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PubMed1983

Multiple leader RNAs and messenger RNAs are transcribed from the La Crosse virus small genome segment.

Nucleotide sequencing has demonstrated that the Small genome segment of Bunyaviruses contains the genetic information for two viral proteins (N and NSs) in overlapping reading frames (Akashi and Bishop, 1983; Cabradilla et al., in press). Using 3' end-labeled genome probes, La Crosse virus (LAC) infected cells were shown to contain three leader RNAs, which start at position 1 and terminate at approximate positions 74, 95, and 115 from the 3' end of the genome. Primer extension and S1 mapping studies have shown that LAC-infected cells contain seven mRNAs, five of which initiate at positions 1, 74, 101, 117, and 123 and two more which initiate at approximate positions 147 and 159 from the 3' end of the Small genome segment. The existence of multiple leader RNAs and mRNAs from the Small genome segment may allow access to both overlapping reading frames by ribosomes that still initiate only at 5' proximal AUGs, without the need to splice the mRNA.

Bunyaviridae↗

PubMed2025

Combinatorial genome engineering of pseudorabies virus Bartha by developing a reverse genetic system based on three overlapping genomic segments.

INTRODUCTION: The 138-kilobase genome of pseudorabies virus vaccine strain Bartha K61 harbors many nonessential genes for replication and exhibits remarkable capacity for incorporating foreign genes for therapeutic applications. However, the large size of the Bartha genome complicates its efficient engineering. OBJECTIVES: Development of a reverse genetic system for pseudorabies virus Bartha based on three overlapping genomic segments to facilitate multiplex genome engineering. METHODS: The 138-kb genome of Bartha was split into three overlapping segments (42 kb, 43 kb, and 53 kb), each cloned in a bacterial artificial chromosome (BAC) to facilitate genome engineering. The infectious virus was reconstituted by transfecting the 3 genomic fragments released from the BACs into Vero cells in which a complete virus genome was assembled using 2-kb overlaps between adjacent pieces. RESULTS: Employing the reverse genetic system, we individually deleted 15 candidate nonessential genes and confirmed that 10 were dispensable for viral growth in cell culture. Deletion of 7 nonessential genes had no impact on viral growth, whereas UL47 deletion reduced viral growth rate and deletions of UL44, UL47, or US3 resulted in smaller viral plaques. A total of 45 viral genomes with double deletions of nonessential genes were constructed, among which 22 were successfully rescued into infectious virions. Fifteen double-deletion mutant viruses had a viral titer comparable with the wild-type Bartha, while the remaining 7 showed a lower titer. Additionally, expressions of the mNeonGreen reporter gene at nonessential gene loci were evaluated. Cells infected with recombinant viruses carrying mNeonGreen at 8 loci showed strong green fluorescence, whereas those with mNeonGreen at 2 loci exhibited very weak fluorescence. CONCLUSION: The reverse genetic system developed in this study enables rapid and combinatorial engineering of viruses with the large DNA genome, and will accelerate development of large DNA virus-based therapeutics including live-attenuated vaccines, vector vaccines, and oncolytic herpesviruses.

Herpesvirus 1, Suid↗

PubMed2002

Sequential partially overlapping gene arrangement in the tricistronic S1 genome segments of avian reovirus and Nelson Bay reovirus: implications for translation initiation.

Previous studies of the avian reovirus strain S1133 (ARV-S1133) S1 genome segment revealed that the open reading frame (ORF) encoding the final sigmaC viral cell attachment protein initiates over 600 nucleotides distal from the 5' end of the S1 mRNA and is preceded by two predicted small nonoverlapping ORFs. To more clearly define the translational properties of this unusual polycistronic RNA, we pursued a comparative analysis of the S1 genome segment of the related Nelson Bay reovirus (NBV). Sequence analysis indicated that the 3'-proximal ORF present on the NBV S1 genome segment also encodes a final sigmaC homolog, as evidenced by the presence of an extended N-terminal heptad repeat characteristic of the coiled-coil region common to the cell attachment proteins of reoviruses. Most importantly, the NBV S1 genome segment contains two conserved ORFs upstream of the final sigmaC coding region that are extended relative to the predicted ORFs of ARV-S1133 and are arranged in a sequential, partially overlapping fashion. Sequence analysis of the S1 genome segments of two additional strains of ARV indicated a similar overlapping tricistronic gene arrangement as predicted for the NBV S1 genome segment. Expression analysis of the ARV S1 genome segment indicated that all three ORFs are functional in vitro and in virus-infected cells. In addition to the previously described p10 and final sigmaC gene products, the S1 genome segment encodes from the central ORF a 17-kDa basic protein (p17) of no known function. Optimizing the translation start site of the ARV p10 ORF lead to an approximately 15-fold increase in p10 expression with little or no effect on translation of the downstream final sigmaC ORF. These results suggest that translation initiation complexes can bypass over 600 nucleotides and two functional overlapping upstream ORFs in order to access the distal final sigmaC start site.

