Using graphic warning labels to counter effects of social cues and brand imagery in cigarette advertising.

Exposure to cigarette advertising can increase the likelihood of youth smoking initiation and may encourage people who already smoke to continue. Requiring prominent, graphic warning labels could reduce these effects. We test whether graphic versus text-only warning labels in cigarette advertisements influence cognitive and emotional factors associated with youth susceptibility to smoking and adult intentions to quit. We conducted two randomized, between-subjects experiments with middle-school youth (n = 474) and adult smokers (n = 451). Both studies employed a two (graphic or text-only warnings) by two (advertisements with social cues or brand imagery) factorial design with a fifth, offset control group (social cue advertisements with the current US Surgeon General's Warning). Graphic warnings outperformed text-only warnings in reducing visual attention to the advertisement, generating visual attention to the warning and arousing more negative affect. Graphic warnings also reduced the appeal of cigarette brands among youth relative to social cue advertisements with the Surgeon General's warnings. None of the warnings (graphic or textual) influenced health risk beliefs. Graphic warning labels on cigarette advertisements appear to have effects similar to those observed on cigarette packs in previous work, with an added benefit of reducing cigarette brand appeal among youth.

Characterization of the transcription map and Rev activity of a highly cytopathic feline immunodeficiency virus.

A highly cytopathic feline immunodeficiency virus, FIV-Oma, was previously isolated from a nondomestic cat. In this report, we describe experiments to characterize its transcription map and examine its Rev activity. The temporal progression of viral gene expression is similar to that of HIV-1. The splicing pattern of viral transcripts was determined by sequence analysis of RT-PCR-amplified viral cDNAs. In vitro transcription and translation of two putative rev cDNAs revealed that they encode at least one 22-kDa protein. The Rev-responsive element (RRE) of FIV-Oma, identified by computer-assisted RNA secondary structure analysis, was inserted into the intron of an HIV-1-derived reporter plasmid and used in a transient transfection assay for Rev activity. Cotransfection of the RRE construct with the two rev cDNA clones significantly increased the expression of the reporter gene linked to the RRE, indicating that both transcripts encode an active Rev protein. The Rev activity of FIV-Oma is 5 to 8 times higher than that of a domestic cat FIV isolate, FIV-PPR. Our experiments also demonstrate the heterologous interaction of FIV-PPR Rev with the FIV-Oma RRE, even though the RREs of the two viruses have very little nucleotide sequence identity.

Proviral organization and sequence analysis of feline immunodeficiency virus isolated from a Pallas' cat.

The nucleotide sequence and genomic organization have been determined for a highly cytopathic feline immunodeficiency virus (FIV) isolated from a Pallas' cat. The 9747-bp provirus of this virus, FIV-Oma, has typical lentivirus organization with LTRs, gag, pol, and env open reading frames (ORFs), putative vif and rev ORFs, and an ORF similar to ORF2/ORFA of domestic cat FIV isolates. Although the FIV-Oma provirus is 300 to 600 bp longer than other FIV proviruses, these additional bases are distributed throughout the genome. Phylogenetic analysis of a conserved region of the pol gene suggests that FIV-Oma is more closely related to some of the puma and lion lentiviruses than it is to domestic cat FIV isolates; however, many regions of the genome exhibit extensive nucleotide sequence divergence. None of the eight molecular proviral clones isolated from a genomic library are infectious, but we have constructed an infectious, cytopathic clone of FIV-Oma from subcloned and PCR-amplified fragments of these proviral clones. This clone will be useful for identifying the genetic determinants of FIV-Oma's biological activities.

Sequence analysis and transcriptional activity of the LTR of OLV-CU1, a North American ovine lentivirus.

Although ovine lentiviruses have been described in the United States since the early part of this century, North American strains of sheep lentiviruses remain relatively uncharacterized at the molecular level. The LTR of a North American ovine lentivirus, OLV-CU1, was found to be closely related at the molecular and functional levels to visna virus, the Icelandic ovine lentivirus. Sequence analysis of the LTR revealed high identity to other ovine and caprine lentiviruses in key regulatory elements of the upstream promoter region (-25 to -115). However, the R region of the LTR was much less homologous. Transcriptional control of OLV-CU1 in transient transcriptional assays required a conserved putative AP-4 region and possibly an AP-1 like element in the upstream promoter region for moderate to high levels of transcription, much like visna virus. In contrast to visna virus, the downstream region beyond the transcriptional start site was required for virus-specific transactivation.

