Structural Characteristics and Nucleotide Sequence Analysis of Genomic RNA from RD-114 Virus and Feline RNA Tumor Viruses

The results of molecular hybridization experiments have demonstrated that the RNA genome of RD-114 virus has extensive nucleotide sequence homology with the RNA genome of Crandell virus, an endogenous type C virus of cats, but only limited homology with the RNA genomes of feline sarcoma virus and feline leukemia virus. The genomic RNAs of RD-114 virus and Crandell virus also had identical sedimentation coefficients of 50S. A structural rearrangement of genomic RNA did not exist within released RD-114 virions, whereas a structural rearrangement of genomic RNA did occur within feline sarcoma virions and feline leukemia virions after release from virus-producing cells.     

Structure, subunit composition, and molecular weight of RD-114 RNA.

The properties and subunit composition of the RNA extracted from RD-114 virions have been studied. The RNA extracted from the virion has a sedimentation coefficient of 52S in a nondenaturing aqueous electrolyte. The estimated molecular weight by sedimentation in nondenaturing and weakly denaturing media is in the range 5.7 X 10(6) to 7.0 X 10(6). By electron microscopy, under moderately denaturing conditions, the 52S molecule is seen to be an extended single strand with a contour length of about 4.0 mum corresponding to a molecular weight of 5.74 X 10(6). It contains two characteristic secondary structure features: (i) a central Y- or T-shaped structure (the rabbit ears) with a molecular weight of 0.3 X 10(6), (ii) two symmetreically disposed loops on each side of and at equal distance from the center. The 52S molecule consists of two half-size molecules, with molecular weight 2.8 X 10(6), joined together within the central rabbit ears feature. Melting of the rabbit ears with concomitant dissociation of the 52S molecule into subunits, has been caused by either one of two strongly denaturing treatments: incubation in a mixture of CH3HgOH and glyoxal at room temperature, or thermal dissociation in a urea-formamide solvent. When half-size molecules are quenched from denaturing temperatures, a new off-center secondary structure feature termed the branch-like structure is seen. The dissociation behavior of the 52S complex and the molecular weight of the subunits have been confirmed by gel electrophoresis studies. The loop structures melt at fairly low temperatures; the dissociation of the 52S molecule into its two subunits occurs at a higher temperature corresponding to a base composition of about 63% guanosine plus cytosine. Polyadenylic acid mapping by electron microscopy shows that the 52S molecule contains two polyadenylic acid segments, one at each end. It thus appears that 52S RD-114 RNA consists of two 2.8 X 10(6) dalton subunits, each with a characteristic secondary structure loop, and joined at the 5' ends to form the rabbit ears secondary structure feature. The observations are consistent with but do not require the conclusion that the two 2.8 X 10(6) dalton subunits of 52S RD-114 RNA are identical.     

Structure and Molecular Length of the Large Subunits of RD-114 Viral RNA

By electron microscopy, the large subunits of RD-114 RNA have a molecular weight of 5.0 x 10(6); they all have a characteristic secondary structure feature close to the middle.     

Isolation of an RD-114-Like Oncornavirus from a Cat Cell Line

A clone of cells derived from a continuous line of cat cells (CCC) spontaneously produced an RNA C-type virus (CCC virus) which did not have the group-specific antigen of the standard strains of feline leukemia viruses but did have that of the RD-114 virus. Single-hit infection of a virus yielding CCC cell with only the feline leukemia virus pseudotype of murine sarcoma virus [MSV(FeLV)] resulted in the release of a pseudotype of MSV coated with the CCC virus envelope. Host range, transmission of virus, helper functions, interference properties, and specific neutralization showed that the CCC and the RD-114 isolates as well as their respective MSV pseudotypes are closely similar if not identical. Parental, virus-negative cells frozen before the existence of RD-114 were chemically induced to yield CCC-like virus de novo. Infection of susceptible human cells with the chemically induced virus resulted in interference with the CCC virus pseudotype of MSV but not with the FeLV pseudotype of MSV.     

Expression of interferon-inducible genes in RD-114 cells.