Amino Acid Sequence↗

PubMed1986

Factors which equalize the representation of genome segments in recombinant libraries.

Genomic segments which contain inverted repetitions longer than 300 bp are frequently lost from recombinant libraries grown on rec+ hosts. We have found that 9% of phage lambda clones that contain 15-20-kb insertions of human or Drosophila DNA are inhibited on rec+ hosts and as a result will become under-represented in amplified genomic libraries. We have therefore examined several factors of both host and vector origin which affect the fidelity of representation of genomic sequences in recombinant DNA libraries constructed in bacteriophage lambda vectors. This loss may be diminished if the vector carries either a chi element or a functional gam gene. The most successful approach, however, involves using a host with mutations in recB, recC, and sbcB, or in recD. We have shown that recombinant clones which require such mutant hosts for growth are somewhat more likely to contain DNA derived from loci in the genome which are polymorphic than are clones recovered on conventional hosts.

Animals↗

PubMed2002

Identification and characterization of a baboon reovirus-specific nonstructural protein encoded by the bicistronic s4 genome segment.

All characterized orthoreoviruses encode a characteristic spike-like protein on their polycistronic S1 genome segments that mediates virus cell attachment. In the case of baboon reovirus (BRV), the polycistronic S-class genome segment corresponds to the smallest S4 segment. We recently determined that the 5'-proximal open reading frame (ORF) of the bicistronic S4 segment encodes a nonstructural protein responsible for virus-induced syncytium formation. Current analysis indicates that the p16 protein encoded by the 3'-proximal ORF of the BRV S4 genome segment shows no sequence similarity to any other protein encoded by the orthoreoviruses, including the well-characterized sigma1/sigmaC reovirus cell attachment protein. Results indicate that p16 is a BRV-specific nonstructural protein that is not required for virus infection in cell culture and is not involved in viral cell attachment. In conjunction with previous studies of the BRV S1, S2, and S3 genome segments, the current results indicate that, unlike all other orthoreoviruses, BRV does not encode a cell attachment protein in its S-class genome segments. Furthermore, cell binding and infectivity studies suggested BRV may not utilize a functional homolog of the prototypical reovirus sigma1/sigmaC cell receptor-binding protein to mediate endocytic uptake by cells.

Animals↗

PubMed1984

Variant dsRNAs associated with transmission-defective isolates of wound tumor virus represent terminally conserved remnants of genome segments.

Variant double-stranded RNAs are often associated with the genome of transmission-defective isolates of wound tumor virus. These RNAs are replicated and packaged into virus particles in systemically infected plants and are transcribed in vitro by the virion-associated transcriptase. Direct physical evidence that the variant RNAs are remnants of particular WTV genome segments was provided by molecular hybridization studies. Subsequently, ribonuclease T1 digestion products of 3'-end-labeled genome and remnant RNAs were analyzed by one- and two-dimensional electrophoretic techniques. One-dimensional partial and complete digestion patterns were indistinguishable, indicating that the guanosine positions relative to the 3' terminus of the corresponding strands of a particular genome segment and its remnant RNA are the same for at least 40 nucleotides from each end. Fingerprints of the 3' terminal ribonuclease T1-resistant fragments were identical, showing that the nucleotide composition of the 3' terminal ends of the corresponding strands of a particular genome segment and its remnant RNA are also identical. These results indicate that variant RNAs associated with transmission-defective WTV isolates are formed by deletion of an internal portion (as much as 85%) of genomic RNA segments yielding terminally conserved genomic remnants that are functional with respect to transcription, replication, and packaging.

Animals↗

PubMed1987

A comparison of different cloned bluetongue virus genome segments as probes for the detection of virus-specified RNA.