Isolation of a highly cytopathic lentivirus from a nondomestic cat.

A feline immunodeficiency virus-like virus (FIV-Oma) isolated from a Pallas' cat (Otocolobus manul) is highly cytopathic in CrFK cells, in contrast to the chronic, noncytolytic infection established by an FIV isolate from a domestic cat (FIV-Fca). The virions have typical lentivirus morphology, density, and magnesium-dependent reverse transcriptase activity. The major core protein is antigenically cross-reactive with that of FIV-Fca; however, FIV-Oma transcripts do not cross-hybridize with FIV-Fca. A conserved region of the FIV-Oma pol gene has 76 to 80% nucleic acid identify with the corresponding pol regions of other feline lentiviruses and 64 to 69% identity with those of human, ovine, and equine lentiviruses.

Characterization of a New York ovine lentivirus isolate.

A lentivirus has been isolated from a Finnish ewe with ovine progressive pneumonia in a closed upstate New York flock. We demonstrated that the virus, designated ovine lentivirus strain CU1 (OLV-CU1), is biologically, biochemically and molecularly related to, but distinct from, previously described sheep and goat lentiviruses. Nine of 32 ewes (from the affected flock) with precipitating antibodies for ovine lentivirus also produced antibodies that were able to neutralize the infectivity of OLV-CU1. The virus replicated in cultured sheep fibroblasts and caused the formation of large multi-nucleated cells. OLV-CU1-specific RNA transcripts found in infected cells and virion antigenic proteins were similar to those of other small ruminant lentiviruses. However, the virus was distinguished from other isolates at the DNA level by nucleic acid hybridization, restriction endonuclease mapping and partial sequencing of the virus genome.

Suppression of lymphocyte blastogenesis to mitogens in cats experimentally infected with feline immunodeficiency virus.

Lymphocytes from normal cats or cats experimentally infected with feline immunodeficiency virus (FIV) were stimulated with phytohemagglutinin, pokeweed mitogen, or concanavalin A. Lymphocytes from infected cats had lower responses than those from uninfected cats. These results support the hypothesis that FIV induces immunosuppression.

The genomic DNA organisation and evolution of a retrovirus-transmissible family of mouse (VL30) genetic elements.

The sequence organisation of endogenous VL30 elements in the mouse genome was investigated by using a cloned representative of a retrovirus-transmissible VL30 cDNA. The majority of dispersed VL30 sequences could be assigned to a proviral-like structure 5.2-5.3 kbp long and bounded by long terminal repeats (LTRs). The existence of a hierarchy of evolutionarily conserved elements was rather limited and sequence heterogeneity between different elements was randomly distributed. However, the retrovirus-transmissible class of VL30 element was found to represent a distinct minority subgroup distinguishable by restriction sites and size (4.6-4.9 kbp long). Analysis of sequence conservation showed that VL30 elements display a more rapid turnover than endogenous murine leukaemia virus-related proviral sequences, and that VL30 LTRs show the most limited evolutionary distribution. Although discrete subsets of VL30 unique sequence were conserved in different rodents, the location of conserved regions was found to be variable, arguing against the presence of a functionally conserved protein coding region. These observations support the hypothesis that high frequency recombination, probably occurring during reverse transcription and the accompanying processes of duplicative transposition and amplification, have been a major determinant in the mode of evolution of the VL30 gene family.

The homologue of the Duchenne locus is defective in X-linked muscular dystrophy of dogs.

Duchenne muscular dystrophy (DMD) is the most common and the most severe of the muscular dystrophies in man. It is inherited as an X-linked recessive trait and is characterized by ongoing necrosis of skeletal muscle fibres with regeneration and eventually fibrosis and fatty infiltration. Although the gene and gene product which are defective in DMD have recently been identified, the pathogenesis of the disease is still poorly understood. A myopathy has been described in the dog which has been shown to be inherited as an X-linked trait and which is therefore a potential model of the human disease. We have studied the phenotypic expression of the disease, canine X-linked muscular dystrophy (CXMD), and have examined the molecular relationship between it and DMD. We report here that dogs with CXMD faithfully mimic the phenotype of Duchenne muscular dystrophy and that they lack the Duchenne gene transcript and its protein product, dystrophin.

Mouse and rat cells undergo rapidly inducible changes in polypeptide secretion following infection with Kirsten murine sarcoma virus.

