Association of Viral Reverse Transcriptase with an Enzyme degrading the RNA Moiety of RNA-DNA Hybrids

DURING replication of RNA tumour viruses, the genetic information contained in the viral RNA seems to be transferred to DNA^1,2. Studies on the enzymatic activities present in the virus particles suggest that this transfer is mediated by an RNA dependent DNA polymerase^3,4. RNA-DNA hybrids have been demonstrated to occur as intermediates in this reaction⁵ and single stranded DNA is generated as an early reaction product⁶, which is then replicated to give a double stranded DNA product^6–8. The mechanism by which the single stranded DNA is displaced from the RNA template is, however, not known.     

Nature of Ribonuclease-resistant Nucleic Acid of Chick Embryo Cells transformed by Schmidt–Ruppin Strain of Rous Sarcoma Virus

THE mode of replication of RNA or RNA-containing tumour viruses is not understood. The recent studies on Rous sarcoma and other RNA-containing oncogenic viruses suggest that the replicative cycle of the RNA of these viruses might not be associated with ribonuclease-resistant structures (double stranded RNAs), but might involve the synthesis of a DNA intermediate specific to viral RNA^1–3. Two groups of workers, however, presented evidence for the presence of a double stranded RNA in 78 Al cell line of rat embryo fibroblasts which had been transformed and chronically infected with the murine sarcoma-leukaemia virus complex (MSV-MLV)^4,5 and it was suggested that the mode of replication of oncogenic viral RNAs was the same as that of non-oncogenic viral RNAs⁴. This apparent discrepancy prompted me to look for ribonuclease-resistant RNA structures in the chick embryo cells transformed by Schmidt-Ruppin Rous sarcoma virus (SR-RSV).     

Inhibitors acting on Nucleic Acid Synthesis in an Oncogenic RNA Virus

IN infection with an oncogenic RNA virus, synthesis of viral RNA seems to be catalysed by an RNA dependent DNA polymerase in the host cell^1–4. Several specific inhibitors of viral DNA polymerases have been found^5–7 and Spiegelman⁸ has shown that the activity of viral enzymes depends strongly on the chemical composition of the template. We report here first a new highly specific poison of the Rauscher murine leukaemia virus (RMLV) DNA polymerases; second, several inactivators of the RNA and DNA template involved in the RMLV enzyme systems; and third, the action of actinomycin D on viral DNA polymerases and on host DNA/RNA polymerase. The results are discussed with respect to the influence of actinomycin D on virus multiplication.     

Host restriction of friend leukemia virus; fate of input virion RNA

Host restriction of oncogenesis by RNA tumor viruses may be studied in vitro by measuring the replication of the lymphatic leukemia component of the Friend Virus Complex (LLV-F) in either NIH-Swiss or Balb/C mouse embryo cells. These cells derive from mice differing at the Fv-1 locus, which controls the replication of all murine RNA leukemia viruses. Studies of early events in the replication of LLV-F were carried out by following the infection of permissive and restrictive mouse embryo cells by ³²P labeled LLV-F. ³²P labeled viral genome RNA rapidly becomes associated with cell nuclei and may be found integrated to the same extent with high molecular weight host DNA of either permissive or restrictive cells. These results suggest that Fv-1 mediated host restriction of LLV-F occurs at a step following integration of viral genome RNA into host DNA.Two other conclusions are suggested by these data. The nucleus appears to be the site of activation and synthesis of DNA of the infecting virus; and the “provirus”, at least transiently, is represented as an RNA-DNA hybrid molecule covalently integrated with host cell DNA.     

Deep sequencing of mycovirus‐derived small RNAs from Botrytis species

RNA silencing is an ancient regulatory mechanism operating in all eukaryotic cells. In fungi, it was first discovered in Neurospora crassa, although its potential as a defence mechanism against mycoviruses was first reported in Cryphonectria parasitica and, later, in several fungal species. There is little evidence of the antiviral potential of RNA silencing in the phytopathogenic species of the fungal genus Botrytis. Moreover, little is known about the RNA silencing components in these fungi, although the analysis of public genome databases identified two Dicer‐like genes in B. cinerea, as in most of the ascomycetes sequenced to date. In this work, we used deep sequencing to study the virus‐derived small RNA (vsiRNA) populations from different mycoviruses infecting field isolates of Botrytis spp. The mycoviruses under study belong to different genera and species, and have different types of genome [double‐stranded RNA (dsRNA), (+)single‐stranded RNA (ssRNA) and (–)ssRNA]. In general, vsiRNAs derived from mycoviruses are mostly of 21, 20 and 22 nucleotides in length, possess sense or antisense orientation, either in a similar ratio or with a predominance of sense polarity depending on the virus species, have predominantly U at their 5′ end, and are unevenly distributed along the viral genome, showing conspicuous hotspots of vsiRNA accumulation. These characteristics reveal striking similarities with vsiRNAs produced by plant viruses, suggesting similar pathways of viral targeting in plants and fungi. We have shown that the fungal RNA silencing machinery acts against the mycoviruses used in this work in a similar manner independent of their viral or fungal origin.     

