Conserved Region of Mammalian Retrovirus RNA

The viral RNAs of various mammalian retroviruses contain highly conserved sequences close to their 3' ends. This was demonstrated by interviral molecular hybridization between fractionated viral complementary DNA (cDNA) and RNA. cDNA near the 3' end (cDNA(3')) from a rat virus (RPL strain) was fractionated by size and mixed with mouse virus RNA (Rauscher leukemia virus). No hybridization occurred with total cDNA (cDNA(total)), in agreement with previous results, but a cross-reacting sequence was found with the fractionated cDNA(3'). The sequences between 50 to 400 nucleotides from the 3' terminus of heteropolymeric RNA were most hybridizable. The rat viral cDNA(3') hybridized with mouse virus RNA more extensively than with RNA of remotely related retroviruses. The related viral sequence of the rodent viruses (mouse and rat) showed as much divergence in heteroduplex thermal denaturation profiles as did the unique sequence DNA of these two rodents. This suggests that over a period of time, rodent viruses have preserved a sequence with changes correlated to phylogenetic distance of hosts. The cross-reacting sequence of replication-competent retroviruses was conserved even in the genome of the replication-defective sarcoma virus and was also located in these genomes near the 3' end of 30S RNA. A fraction of RD114 cDNA(3'), corresponding to the conserved region, cross-hybridized extensively with RNA of a baboon endogenous virus (M7). Fractions of similar size prepared from cDNA(3') of MPMV, a primate type D virus, hybridized with M7 RNA to a lesser extent. Hybridization was not observed between Mason-Pfizer monkey virus and M7 if total cDNA's were incubated with viral RNAs. The degree of cross-reaction of the shared sequence appeared to be influenced by viral ancestral relatedness and host cell phylogenetic relationships. Thus, the strikingly high extent of cross-reaction at the conserved region between rodent viruses and simian sarcoma virus and between baboon virus and RD114 virus may reflect ancestral relatedness of the viruses. Slight cross-reaction at the site between type B and C viruses of rodents (mouse mammary tumor virus and RPL virus, 58-2T) or type C and D viruses of primates (M7, RD114, and Mason-Pfizer monkey virus) may have arisen at the conserved region through a mechanism that depends more on the phylogenetic relatedness of the host cells than on the viral type or origin. Determining the sequence of the conserved region may help elucidate this mechanism. The conserved sequences in retroviruses described here may be an important functional unit for the life cycle of many retroviruses.     

Probing the structure and function of viral RNA genomes

The majority of human, animal and plant viral pathogens possess genomes composed of RNA. The strategies evolved for expression and replication of viral RNA genomes can differ significantly from those utilized for expression and replication of host-cell genetic material. Consequently, knowledge of the molecular details of these strategies can lead to a clearer understanding of the origin, evolution and control of viral pathogens. We describe recent progress in identifying important structural and functional domains of the RNA genomes and associated replicative enzymes for two very different viruses: vesicular stomatitis virus, which possesses a single-stranded RNA genome of negative polarity, and wound tumor virus, which contains a genome composed of 12 discrete segments of double-stranded RNA.     

Rat sequences of the Kirsten and Harvey murine sarcoma virus genomes: nature, origin, and expression in rat tumor RNA.

Two murine sarcoma viruses, the Kirsten and the Harvey, were isolated by passage of mouse type C leukemia viruses through rats. These sarcoma viruses have genomes containing portions of their parental type C mouse leukemia virus genomes, in stable association with specific rat cellular sequences that we find to be quite likely not those of a rat type C leukemia virus. To determine if these murine sarcoma viruses provide a model relevant to the events occurring in spontaneous tumors, we have hybridized DNA and RNA prepared from rat tumors and normal rat tissues to [3H]DNA prepared from the Kirsten murine sarcoma virus. We have also hybridized these rat tissue nucleic acids to [3H]DNA prepared from a respresentative endogenous rat type C leukemia virus, the WFU (Wistar-Furth). Sarcoma-viral rat cellular sequences and endogenous rat leukemia viral sequences were detected in the DNA of both tumor and normal tissues, with no evidence of either gene amplification or additional sequences being present in tumor DNA. Sarcoma-viral rat cellular sequences and endogenous rat leukemia viral sequences were detected at elevated concentrations in the RNA of many rat tumors and in specific groups of normal tissues.     

The genome-associated,specific RNA binding proteins of avian and mammalian type C viruses

A structural protein purified from the Rous sarcoma virus (RSV) can specifically bind in vitro to purified avian, but not mammalian, type C viral RNA. Following ultraviolet irradiation of viral particles under conditions which stabilize the polyploid 70S viral RNA, the same polypeptide can be directly purified from the RSV genome. Based on its electrophoretic mobility in polyacrylamide gels containing sodium dodecylsulfate, the RNA binding protein has been identified as the major phosphoprotein (p19) of avian type C viruses. Similar experiments show that the major phosphoproteins of mammalian type C viruses (p12 for murine viruses and p16 for endogenous primate viruses) are also the specific RNA binding proteins and, similarly, are found closely associated with the 70S RNA genomes in the intact viral particles.     