The seven largest double-stranded (ds) RNA genome segments of bluetongue virus (BTV) serotype 4 as well as genome segment 8 of BTV10 have been cloned into pBR322. The length of the cloned genes indicates that, with the exception of genome segment 1, the entire gene has been cloned in each case. A method is described for isolating different sized cDNA transcripts on alkaline sucrose gradients with very good recovery. The eight cloned genes were compared as 32P-labeled probes for the detection of dsRNA from 21 different BTV serotypes. The S1, S3, S4, S5, and S8 genes were identified as being highly conserved. Of these, S5, which codes for nonstructural protein NS1, gave the best hybridization signal with all the dsRNA isolates. All the probes hybridized significantly weaker with the dsRNA of BTV isolates from Australia and Pakistan than with dsRNA from other serotypes. Genome segment 7, which codes for the group-specific antigen P7, was not highly conserved. Even more variation was shown by genome segment 6 which codes for outer capsid polypeptide P5. S2 which codes for protein P2 is the obvious choice for a serotype-specific probe. Hybridization of this probe with dsRNA from other serotypes reflects the cross-neutralization between BTV4 and these serotypes. The hybridization results can also be used to define the relatedness of BTV4 to other serotypes. None of the probes hybridized with dsRNA from any of the other orbiviruses investigated.

Bluetongue virus↗

PubMed2001

Cloning and Sequencing of the Genome Segment S9 of Rice Black-streaked Dwarf Virus.

Genome segment S9's of rice black-streaked dwarf virus (RBSDV) of three Chinese isolates were amplified by RT-PCR and sequenced, and were found to be consisted of 1 900 nt (RBSDV-Zj S9 EMBL accession number AJ297430),1 898 nt (RBSDV-Heb S9 EMBL accession number AJ297429) and 1 900 nt (RBSDV-Hub S9 EMBL accession number AJ291706), respectively. Genome segment S9's of three Chinese Isolates shared 98.5%--98.8% sequence homology and all contained two open reading frames (ORF), which encoded two polypeptides with moleclular weights of 40 kD and 24 kD, respectively. Amino acid sequence comparison of the polypeptides encoded by the second ORF of the corresponding genomic segment of five isolates, including three Chinese isolates, Japanese isolate, and Italian MRDV (maize rough dwarf virus), were highly conserved.

Journal Article↗

PubMed1988

Identification of the simian rotavirus SA11 genome segment 3 product.

Previous studies on rotavirus gene-coding assignments failed to clearly identify the simian rotavirus SA11 genome segment 3 protein product. This question was reexamined by using new conditions of electrophoresis with improved resolution of proteins in the high-molecular-weight range. Our results showed that the SA11 genome segment 3 codes for a protein with an apparent mol wt of 88,000. This protein normally comigrates with the protein product of genome segment 4. The gene 3 protein was located in viral core particles by comparing the electrophoretic patterns of [35S]methionine-labeled viral polypeptides from infected cells and from purified double-shelled, single-shelled, and core particles. To confirm the identity of the gene 3 product, we also studied two reassortant viruses in which genome segment 3 was reassorted from each of two parental virus strains (SA11 and NCDV). The gene 3 and gene 4 products of these viruses were identified by (i) their separation by two different polyacrylamide gel systems, (ii) their location in distinct particle types, (iii) their differential sensitivity to trypsin digestion, and (iv) their distinctive protease peptide maps. We propose that the genome segment 3 product be called VP3 and that the gene 4 product be named VP4 from now on.

Animals↗

PubMed1998

Development of a mouse model system, coding assignments and identification of the genome segments controlling virulence of African horse sickness virus serotypes 3 and 8.

Attenuated (att) and wild type (wt) strains of the nine AHSV serotypes were evaluated for virulence in adult Balb C mice. Although most were avirulent in this system, isolates of AHSV 1att, 3wt, 3att, 4wt, 5att, 7att and 8att caused some mortality when administered via an intranasal route. After plaque cloning, only the attenuated vaccine strain of AHSV 7att caused any mortality via an intravenous route. AHSV 3att and AHSV 8wt were virulent (V) and avirulent (AV) (respectively) in the mouse model and were selected as parental strains for production of genome segment reassortants. These progeny virus strains were plaque cloned, then characterised to identify the genome segments that influence virulence of AHSV in the mouse model. Three virulence phenotypes were observed: fully virulent (V); fully avirulent (A); and a novel intermediate virulence (N) not expressed by either parental strain. Genome segment 2 (encoding outer capsid protein VP2) from the avirulent parent appeared to have a controlling influence in production of the A phenotype. Reassortants with the V phenotype all contained segment 2 from the virulent parent, however in each case they also contained genome segments 5 and 10, also from AHSV 3 (V). Genome segments 5 and 10 encode the smaller outer capsid protein VP5 and the non structural proteins NS3/NS3a, respectively. A combination of genome segments 2, 5 and 6 from the avirulent parent and segment 10 from the virulent parent were found in each of the virus strains with the N phenotype. However, comparison of two reassortants (A79 and A790), which differ only in a single segment, showed that replacement of genome segment 10 from the avirulent parent with that from the virulent parent, conferred the N phenotype on A790.

