RNA and Cell Volume Distribution Analysis of VEE Virus Infected Mosquito Cell Cultures

The subject of this investigation is the Aedes albopictus (Aal) cell line infected with Venezuelan equine encephalomyelitis (VEE) virus, a group A arbovirus. This cell-virus system is an ideal choice for both types of analysis. Certain of the group A arboviruses have been looked at as models for ribovirus replication and thus have several advantages for an FNA study. The genome is a single strand of RNA containing only about a dozen cistrons, the virions contain only one polypeptide in the nucleocapsid and one in the envelope, and infectious RNA is easily isolated and titrated by sensitive plaque assay. In addition, VEE infected Aal cells exhibit a noncytocidal response and are thus ideally suited for the volume distribution profile study. The A. albopictus cell s were employed for several reasons. First, the A albopictus mosquito can function as a vector for the VEE virus. Second, the A. albopictus cell line was a recent development and little was known about virus replication in these cells. Third, these cells do not exhibit CPE upon infection with VEE. Fourth, this cell-virus system provided a model by which other noncytocidal cell-virus systems could be compared. There is a definite need for the RNA analysis. The mechanism of single-strand RNA virus replication has been under intensive study in recent years; the results of which could possibly lead to the means by which RNA virus infections could be prevented. Certainly, some of the most destructive viruses, in terms of human misery and economic considerations, are the single-strand RNA viruses. These include polio, rabies, yellow fever, influenza, mumps, measles, and encephalitis viruses. In addition, a large number of the known animal cancer viruses are single-strand RNA viruses. Virtually all the experimental evidence supporting the current hypothesis of ribovirus replication is based on virus infections in cell lines exhibiting cytocidal response. The function of the RNA analysis in this investigation is to determine if the mode of ribovirus replication in cells exhibiting the noncytocidal response is similar to the mechanism of ribovirus replication in cells exhibiting a cytocidal response. In order to accomplish this goal, infected Aal cells were treated with Actinomycin D to inhibit DNA dependent RNA replication, treated with tritiated uridine supplemented media, and allowed to incubate. At various times post-infection, the virus-specific RNA was extracted from the cells with phenol-SDS. The different types of RNA isolated was determined by polyacrylamide gel electrophoresis of the molecules. The gels were then sectioned, and each section assayed for radioactive RNA species

Resolution of a common RNA sequencing ambiguity by terminal deoxynucleotidyl transferase

One of the more common ambiguities which arise when using reverse transcriptase and dideoxynucleotide-chain termination to sequence RNA is a radioactive band of cDNA that extends over all four lanes on a sequencing gel. The adjacent sequences both above and below the band are not affected. Assuming then, that these ambiguities are caused by the termination of the DNA polymerase activity of reverse transcriptase for reasons other than the insertion of a dideoxynucleotide in the growing cDNA chain, terminal deoxynucleotidyl transferase should be able to continue to add deoxynucleotides to these products after the sequencing reaction is complete. It does, clearing the improperly terminated cDNA from these pileup sites, revealing the correct sequence. This technique can also be used to identify the template RNA's 5'-terminal base, although far more units of terminal deoxynucleotidyl transferase are required.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26058/1/0000132.pd

Identification of sequence changes in the cold-adapted, live attenuated influenzavaccine strain, A/Ann Arbor/6/60 (H2N2)

Nucleotide sequences have been obtained for RNA segments encoding the 13132, P131, PA, NP, M1, M2, NS1, and NS2 proteins of the influenza A/Ann Arbor/6/60 (H2N2) wild-type (wt) virus and its cold-adapted (ca) derivative that has been used for preparing investigational live attenuated vaccines. Twenty-four nucleotide differences between the ca and wt viruses were detected, of which 11 were deduced to code for amino acid substitutions in the ca virus proteins. One amino acid substitution each was predicted for the PB2, M2, and NS1 proteins. Two amino acid substitutions were predicted for the NP and the PA proteins. Four substitutions were predicted for the PB1 protein. The biological significance of mutations in the PB2, PB1, PA, and M2 genes of the ca virus is suggested by currently available genetic data, a comparison with other available influenza gene sequences, and the nature of the predicted amino acid changes. In addition, the sequence data confirm the close evolutionary relationship between the genomes of influenza A (H2N2) and influenza A (H3N2) viruses.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27049/1/0000039.pd

Sequence comparison of wild-type and cold-adapted B/Ann Arbor/1/66 influenza virus genes

Consensus sequences for both wt and ca B/Ann Arbor/1 /66 viral PB2, PB1, PA, NP, M, and NS genes were directly determined from vRNA using a combination of chemical and chain-termination sequencing methods. There were 105 sites of difference between the wt and ca sets of these six RNA genes. The differences resulted in 26 amino acid substitutions distributed over the six proteins. The sequence changes were compared to the sequences of other known influenza type B wt viruses to pinpoint those changes that were unique to the ca B/Ann Arbor/1/66 virus. Of the 26 amino acid differences, only 11 were unique to the cold-adapted virus. These unique sites were distributed among five of the six genes. The NS protein had no amino acid substitutions. The sequence changes are discussed in terms of their probable mode of origin and selection, and in terms of their importance to the cold-adapted, temperature-sensitive, and attenuation phenotypes of ca B/AA/1 /66 virus. The sequence and organization of the PB2 gene and predicted protein are also given. The PB2 gene was 2396 nucleotides long, and it encoded a predicted protein of 770 amino acids with a molecular weight of 88,035 Da for the wt virus and 88,072 Da for the ca virus. Both proteins were predominantly hydrophilic, and each had an overall charge of +24.5 at pH 7.0.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27362/1/0000387.pd

Influenza in Migratory Birds and Evidence of Limited Intercontinental Virus Exchange