Genetic individuality of intracisternal A-particles of Mus musculus.

The nucleic acid sequence relationship between mouse intracisternal type A-particles and type C and B viruses was examined by reciprocal complementary DNA-RNA hybridization; complementary DNAs prepared from the RNAs of intracisternal A-particles were hybridized with high-molecular-weight RNAs from a variety of murine tumor viruses, and complementary DNAs representing a variety of RNA tumor virus genomes were hybridized with the high-molecular-weight RNAs from A-particles. The criterion for homology between two types of virus was that the heterologous hybridization reaction occurs over the same RNA concentration range as the homologous reacton. The results of these hybridizations indicate that there are no major sequence homologies between the RNA of intracisternal A-particles and the RNA of representative members of type B and C viruses of Mus musculus.     

Capsid protein synthesis from replicating RNA directs specific packaging of the genome of a multipartite, positive-strand RNA virus

Flock house virus (FHV) is a bipartite, positive-strand RNA insect virus that encapsidates its two genomic RNAs in a single virion. It provides a convenient model system for studying the principles underlying the copackaging of multipartite viral RNA genomes. In this study, we used a baculovirus expression system to determine if the uncoupling of viral protein synthesis from RNA replication affected the packaging of FHV RNAs. We found that neither RNA1 (which encodes the viral replicase) nor RNA2 (which encodes the capsid protein) were packaged efficiently when capsid protein was supplied in trans from nonreplicating RNA. However, capsid protein synthesized in cis from replicating RNA2 packaged RNA2 efficiently in the presence and absence of RNA1. These results demonstrated that capsid protein translation from replicating RNA2 is required for specific packaging of the FHV genome. This type of coupling between genome replication and translation and RNA packaging has not been observed previously. We hypothesize that RNA2 replication and translation must be spatially coordinated in FHV-infected cells to facilitate retrieval of the viral RNAs for encapsidation by newly synthesized capsid protein. Spatial coordination of RNA and capsid protein synthesis may be key to specific genome packaging and assembly in other RNA viruses.     

Mechanisms for RNA Capture by ssDNA Viruses: Grand Theft RNA

Viruses contain three common types of packaged genomes; double-stranded DNA (dsDNA), RNA (mostly single and occasionally double stranded) and single-stranded DNA (ssDNA). There are relatively straightforward explanations for the prevalence of viruses with dsDNA and RNA genomes, but the evolutionary basis for the apparent success of ssDNA viruses is less clear. The recent discovery of four ssDNA virus genomes that appear to have been formed by recombination between co-infecting RNA and ssDNA viruses, together with the high mutation rate of ssDNA viruses provide possible explanations. RNA–DNA recombination allows ssDNA viruses to access much broader sequence space than through nucleotide substitution and DNA–DNA recombination alone. Multiple non-exclusive mechanisms, all due to the unique replication of ssDNA viruses, are proposed for this unusual RNA capture. RNA capture provides an explanation for the evolutionary success of the ssDNA viruses and may help elucidate the mystery of integrated RNA viruses in viral and cellular DNA genomes.     

A replication silencer element in a plus-strand RNA virus

Replication represents a key step in the infectious cycles of RNA viruses. Here we describe a regulatory RNA element, termed replication silencer, that can down-regulate complementary RNA synthesis of a positive-strand RNA virus via an RNA-RNA interaction. This interaction occurs between the 5-nucleotide-long, internally positioned replication silencer and the extreme 3'-terminus of the viral RNA comprising part of the minimal minus-strand initiation promoter. Analysis of RNA synthesis in vitro, using model defective interfering (DI) RNA templates of tomato bushy stunt virus and a partially purified, RNA-dependent RNA polymerase preparation from tombusvirus-infected plants, revealed that this interaction inhibits minus-strand synthesis 7-fold. This functional interaction was supported further by: (i) RNA structure probing; (ii) phylogenetic analysis; (iii) inhibition of activity by short complementary DNAs; and (iv) compensatory mutational analysis. The silencer was found to be essential for accumulation of DI RNAs in protoplasts, indicating that it serves an important regulatory role(s) in vivo. Because similar silencer-promoter interactions are also predicted in other virus genera, this type of RNA-based regulatory mechanism may represent a widely utilized strategy for modulating replication.     