Evidence for genetic relationship between RNA and DNA viruses from the sequence homology of a putative polymerase gene of bluetongue virus with that of vaccinia virus: conservation of RNA polymerase genes from diverse species.

The nucleotide sequence of segment 1 of the double stranded RNA genome of bluetongue virus serotype 10 (BTV-10), encoding the largest viral core protein, VP1, has been determined. Linear sequence analysis of the predicted amino acid sequence of the 149-K Da protein, a putative component of the viral RNA-directed RNA polymerase, revealed extensive homology with the vaccinia virus 147K Da DNA-directed RNA polymerase subunit. Similar homologies were detected between the VP1 polypeptide and the beta chain subunit of Escherichia coli and common tobacco chloroplast RNA polymerases, yeast RNA polymerase II and III and fruit fly polymerase II.     

DNA Polymerases of Myeloma: Template and Primer Specificities of Two Enzymes

INTEREST in the viral RNA-dependent DNA polymerases^1,2 has led to an examination of mammalian DNA polymerases and to the characterization in certain systems of DNA polymerases which can transcribe ribopolymer chains of appropriate ribopolymer-oligodeoxyribopolymer hybrids^3,4. The nature of these enzymes and their function in cells are unclear. We have reported the separation, by chromatography on DEAE-cellulose, of the DNA polymerases of the murine myeloma tumour into two separate fractions which we have designated as fractions D-I and D-II (ref. 5). Each of these fractions contains an activity capable of utilizing activated DNA and of transcribing the ribopolymer strand of poly rA oligo dT. We describe here the separation of two DNA polymerase enzymes in fraction D-I.     

DNA Polymerase Activity associated with Purified Kilham Rat Virus

RNA tumour viruses contain an enzyme which can transcribe DNA from an RNA template^1,2, an endonuclease and a DNA-dependent DNA polymerase activity^3,4. RNA polymerase has been reported in vaccinia virus^5,6, reovirus^7,8 and cytoplasmic polyhidrosis virus⁹. I wish to describe a DNA polymerase activity associated with a highly purified preparation of the parvovirus, Kilham rat virus (KRV), which is thus the first report of a DNA polymerase associated with a DNA virus. KRV, a small virus first isolated from a rat sarcoma¹⁰, is antigenically related to the H viruses isolated from human transplantable tumours¹¹. Those parvoviruses which have been characterized all contain single stranded DNA with molecular weights of 1.5 to 2.5 × 10⁶ (refs. 12,13 and 14).     

A rapid in vitro assay for HIV DNA integration.

Retroviruses synthesize a double stranded DNA copy of their RNA genome after infection of a permissive cell and subsequent integration of this DNA copy into the host genome is necessary for normal viral replication. Integration occurs by a specialized DNA recombination reaction, mediated by the viral IN protein. Because this reaction has no known cellular counterpart, it is a particularly attractive target in the search for specific inhibitors with low toxicity that may serve as therapeutic antiviral agents. We present a simple assay system that is suitable for screening potential inhibitors of HIV DNA integration. Only short oligonucleotides matching one end of HIV DNA and purified HIV IN protein are required as substrates. Furthermore, since each step of the assay can be carried out in the wells of microtiter plates, large numbers of reactions can be processed simultaneously.     

Structural and Functional Studies of the Phage Sf6 Terminase Small Subunit Reveal a DNA-Spooling Device Facilitated by Structural Plasticity

In many DNA viruses, genome packaging is initiated by the small subunit of the packaging terminase, which specifically binds to the packaging signal on viral DNA and directs assembly of the terminase holoenzyme. We have experimentally mapped the DNA-interacting region on Shigella virus Sf6 terminase small subunit gp1, which occupies extended surface areas encircling the gp1 octamer, indicating that DNA wraps around gp1 through extensive contacts. High‐resolution structures reveal large-scale motions of the gp1 DNA-binding domain mediated by the curved helix formed by residues 54–81 and an intermolecular salt bridge formed by residues Arg67 and Glu73, indicating remarkable structural plasticity underlying multivalent, pleomorphic gp1:DNA interactions. These results provide spatial restraints for protein:DNA interactions, which enable construction of a three-dimensional pseudo-atomic model for a DNA-packaging initiation complex assembled from the terminase small subunit and the packaging region on viral DNA. Our results suggest that gp1 functions as a DNA-spooling device, which may transform DNA into a specific architecture appropriate for interaction with and cleavage by the terminase large subunit prior to DNA translocation into viral procapsid. This may represent a common mechanism for the initiation step of DNA packaging in tailed double‐stranded DNA bacterial viruses.     