African Horse Sickness↗

PubMed2006

Phylogenetic analysis reveals a correlation between the expansion of very virulent infectious bursal disease virus and reassortment of its genome segment B.

Infectious bursal disease virus (IBDV) is a birnavirus causing immunosuppressive disease in chickens. Emergence of the very virulent form of IBDV (vvIBDV) in the late 1980s dramatically changed the epidemiology of the disease. In this study, we investigated the phylogenetic origins of its genome segments and estimated the time of emergence of their most recent common ancestors. Moreover, with recently developed coalescence techniques, we reconstructed the past population dynamics of vvIBDV and timed the onset of its expansion to the late 1980s. Our analysis suggests that genome segment A of vvIBDV emerged at least 20 years before its expansion, which argues against the hypothesis that mutation of genome segment A is the major contributing factor in the emergence and expansion of vvIBDV. Alternatively, the phylogeny of genome segment B suggests a possible reassortment event estimated to have taken place around the mid-1980s, which seems to coincide with its expansion within approximately 5 years. We therefore hypothesize that the reassortment of genome segment B initiated vvIBDV expansion in the late 1980s, possibly by enhancing the virulence of the virus synergistically with its existing genome segment A. This report reveals the possible mechanisms leading to the emergence and expansion of vvIBDV, which would certainly provide insights into the scope of surveillance and prevention efforts regarding the disease.

Animals↗

PubMed1983

Nucleotide sequence of reovirus genome segment S3, encoding non-structural protein sigma NS.

This report describes the complete nucleotide sequence of human reovirus (Dearing strain) genome segment S3. Previous studies indicated that this RNA encodes the major non-structural viral polypeptide sigma NS, a protein that binds ssRNAs (Huisman & Joklik, Virology 70, 411-424, 1976) and has a poly(C)-dependent poly(G) polymerase activity (Gomatos et al., J. Virol. 39, 115-124, 1981). The genome segment consists of 1,198 nucleotides and possesses an open reading frame that extends 366 codons from the first AUG triplet (residues 28-30). There is no significant sequence homology between the plus strand of genome segment S3 and that of genome segment S2 determined previously (Cashdollar et al., PNAS 79, 7644-7648, 1982). However, S3 RNA has significant dyad symmetry and regions that can potentially hybridize (delta G = -26 KCal/mole) with S2 RNA. From the predicted amino acid sequence a possible secondary structure for sigma NS protein was determined. Structural features of reovirus RNA and sigma NS are discussed in relation to their role(s) in viral genome assembly.

Base Sequence↗

PubMed1998

Molecular epidemiology of African horse sickness virus based on analyses and comparisons of genome segments 7 and 10.

This paper describes a method to rapidly identify African horse sickness virus (AHSV), using a single tube reverse transcription polymerase chain reaction (PCR). This method was used to amplify cDNA copies of genome segments 7 and 10 from several different AHSV strains, of different serotypes, which were then analysed by sequencing and/or endonuclease digestion. AHSV VP7 (encoded by genome segment 7) is one of the two major capsid proteins of the inner capsid layer, forming the outer surface of the core particle. VP7 is highly conserved and is the major serogroup specific antigen common to all nine AHSV serotypes. Digestion of the 1179 bp cDNA with restriction enzymes, allowed differentiation of several strains of different serotypes and identified six distinct groups containing AHSV-1, 3, 6 and 8; AHSV-2; AHSV-4; AHSV-5; AHSV-7; and AHSV-9. Differences were detected between wild type viruses and vaccine strains that had been attenuated by multiple passage in suckling mouse brain or in tissue cultures. RFLP analysis was also used to study variation the 758 bp cDNA copies of AHSV genome segment 10, which encodes the two small non-structural membrane proteins NS3 and NS3a. In this way it was possible to distinguish each of the strains tested, except AHSV 4 (USDA) and AHSV 9 (USDA). However, these isolates could be distinguished by RFLP analysis of genome segment 7 cDNA. Using sequence analysis of genome segment 10 we were able to classify the virus isolates into three groups: AHSV-1, 2 and 8; AHSV-3 and 7; AHSV 4, 5, 6 and 9. These studies confirmed that the virus which first appeared in central Spain in July 1987, subsequently spread into northern Morocco in October 1989.