RD-114 is a cell line which is partially responsive to interferon (IFN). Although both IFN-alpha and IFN gamma inhibit production of the resident retrovirus, they do not inhibit replication of other viruses, such as vesicular stomatitis virus and encephalomyocarditis virus, in these cells. In the studies reported here, we studied the characteristics of induction of seven IFN-inducible mRNAs in RD-114 cells. We observed that mRNAs 561, 6-16, 1-8, 2A, and 6-26 have similar induction characteristics in RD-114 cells and in HeLa cells, a fully responsive line. mRNA 2'-5'-oligo-adenylate synthetase (2-5(A) synthetase), however, was induced more efficiently by IFN-alpha in HeLa cells than in RD-114 cells. The same was true for the induction of metallothionein II mRNA by IFN-gamma. However, the latter mRNA was induced equally strongly in both lines when ZnCl2 was used as the inducer, suggesting that the gene is not defective in RD-114 cells. Although IFN-alpha induced 2-5(A) synthetase mRNA poorly and IFN-gamma did not induce it at all in these cells, a mixture of IFN-alpha and IFN-gamma induced this mRNA quite effectively, to a level of induction comparable to that in HeLa cells. Only 1 U of IFN-gamma per ml was sufficient to elicit this synergism, and the data suggested that an IFN-gamma-inducible protein was needed for this process. Induction of mRNA 561 by IFN-alpha in RD-114 cells, unlike that in HeLa cells, did not need ongoing protein synthesis. Once induced, this mRNA turned over rapidly in both cell lines, and this turnover could be slowed down by inhibiting protein synthesis in either cell line. IFN-induced mRNAs, such as 561 and 1-8, were polysome associated in IFN-treated RD-114 cells, suggesting that they were actively translated. Therefore, it is unlikely that the products of these IFN-inducible genes, by themselves, mediate the inhibition of replication of those viruses which are insensitive to IFN action in RD-114 cells.     

Nucleotide composition of RNA by polyacrylamide gel electrophoresis

A procedure using polyacrylamide gel electrophoresis has been devised for determining the nucleotide composititon of small amounts (50 μg) of RNA. The procedure permits analysis of the separated nucleotides in the gel matrix, thereby avoiding sample manipulation and allowing for greater reproducibility.     

Experimental infection of dogs with a feline endogenous retrovirus RD-114

Background  

The feline endogenous retrovirus RD114 is contained in the genome of cats. The virus may contaminate live canine vaccines based on cultured feline cells. The in vivo infectivity, acute and subacute pathogenicity, and viral proliferation of the RD114 virus were evaluated by experimental infection of dogs.     

Genetic mapping of endogenous RD-114 retroviral sequences of domestic cats.

The RD-114 family of endogenous retroviral sequences in domestic cats has been shown to consist of approximately 20 copies of genetically divergent virogenes per haploid genome. The chromosomal localization for four endogenous sequences (RDV1-4) was accomplished by correlating the occurrence of specific feline chromosomes with diagnostic viral DNA fragments in a panel of cat X rodent somatic cell hybrids. Analysis of the hybrid panel revealed that endogenous RD-114 sequences are dispersed on multiple cat chromosomes, that certain proviral segments are polymorphic with respect to the presence or absence of virus, and that a restriction fragment characteristic of inducible RD-114 resides on a single feline chromosome (B3), probably at a single locus.     

Heteroduplex study of the sequence relations between RD-114 and baboon viral RNAs.

The regions of sequence homology and nonhomology between the RNA genomes of RD-114 and baboon endogenous type C viruses have been mapped by an electron microscope heteroduplex study. Short complementary DNA (cDNA) copies (approximately 150 to 200 nucleotides in length) of RD-114 RNA were prepared by an endogenous synthesis; labels of polydeoxythymidylic acid [poly(dT)] were attached to the 3' ends of the cDNA molecules by a reaction catalyzed by deoxynucleotidyl terminal transferase. The cDNA-poly(dT) was hybridized to RD-114 RNA and to baboon viral RNA dimer (50 to 70S) units, and the position- of the poly(dT) labels were observed by electron microscopy. With RD-114, labels were distributed uniformly along the genome. With baboon virus RNA (monomer length, 9.5 kilobases [kb]), the regions of high homology with RD-114 cDNA were observed to lie in the intervals from 1.5 to 2.5 kb and from 3.7 to 5.5 kb from the 5' end. The relations of these heteroduplex maps to the known antigenic similarities and differences among the several viral proteins and to the genetic maps of the viruses are discussed.     

Molecular genetic characterization of the RD-114 gene family of endogenous feline retroviral sequences.