A comparison of the enzymatic responses of the DNA polymerases from four RNA tumor viruses.

DNA polymerases from avian, feline, murine and simian RNA tumor viruses exhibit substantial differences in optimal assay conditions and vary widely in their template-primer preferences. Avian DNA polymerase utilizes both natural and synthetic template-primers efficiently in the presence of Mg⁺⁺ as well as Mn⁺⁺. By contrast, the mammalian viral DNA polymerases are much more responsive to poly(A)·oligo(dT) than to other template-primers, and exhibit up to 20-fold greater activity with Mn⁺⁺ than with Mg⁺⁺. In addition, simian sarcoma virus DNA polymerase shows no detectable response to poly(C)·oligo(dG) over a wide variety of conditions stimulatory to the other viral enzymes.     

A measurement of the fraction of chloroplast DNA transcribed in Euglena

The fraction of the chloroplast DNA transcribed in the single celled alga $\text{Euglena}$ has been determined by RNA-DNA hybridization. A vast excess of total cell RNA from cells which were rapidly dividing in the light was hybridized in liquid to [¹²⁵I] — chloroplast DNA, and the resulting duplexes separated on hydroxyapatite columns. The contribution of DNA-DNA duplex formation was determined separately and was used to calculate that portion of the duplex which was actually a RNA-DNA hybrid. Sixteen percent of the single stranded chloroplast DNA forms a duplex with this RNA suggesting that 32 percent of the double stranded DNA molecule is being transcribed into RNA under these conditions of cell growth.     

Nucleotide Composition of RNA hybridized to Homologous DNA from Cells transformed by Avian Tumour Viruses

PART of the evidence which indicates that RNA tumour viruses replicate through a DNA intermediate¹ was the detection of DNA which is complementary to the viral RNA in leukaemic cells transformed by avian myeloblastosis virus (AMV)² and in cells transformed in vitro by avian sarcoma viruses, Schmidt-Ruppin (SR-RSV) and B-77 (ref. 3). If this DNA serves as a template for the viral RNA, it must be a copy of the entire viral genome. One of the necessary requirements for this function is that the homologous DNA has the same nucleotide composition as the viral RNA. In this study, the average base composition of the RNA which had been hybridized to homologous DNA from transformed cells was compared with the base composition of the input viral RNA. Two experimental conditions had to be met: (1) the recovery of all the ribonucleotides which had been hybridized and (2) the absence of partially hybridized ribonucleotide sequences. The first requirement called for the deletion of the treatment of DNA-RNA hybrids with pancreatic ribonuclease fraction A and ribonuclease T₁ which had been used in our previous experiments because such a treatment can cause the non-random loss of hybridized nucleotides⁴. The second requirement called for a hybridization and washing procedure in which only specifically hybridized ribonucleotide sequences would remain bound to the filters. Both of these conditions were met by using fragmented viral RNA and a modified washing procedure which excluded the use of ribonuclease. The results show that the average nucleotide composition of the hybridized RNA is identical to that of the input viral RNA.     

The use of retroviral vectors for gene therapy-what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery

Retroviral vector-mediated gene transfer has been central to the development of gene therapy. Retroviruses have several distinct advantages over other vectors, especially when permanent gene transfer is the preferred outcome. The most important advantage that retroviral vectors offer is their ability to transform their single stranded RNA genome into a double stranded DNA molecule that stably integrates into the target cell genome. This means that retroviral vectors can be used to permanently modify the host cell nuclear genome. Recently, retroviral vector-mediated gene transfer, as well as the broader gene therapy field, has been re-invigorated with the development of a new class of retroviral vectors which are derived from lentiviruses. These have the unique ability amongst retroviruses of being able to infect non-cycling cells. Vectors derived from lentiviruses have provided a quantum leap in technology and seemingly offer the means to achieve significant levels of gene transfer in vivo.     

Coexpression of Escherichia coli RNase III in silkworm cells improves the efficiency of RNA interference induced by long hairpin dsRNAs

Abstract Long hairpin dsRNA transcribed from chromosomal DNA can induce RNA interference in Bombyx mori cells, although its gene silencing efficiency is lower than that of exogenously introduced double‐stranded RNAs (dsRNAs). To solve this problem, we monitored the nuclear cytoplasmic translocation of the transcribed hairpin dsRNA and analyzed the processing efficiency into mature small interfering RNA (siRNA). Northern blot analysis revealed that the transcribed hairpin dsRNAs were spliced and transported into the cytoplasm, but were not effectively diced into siRNAs. Interestingly, RNAi with hairpin dsRNAs from genome‐integrated IR transgene was stimulated by the coexpression of Escherichia coli RNase III, although this exogenous enzyme seemed to bring about nonspecific cleavage of cellular mRNA.