examined the abilities of viral RNAs with a lethal mutation in the 5'-terminal structural elements to synthesize negative- and positive-strand RNAs. The results showed that the formation of three stem-loops and the pseudoknot structure at the 5' end of the genome is required for negative-strand RNA synthesis. In addition, specific nucleotide sequences in the stem of SL-A or its complementary sequences at the 3' end of the negative-strand were shown to be critical for the initiation of positive-strand RNA synthesis but not for that of negative-strand synthesis. Thus, the 5' end of the Aichi virus genome encodes elements important for not only negative-strand synthesis but also positive-strand synthesis.     

Sequence, necessary for initiating RNA synthesis, in the 3'-noncoding region of the classical swine fever virus genome

Classical swine fever virus (CSFV) is the causative agent of swine fever, which represents an economically important disease in hogs. We previously made a prediction about the recognition sites of replication initiation of CSFV by using the information content method, and it was predicted that the 21 nucleotides located at 3' end of the CSFV genome 3'UTR were essential to CSFV replication. In this paper, we experimentally studied these 21 nucleotides by site-directed mutagenesis. It was found that the 3'UTRs with the 21 nucleotides had the function of initiating RNA synthesis, while the 3'UTRs without the 21 nucleotides did not. The 21 nucleotides alone, without the rest of 3'UTR, were able to initiate RNA synthesis, though with a slump. It was demonstrated that the 21 nucleotides were essential to the replication of CSFV genome. The other part of 3'UTR was also required for sufficient RNA synthesis. It is highly likely that the 21 nucleotides were the necessary site for the CSFV genome replication initiation, and that the elements required for sufficient RNA synthesis were in the other part of 3'UTR. It was assumed that the CSFV replicase bound to the site and initiated the replication of the CSFV genome. In the 21 nucleotides, it was found that the mutation of position 216 and destruction of the 3' terminus in the 3'UTR precluded initiation of RNA synthesis, that the mutation of position 212 did not affect the capacity for initiation of RNA synthesis but attenuated the synthesis of RNA. Among the four mutants of 3'UTR at position 219, three produced the 3'UTR without initiation of RNA synthesis, and the other one produced the 3'UTR with initiation of less RNA synthesis. Therefore, it could be concluded that T216 was the most important while T212 was the least important, and that G219 and C228 were also important for RNA synthesis. The normal base component within the 21 nucleotides was essential to sufficient RNA synthesis.     

Kelp fly virus: a novel group of insect picorna-like viruses as defined by genome sequence analysis and a distinctive virion structure

The complete genomic sequence of kelp fly virus (KFV), originally isolated from the kelp fly, Chaetocoelopa sydneyensis, has been determined. Analyses of its genomic and structural organization and phylogeny show that it belongs to a hitherto undescribed group within the picorna-like virus superfamily. The single-stranded genomic RNA of KFV is 11,035 nucleotides in length and contains a single large open reading frame encoding a polypeptide of 3,436 amino acids with 5' and 3' untranslated regions of 384 and 343 nucleotides, respectively. The predicted amino acid sequence of the polypeptide shows that it has three regions. The N-terminal region contains sequences homologous to the baculoviral inhibitor of apoptosis repeat domain, an inhibitor of apoptosis commonly found in animals and in viruses with double-stranded DNA genomes. The second region contains at least two capsid proteins. The third region has three sequence motifs characteristic of replicase proteins of many plant and animal viruses, including a helicase, a 3C chymotrypsin-like protease, and an RNA-dependent RNA polymerase. Phylogenetic analysis of the replicase motifs shows that KFV forms a distinct and distant taxon within the picorna-like virus superfamily. Cryoelectron microscopy and image reconstruction of KFV to a resolution of 15 A reveals an icosahedral structure, with each of its 12 fivefold vertices forming a turret from the otherwise smooth surface of the 20-A-thick capsid. The architecture of the KFV capsid is unique among the members of the picornavirus superfamily for which structures have previously been determined.     

A poliovirus replicon containing the chloramphenicol acetyltransferase gene can be used to study the replication and encapsidation of poliovirus RNA.

A poliovirus replicon, FLC/REP, which incorporates the reporter gene chloramphenicol acetyltransferase (CAT) in place of the region encoding the capsid proteins VP4, VP2, and part of VP3 in the genome of poliovirus type 3, has been constructed. Transfection of cells indicates that the FLC/REP replicon replicates efficiently and that active CAT enzyme is produced as a CAT-VP3 fusion protein. The level of CAT activity in transfected cells broadly reflects the level of FLC/REP RNA. A series of mutations in the 5' noncoding region of poliovirus type 3 were introduced into FLC/REP, and their effects were monitored by a simple CAT assay. These experiments helped to define further the stem-loop structures in the 5' noncoding region which are essential for RNA replication. The CAT-containing poliovirus replicon could also be packaged into poliovirus capsids provided by helper virus and was stable as a subpopulation of virus particles over at least four passages. The location of the CAT gene in FLC/REP excluded the presence of an encapsidation signal in the region of the poliovirus genome comprising nucleotides 756 to 1805.     