Characteristic changes in the secreted polypeptides of Kirsten murine sarcoma virus (KiMSV) transformed mouse and rat cell lines could be detected 48 hours after infection of phenotypically normal cells with this virus and correlated with detection of the KiMSV encoded polypeptide p21.

Expression and transmission of a rodent retrovirus-like (VL30) gene family.

A transcriptionally active sub-set of the dispersed mouse VL30 family of proviral genetic elements was shown to be highly transmissible as a murine leukaemia virus pseudotype. Newly acquired VL30 proviruses (present at 1 to 2 copies per cell) were shown to be transcriptionally active. These data substantiate the hypothesis that this process of duplicative transposition may have played a major role in the evolution of the gene family and also demonstrate that VL30 elements would be capable of mediating oncogene activation by a promoter-insertion-type mechanism during leukaemia virus-induced tumourgenesis.

Loss of epidermal growth factor receptors and release of transforming growth factors do not correlate with sarcoma virus-transformation in clonally-related NIH/3T3-derived cell lines.

Transformation of NIH/3T3 cells by Kirsten murine sarcoma virus (MSV) caused a dramatic reduction in the number of cell-surface receptors for epidermal growth factor (EGF). However, the number of EGF receptors remained at a very low level in a non-tumourigenic revertant cell line isolated from the virus-transformed cells, indicating that an increase in EGF receptors is not a requirement for the phenotypic reversion of Kirsten MSV-transformed 3T3 cells. Serum-free conditioned medium from normal and virus-transformed cell lines contained similar amounts of cell growth-promoting activity as assayed by the ability to stimulate DNA synthesis in quiescent Swiss 3T3 cell cultures. However, the concentrated conditioned medium from these cell lines showed no evidence of beta-transforming growth factor (TGF) activity as assayed by promotion of anchorage-independent growth of untransformed normal rat kidney (NRK) fibroblasts in agarose. The cellular release of alpha-TGF activity was assayed by measuring the ability of concentrated conditioned medium to inhibit the binding of 125I-EGF to Swiss 3T3 cells. Conditioned medium protein from the virus-transformed cell line inhibited 125I-EGF binding but only to the same extent as conditioned medium protein prepared from the untransformed cell line. The alpha-TGF secretion by these cell lines was estimated to be 30-45-fold lower than the level of alpha-TGF released by a well-characterized alpha-TGF-producing cell line (3B11). These results suggest that the induction of TGF release is not a necessary event in the transformation of NIH/3T3 cells by Kirsten MSV.

Genesis of Kirsten murine sarcoma virus: sequence analysis reveals recombination points and potential leukaemogenic determinant on parental leukaemia virus genome.

The genome of Kirsten murine sarcoma virus was formed by recombination between Kirsten murine leukaemia virus sequences, and rat sequences derived from a retrovirus-like '30S' (VL30) genetic element encompassing the Kras oncogene. Using cloned DNAs we have determined the nucleotide sequences of the long terminal repeats and adjacent regions, extending across the points of recombination on the sarcoma and leukaemia virus genomes. Our results suggest that discrete regions of homology and other cryptic sequence features, may have constituted recombinational hot-spots involved in the genesis of the Kirsten murine sarcoma virus genome. We have also compared the sequence of the Kirsten murine leukaemia virus p15 env and adjacent long terminal repeat with the corresponding regions of the AKV and Gross A murine leukaemia virus genomes. This comparison has identified a leukaemogenic determinant in the U3 domain of the long terminal repeat, possibly within a enhancer-like sequence element.

Expression of Kirsten murine sarcoma virus in transformed nonproducer and revertant NIH/3T3 cells: evidence for cell-mediated resistance to a viral oncogene in phenotypic reversion.

Expression of the provirus in a clonally related series of Kirsten murine sarcoma virus-transformed NIH/3T3 nonproducer cell lines was examined at both the transcriptional and translational levels. All cells expressed high levels of genome-sized viral RNA with little variation between cell lines despite differences in provirus integration site and copy number. Expression of K-ras RNA was estimated to be at least 10- to 20-fold higher than that of the mouse cellular homolog of the viral transforming gene. Levels of the virus-coded transforming protein, p21, were similarly elevated, with little variation between nonproducer cells. In two revertant cell lines containing a normal provirus and a rescuable transforming gene, no impairment in expression at either the transcriptional or translational level was found. After superinfection with Kirsten murine sarcoma virus, one revertant became more tumorigenic, whereas the other remained nontumorigenic. These results show that cell transformation by Kirsten murine sarcoma virus is invariably associated with elevated expression of the virus-coded oncogene and that one of the revertants is resistant to the action of the viral transforming gene.