Migratory waterfowl of the world are the natural reservoirs of influenza viruses of all known subtypes. However, it is unknown whether these waterfowl perpetuate highly pathogenic (HP) H5 and H7 avian influenza viruses. Here we report influenza virus surveillance from 2001 to 2006 in wild ducks in Alberta, Canada, and in shorebirds and gulls at Delaware Bay (New Jersey), United States, and examine the frequency of exchange of influenza viruses between the Eurasian and American virus clades, or superfamilies. Influenza viruses belonging to each of the subtypes H1 through H13 and N1 through N9 were detected in these waterfowl, but H14 and H15 were not found. Viruses of the HP Asian H5N1 subtypes were not detected, and serologic studies in adult mallard ducks provided no evidence of their circulation. The recently described H16 subtype of influenza viruses was detected in American shorebirds and gulls but not in ducks. We also found an unusual cluster of H7N3 influenza viruses in shorebirds and gulls that was able to replicate well in chickens and kill chicken embryos. Genetic analysis of 6,767 avian influenza gene segments and 248 complete avian influenza viruses supported the notion that the exchange of entire influenza viruses between the Eurasian and American clades does not occur frequently. Overall, the available evidence does not support the perpetuation of HP H5N1 influenza in migratory birds and suggests that the introduction of HP Asian H5N1 to the Americas by migratory birds is likely to be a rare event

A novel retinoblastoma therapy from genomic and epigenetic analyses.

Retinoblastoma is an aggressive childhood cancer of the developing retina that is initiated by the biallelic loss of RB1. Tumours progress very quickly following RB1 inactivation but the underlying mechanism is not known. Here we show that the retinoblastoma genome is stable, but that multiple cancer pathways can be epigenetically deregulated. To identify the mutations that cooperate with RB1 loss, we performed whole-genome sequencing of retinoblastomas. The overall mutational rate was very low; RB1 was the only known cancer gene mutated. We then evaluated the role of RB1 in genome stability and considered non-genetic mechanisms of cancer pathway deregulation. For example, the proto-oncogene SYK is upregulated in retinoblastoma and is required for tumour cell survival. Targeting SYK with a small-molecule inhibitor induced retinoblastoma tumour cell death in vitro and in vivo. Thus, retinoblastomas may develop quickly as a result of the epigenetic deregulation of key cancer pathways as a direct or indirect result of RB1 loss

Cloning of the human equilibrative, nitrobenzylmercaptopurine riboside (NBMPR)-insensitive nucleoside transporter ei by functional expression in a transport-deficient cell line

Mammalian cells obtain nucleic acid precursors through the de novo synthesis of nucleotides and the salvage of exogenous nucleobases and nucleosides. The first step in the salvage pathway is transport across the plasma membrane. Several transport activities, including equilibrative and concentrative mechanisms, have been identified by their functional properties. We report here the functional cloning of a 2.6-kilobase pair human cDNA encoding the nitrobenzylmercaptopurine riboside (NBMPR)- insensitive, equilibrative nucleoside transporter ei by functional complementation of the transport deficiency in a subline of CEM human leukemia cells. Expression of this cDNA conferred an NBMPR-insensitive, sodium-independent nucleoside transport activity to the cells that exhibited substrate specificity and inhibitor sensitivity characteristic of the ei transporter. The cDNA contained a single open reading frame that encoded a 456-residue protein with 11 potential membrane-spanning regions and two consensus sites for N-glycosylation in the first predicted extracellular loop. The predicted protein was 50% identical to the recently cloned human NBMPR-sensitive, equilibrative nucleoside transporter ENT1 and thus was designated ENT2. Surprisingly, the carboxyl-terminal portion of the ENT2 protein was nearly identical to a smaller protein in the GenBank(TM) data base (human HNP36, 326 residues) that has been identified as a growth factor- induced delayed early response gene of unknown function. Comparison of the ENT2 and HNP36 nucleotide sequences suggested that HNP36 was translated from a second start codon within the ENT2 open reading frame. Transient expression studies with the full-length ENT2 and a 5\u27-truncated construct that lacks the first start codon (predicted protein 99% identical to HNP36) demonstrated that only the full-length construct conferred uridine transport activity to the cells. These data suggest that the delayed early response gene HNP36 is a truncated form of ENT2 and that the full-length open reading frame of ENT2 is required for production of a functional plasma membrane ei transporter

Nucleotide sequences of the PA and PB1 genes of B/Ann Arbor/1/66 virus: comparison with genes of B/Lee/40 and type A influenza viruses

The complete sequences of the PA and PB1 genome RNA segments of B/Ann Arbor/1/66 virus have been determined. The PA vRNA is 2308 bases long. Its complementary RNA has a single open reading frame of 2187 bases, capable of encoding a PA protein of 726 amino acids with a molecular weight of 83,175 Da. The predicted PA polypeptide has an overall net charge of -- 7.5 at pH 7.0. The PB1 vRNA is 2369 bases long. Its complementary RNA has a single open reading frame of 2277 bases, capable of encoding a PB1 protein of 752 amino acids with a molecular weight of 84,332 Da. The predicted PB1 polypeptide has an overall net charge of + 18.5 at pH 7.0. Sequence homology comparisons of the PA and PB1 polypeptides from B/Ann Arbor/1/66 virus to the PA and PB1 polypeptides of type A influenza virus reveal respective homologies of approximately 38 and 60%. This high cross-type homology (61%) was previously reported for the PB1 protein of B/Lee/40 virus (Kemdirim et al., 1986). The cross-type homology for the PA protein is similar to that of other non-polymerase proteins, but is substantially lower than that seen for the PB1 protein. Thus, the high cross-type homology that exists for the PB1 gene does not appear to be a characteristic of all polymerase genes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26645/1/0000187.pd