Detection of Mouse Mammary Tumor Virus RNA in BALB/c Tumor Cell Lines of Nonviral Etiologies

A complementary DNA (cDNA) probe to mouse mammary tumor virus (MMTV) RNA was synthesized using calf thymus DNA oligonucleotides as a random primer. This probe was then used to study the expression of MMTV RNA in cell lines from BALB/c tumors induced in vivo either spontaneously or in response to viral, chemical, or hormonal stimuli. The cDNA had a length of approximately 400 to 500 nucleotides and specifically hybridized to MMTV RNA and BALB/c lactating mammary gland RNA, but not to Moloney leukemia virus RNA. Calf thymus DNA-primed cDNA could protect 50% of iodinated MMTV RNA from S1 nuclease digestion at cDNA-RNA ratios of 1:1 and 90% of labeled viral RNA at ratios of 10:1. Thermal denaturation of MMTV RNA-cDNA hybrids yielded a T(m) of 88.5 degrees C, indicative of a well-base-paired duplex. Screening of mouse mammary tumor cells for MMTV sequences revealed that three out of five lines of BALB/c origin had undetectable levels of viral RNA (相似文献   

虫媒黄病毒亚基因组RNA

黄病毒是一大科人类致病性的单股正链RNA病毒。黄病毒包括登革病毒、西尼罗脑炎病毒及日本脑炎病毒等成员,主要径通过节肢动物的叮咬进行传播,即为虫媒黄病毒。研究发现,在虫媒黄病毒复制过程中,除病毒基因组正链RNA、互补的负链RNA及两者的杂合RNA分子外,在病毒感染细胞后还能产生一种病毒亚基因组RNA(subgenomic RNA,sgRNA)。近年对这种sgRNA展开了比较多的研究,结果表明,其产生机制与已知的其他病毒sgRNA产生机制并不相同。该sgRNA的产生与虫媒黄病毒基因组3’非编码区所形成的保守二级结构有关,同时宿主核酸酶对其的不完全降解亦有重要作用。虫媒黄病毒基因组3’非编码区中带有多个与病毒复制相关的RNA元件,而sgRNA的发现有助于全面地认识病毒RNA与宿主RNA代谢途径间的相互作用,为最终阐明病毒的致病机制奠定基础。     

THE PENETRATION OF REOVIRUS RNA AND INITIATION OF ITS GENETIC FUNCTION IN L-STRAIN FIBROBLASTS

Reovirus type 3 is phagocytized by L cells and rapidly sequestered inside lysosomes. Hydrolases within these organelles are capable of stripping the viral coat proteins, but they fail to degrade the double-stranded RNA genome. These observations support the view that sojourn of reovirus in lysosomes, when the lytic enzymes uncoat its genome, is an obligatory step in the sequence of infection. Although the mechanism for transferring the uncoated RNA out of lysosomes remains to be elucidated, evidence is presented suggesting that progeny genomes are bound to site(s) possessing the fine structure of viral inclusions or factories. It appears that both the synthesis of single- and double-stranded viral RNA and the morphogenesis of progeny virus particles occur in such factories.     

Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus.

To identify RNA and protein sequences involved in packaging of human immunodeficiency virus type 1 (HIV-1), various mutations were introduced into the viral genome. Portions of the human immunodeficiency virus type 1 genome between the first splice donor site and the gag initiation codon were deleted to investigate the RNA packaging site (psi). Point mutations that alter cysteine residues in one or both zinc finger motifs of p7, a cleavage product of the gag precursor, were created to study the role of the gag zinc fingers in packaging. The psi site mutants and the gag mutants exhibited similar phenotypes. Cells transfected with the mutant genomes, while expressing normal levels of human immunodeficiency virus type 1 RNA and proteins, produced viral particles that were normal in protein content but lacked detectable viral RNA. These mutant virions were unable to productively infect cells. The combination of human immunodeficiency virus type 1 packaging mutations should minimize fortuitous assembly of infectious virus and may provide a means to produce noninfectious particles for candidate vaccines.     

Molecular evidence for a type C retrovirus etiology of the lymphoproliferative disease of turkeys.

Recently, we isolated from the blood of lymphoproliferative disease (LPD)-affected turkeys a type C retrovirus distinct from the avian leukosis-sarcoma virus complex and the reticuloendotheliosis virus group. We present molecular evidence for the implication of this virus in the LPD of turkeys. Using complementary DNA of LPD viral RNA, we found that the LPD viral genome is specifically and efficiently transcribed (2,500 copies per cell) in LPD tumor cells. Moreover, the LPD tumor cells contained newly inserted LPD viral information (5 to 10 copies per haploid genome), which was not present before the infection. From the absence of LPD virus-specific sequences in the normal cell genome of turkeys, it was concluded that the LPD virus is not an endogenous virus of turkeys. DNA-DNA annealing experiments revealed that the degree of sequence homology between LPD viral complementary DNA and cellular DNA of turkeys was not higher than that between LPD viral complementary DNA and cellular DNA of other species, thus indicating that the virus does not originate from turkeys.