Unusual long terminal repeat sequence of a retrovirus transmissible mouse (VL 30) genetic element: identification of functional domains.

We have determined the nucleotide sequence and mapped the transcriptional boundaries in the long terminal repeats (LTRs) and adjacent regions of a retrovirus transmissible virus-like 30S ( VL30 ) mouse genetic element. The 572 base pair LTRs contain transcriptional regulatory sequences and are bounded by short imperfect repeats, with a minus strand tRNAgly primer binding site and a purine rich plus strand primer site flanking each of their inner boundaries. The 3' end of each LTR consists of an extensive 80 base pair redundancy of tRNA primer site and inverted repeat sequences while 41 and 47 base pair imperfect tandem repeats are present between the 5' capping site and the putative polyadenylation signal. Comparison with other retrovirus-like LTR sequences suggests possible modes of recombination that could occur between VL30 and other genetic elements.

Characterization and cloning of the Kirsten murine leukemia virus genome.

We have characterized the intracellular and circular unintegrated proviral DNA species of Kirsten murine leukemia virus by restriction mapping using the Southern blotting technique. These studies show the 8.5 kilobase pair genome to possess long terminal repeats (0.5 kilobase pairs in length) which are indistinguishable from those of the derivative Kirsten murine sarcoma virus. In addition, we have identified a 3'-located region in Kirsten murine leukemia virus which is very similar to the putative leukemogenic region of Gross murine leukemia virus. We also report the cloning of the leukemia virus genome using DNA obtained from the endogenous reverse transcriptase reaction of detergent disrupted virions.

Integration of proviral DNA in Kirsten murine sarcoma virus-infected mouse fibroblasts.

The structure and sites of integration of proviral DNA were studied in 19 clonally related Kirsten murine sarcoma virus-transformed non-producer NIH/3T3 cell lines. The majority of these cell lines contained a single provirus, inserted colinearly with respect to unintegrated linear viral DNA, and lacking detectable methylation at MspI/HpaII sites. Although all proviruses were located at distinct integration sites in the host cell genome, the possible existence of similarities between some adjacent host flanking sequences, suggested from restriction mapping data, could not be ruled out. In three phenotypically reverted cell lines no change in either proviral DNA or adjacent host flanking sequences was detectable. In addition, the revertant proviruses lacked detectable methylation at MspI/HpaII sites. These findings suggest that changes in cellular function(s) may be responsible for loss of transformed phenotype in these cells.

Virus-like 30S RNA is selectively packaged by Kirsten leukemia virus in virions of smaller size class. Brief report.

Virus particles containing Kirsten murine leukemia virus 38 S RNA and a pseudotyped virus-like cellular 30 S RNA, were partially separable. This provides a means for enriching a population of virions for those containing 30 S RNA.

A novel approach to cloning transcriptionally active retrovirus-like genetic elements from mouse cells.

A family of dispersed, moderately repeated mouse genetic elements is expressed as retrovirus-like 30S RNA species (VL30 RNA) which can be transmitted to other cells when packaged as a pseudovirion complex by murine leukemia viruses (MuLV). Using the endogenous reverse transcriptase reaction of VL30 RNA-containing MuLV particles, full-length VL30 DNA was synthesized and cloned in pAT153. Analysis of a number of clones identified long terminal repeat structures (LTRs) characteristic of retrovirus proviruses and transposable genetic elements. Whilst the unique region of all clones was identical, the LTRs displayed some heterogeneity. Comparison of the unique region of cloned VL30 DNA with mouse genomic VL30 sequences showed the retrovirus-derived clones to be encoded by only a few members of the divergent VL30 gene family. These findings thus demonstrate a method for cloning a defined sub-class of retrovirus-like cellular genes which are both transcriptionally active and transmissible by a retrovirus.

Biochemical characterization of a deleted Kirsten sarcoma virus genome. Brief report.

A Kirsten sarcoma virus transformed mouse cell line was found to contain a deleted provirus. RNA from the virus produced by these cells was characterized by hybridisation protection oligonucleotide fingerprinting and was found to be a simple deletion.