African Horse Sickness↗

PubMed1989

The outer capsid protein of rice dwarf virus is encoded by genome segment S8.

The nucleotide sequence of DNA complementary to the eighth largest (S8) of the 12 genome segments of rice dwarf virus was determined. This genome segment is 1424 nucleotides in length and has a single long open reading frame extending 1260 nucleotides from the first AUG triplet (residues 24 to 26). The predicted translational product comprises 420 amino acids and has an Mr of 46,422. The amino acid sequences of several peptide fragments of the major outer capsid protein were found to be contained in the predicted translational product of the above nucleotide sequence. This protein, previously reported to be 43K, is encoded by genome segment S8 and therefore renamed the 46K protein.

Amino Acid Sequence↗

PubMed1998

Directed changes in the number of double-stranded RNA genomic segments in bacteriophage phi6.

Bacteriophage Phi6 has a genome of three segments of double-stranded RNA. The segments are designated S, M, and L. Each segment has a unique packaging site, pac, near the 5' end of the plus strand. The plus strands of the segments are normally packaged in the order S, M, L. Chimeras of segment M and S in which segment M is at the 5' end of the plus strand can be stably incorporated into the virion; however, an independent segment S must be included along with normal segment L, even if it contains no active genes. A chimera of segment M and S in which segment S is at the 5' end of the plus strand can be stably incorporated into the virion along with normal segment L to form a two-segment genome. A chimera of segments S, M, and L in which the packaging sequence is that of S can also form a stable nonsegmented genome. These findings are consistent with a model that we have proposed for the packaging of the Phi6 genome.

Bacteriophage phi 6↗

PubMed1982

The double-stranded RNA genome segments of cytoplasmic polyhedrosis virus are independently transcribed.

The in vitro transcription of the genome segments of insect cytoplasmic polyhedrosis virus was examined for a possible mechanism of coordinate control. Initiation of the first transcription cycle, primed by preincubation in the absence of CTP, was synchronous for all 10 viral genome segments. The time required for the completion of each genome transcript was proportional to the chain length, and all 10 mRNA's were synthesized in molar amounts inversely proportional to their molecular weights. The rate of chain elongation for all mRNA's was approximately 6 nucleotides per s. The combined results indicate that each of the viral genome segments is transcribed independently and suggest that RNA chain elongation, not initiation, is rate limiting for cytoplasmic polyhedrosis virus mRNA synthesis in vitro.

Animals↗

PubMed1989

Nucleotide sequence of rice dwarf virus genome segment 9.

The complete nucleotide sequence of the phytoreovirus rice dwarf virus (RDV) genome segment 9 is presented. It consisted of 1305 nucleotides and had an open reading frame that codes for a putative polypeptide of 351 amino acids. Mr of the protein was calculated to be 38,598. The terminal nucleotides 5' GGUAAA--GAU 3' were the same as those of RDV genome segment 10. The fourth nucleotide from the 3' end of an expected conserved sequence was a C rather than the U found in the previously sequenced genome segment 10. A structure similar to the segment-specific inverted repeat of wound tumour virus was also found in the terminal region of segment 9.

Amino Acid Sequence↗

PubMed1990

Fine-mapping of DNA damage and repair in specific genomic segments.

The susceptibility of various genomic regions to DNA damage and repair is heterogeneous. While this can be related to factors such as primary sequence, physical conformation, and functional status, the exact mechanisms involved remain unclear. To more precisely define the key features of a genomic region targeted for these processes, a useful tool would be a method for fine-mapping gene-specific DNA damage and repair in vivo. Here, a polymerase chain reaction-based assay is described for measuring DNA damage and repair in small (less than 500 bp) genomic segments of three transcriptionally active but functionally distinct loci (rearranged immunoglobulin heavy chain variable region [Ig VDJ], low-density lipoprotein receptor gene, and N-ras proto-oncogene) in human tonsillar B lymphocytes. Analysis of ultraviolet (254 nm)-induced DNA damage revealed single-hit kinetics and a similar level of sensitivity (D50% approximately 6000 joule/m2) in all three regions, indicating that a single photoproduct was sufficient to fully block PCR amplification. A similar time period per unit length was required for repair of this DNA damage (average t1/2 per fragment length = 23.5 seconds per bp). DNA damage and repair was also detectable with the base adducting agent, 4-nitroquinoline-1-oxide. However, in this case IgVDJ differed from segments within the other two loci by its relative inaccessibility to alkylation. This assay thus permits high-resolution mapping of DNA damage and repair activity.

B-Lymphocytes↗