Sequence and structural elements at the 3' terminus of bovine viral diarrhea virus genomic RNA: functional role during RNA replication

Bovine viral diarrhea virus (BVDV), a member of the genus Pestivirus in the family Flaviviridae, has a positive-stranded RNA genome consisting of a single open reading frame and untranslated regions (UTRs) at the 5' and 3' ends. Computer modeling suggested the 3' UTR comprised single-stranded regions as well as stem-loop structures-features that were suspected of being essentially implicated in the viral RNA replication pathway. Employing a subgenomic BVDV RNA (DI9c) that was shown to function as an autonomous RNA replicon (S.-E. Behrens, C. W. Grassmann, H. J. Thiel, G. Meyers, and N. Tautz, J. Virol. 72:2364-2372, 1998) the goal of this study was to determine the RNA secondary structure of the 3' UTR by experimental means and to investigate the significance of defined RNA motifs for the RNA replication pathway. Enzymatic and chemical structure probing revealed mainly the conserved terminal part (termed 3'C) of the DI9c 3' UTR containing distinctive RNA motifs, i.e., a stable stem-loop, SL I, near the RNA 3' terminus and a considerably less stable stem-loop, SL II, that forms the 5' portion of 3'C. SL I and SL II are separated by a long single-stranded intervening sequence, denoted SS. The 3'-terminal four C residues of the viral RNA were confirmed to be single stranded as well. Other intramolecular interactions, e.g., with upstream DI9c RNA sequences, were not detected under the experimental conditions used. Mutagenesis of the DI9c RNA demonstrated that the SL I and SS motifs do indeed play essential roles during RNA replication. Abolition of RNA stems, which ought to maintain the overall folding of SL I, as well as substitution of certain single-stranded nucleotides located in the SS region or SL I loop region, gave rise to DI9c derivatives unable to replicate. Conversely, SL I stems comprising compensatory base exchanges turned out to support replication, but mostly to a lower degree than the original structure. Surprisingly, replacement of a number of residues, although they were previously defined as constituents of a highly conserved stretch of sequence of the SS motif, had little effect on the replication ability of DI9c. In summary, these results indicate that RNA structure as well as sequence elements harbored within the 3'C region of the BVDV 3' UTR create a common cis-acting element of the replication process. The data further point at possible interaction sites of host and/or viral proteins and thus provide valuable information for future experiments intended to identify and characterize these factors.     

Nucleotide sequence of the genome and complete amino acid sequence of a polyprotein of the tick-borne encephalitis virus

We have cloned and sequenced RNA encoding all virion and nonstructural proteins of tick-borne encephalitis virus (TBEV). Its length is 10,477 bases with a single open reading frame (nucleotides 127-10,363) encoding 3412 amino acids. The 5'- and 3'-noncoding regions have stem- and- loop structure. The polyprotein precursor is proteolytically cleaved, apparently, by a mechanism resembling that proposed for the expression of polyproteins of other flaviviruses, such as yellow fever, West Nile and Kunjin viruses. The deduced TBEV gene order is 5'-C-preM (M)-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5++ +-3'. The genome and the polyprotein of TBEV and other flaviviruses appears to be structurally similar, although these flaviviruses are transmitted to and from their vertebrate hosts by different carriers, such as ticks or mosquitoes. Analysis of sequence homologies of polyproteins of flaviviruses suggests that TBEV is more closely related to yellow fever virus than to other serological subgroups of flaviviruses (West Nile or Dengue viruses). The hydrophobic profiles of the flaviviruses are highly conservative. Nonstructural proteins NS2A, NS2B, NS4A and NS4B are extremely hydrophobic, suggesting that they are likely to be associated with cellular membranes. Proteins E, NS1, NS3 and NS5 are the most conservative and may be involved in general enzymatic activities related to viral replication and virion assembly.     

Messenger RNA structure participating in the initiation of synthesis of cucumber mosaic virus coat protein

The sequence of the 5'-terminal 106 nucleotides of cucumber mosaic virus (strain Y) RNA 4, the mRNA coding for viral coat protein, has been determined. The first AUG was located at 77 nucleotides from the 5'-terminus and was confirmed to be an initiation codon by analysis of the N-terminal amino acid sequence of the protein. The nucleotide sequence (positions 77-106) beyond the AUG codon predicted the sequence of ten amino acids corresponding to the N-terminal region of the protein, which exactly matched the determined amino acid sequence containing an acetyl methionine as the N-terminal amino acid. The distance of the initiation codon AUG from the cap structure was 76 nucleotides and the longest among the mRNAs for coat protein of plant viruses so far reported (9-36 nucleotides). This noncoding region is rich in U residues (40%) and the number of G residues (21 nucleotides) is the largest among these mRNAs (usually 1 or 2 residues). A possible secondary structure is postulated for the region, which might be implicated in efficient translation of the RNA 4 in vivo.