RD-114 is a replication-competent, xenotropic retrovirus which is homologous to a family of moderately repetitive DNA sequences present at ca. 20 copies in the normal cellular genome of domestic cats. To examine the extent and character of genomic divergence of the RD-114 gene family as well as to assess their positional association within the cat genome, we have prepared a series of molecular clones of endogenous RD-114 DNA segments from a genomic library of cat cellular DNA. Their restriction endonuclease maps were compared with each other as well as to that of the prototype-inducible RD-114 which was molecularly cloned from a chronically infected human cell line. The endogenous sequences analyzed were similar to each other in that they were colinear with RD-114 proviral DNA, were bounded by long terminal redundancies, and conserved many restriction sites in the gag and pol regions. However, the env regions of many of the sequences examined were substantially deleted. Several of the endogenous RD-114 genomes contained a novel envelope sequence which was unrelated to the env gene of the prototype RD-114 env gene but which, like RD-114 and endogenous feline leukemia virus provirus, was found only in species of the genus Felis, and not in other closely related Felidae genera. The endogenous RD-114 sequences each had a distinct cellular flank which indicates that these sequences are not tandem but dispersed nonspecifically throughout the genome. Southern analysis of cat cellular DNA confirmed the conclusions about conserved restriction sites in endogenous sequences and indicated that a single locus may be responsible for the production of the major inducible form of RD-114.     

Two active forms of RD-114 virus DNA polymerase in infected cells.

Two forms of DNA polymerase are present in RD-114-infected human, dog, and mink cells, but are not detectable in uninfected cells. The two enzymes are indistinguishable catalytically and immunologically, but differ with respect to molecular weight and elution position from (dT)12-18-cellulose and phosphocellulose. The large enzyme (equivalent 95,000 daltons) is found in the infected cells, but not the virions produced by these cells. The virions contain only the smaller enzyme (equivalent 70,000 daltons). The larger form may represent a mammalian viral equivalent to the beta subunit of avian RNA tumor virus DNA polymerase.     

Endogenous RD-114 virus genome expression in malignant tissues of domestic cats.

Endogenous xenotropic cat type C virus (RD-114)- and infectious feline leukemia virus (FeLV)-specific gene expressions were measured in spontaneous sarcomas carcinomas, and nonmalignant cat tissues by molecular hybridization for virus-specific RNA and competition radio-immunoassays for the major internal protein (p30) of these two viruses. The results indicate that RD-114 gene expression in sarcomas and carcinomas at both RNA and p30 levels is significantly higher than histologically normal tissues from cats free of cancer. In contrast, the levels of FeLV viral RNA and p30 are fount to be low or undetectable in the majority of these tumored and normal tissues examined. Whereas variability in the amounts of RD-114 OR FeLV RNA and p30 expressed is found in tissues from different cats, their expression is fairly uniform in multiple malignant tissues of the same cat. The finding of widespread occurrence of elevated RD-114 gene expression in sarcomas and carcinomas is consistent with our similar observation with natural lymphomas of domestic cats and suggests that expression of certain functions of this endogenous virus may be etiologically involved in the development of many different spontaneous neoplasms of cats.     

Viral-specific antigen synthesis following de novo RD-114 virus infection of stationary cells

The cell cycle dependence of retrovirus replication was studied. Canine sarcoma (D-17) cells were infected de novo with the xenotropic feline retrovirus RD-114 under conditions previously reported to simultaneously inhibit virus replication and cell DNA synthesis and/or cell division. RD-114 viral antigen synthesis was observed under conditions previously reported to be inhibitory to avian and murine oncornavirus replication, including confluency and serum deprivation, X-irradiation, mitomycin C pretreatment, colchicine, and ethidium bromide treatments of cells. Several mechanisms that could account for viral antigen synthesis under the restrictive conditions used are discussed.     

Baboon Virus Isolate M-7 with Properties Similar to Feline Virus RD-114

A virus (M-7) isolated from baboon placental tissue demonstrates many similarities to endogenous feline virus RD-114. Immunodiffusion analysis shows a group-specific antigen (gs-1) line of identity between M-7 and RD-114. Anti-RD-114 DNA polymerase IgG inhibits M-7 polymerase by 57% compared to 97% for RD-114. M-7 virus has helper activity as demonstrated by rescue of murine sarcoma virus (MSV) from sarcoma-positive leukemia-negative human amnion cells. The host range of the rescued M-7 pseudotype of MSV, MSV (M-7), is similar to that of RD-114 virus. MSV (M-7) is also able to transform baboon cells and causes no detectable transformation of feline cells without addition of helper feline leukemia virus. Interference properties of M-7 and RD-114 virus are identical. Virus-specific neutralizing antisera, although partially cross-reacting, can distinguish MSV (M-7) from MSV (RD-114). These similarities and differences between RD-114 and M-7 viruses are best explained as type-specific differences between two viruses within the same strain.