Infidelity of DNA synthesis: a general property of RNA tumor viruses

We have determined the frequency with which a non-complementary base-paired nucleotide is incorporated by the DNA polymerase of Rauscher leukemia virus using synthetic polynucleotides as templates. The observed error rates are very similar to those error rates previously reported with the DNA polymerase from avian myeloblastosis virus. This similarity suggests that a high level of infidelity may be a common characteristic of RNA tumor viruses. This high error rate may be relevant to the mode of action of the polymerase during carcinogenesis.     

Transcription of 70S RNA by DNA polymerases from mammalian RNA viruses.

DNA polymerases purified by the same procedure from four mammalian RNA viruses, simian sarcoma virus type 1, gibbon ape lymphoma virus, Mason-Pfizer monkey virus, and Rauscher murine leukemia virus are capable of transcribing heteropolymeric regions of viral 70S RNA without any other primer. In this reconstituted system the enzymes from simian sarcoma virus type 1, Mason-Pfizer monkey virus, and Rauscher murine leukemia virus transcribe viral 70S RNA almost as efficiently as the DNA polymerase from the avian myeloblastosis virus, but gibbon ape lymphoma virus DNA polymerase is approximately three-to fivefold less efficient. Although there is a substantial difference among the sizes of these DNA polymerases (160,000 daltons for the avian myeloblastosis virus enzyme, 110,000 daltons for the Mason-Pfizer monkey virus enzyme, and 70,000 daltons for the mammalian type C viral polymerases), the ability to transcribe viral 70S RNA is a characteristic common to these enzymes.     

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 closely related group of RNA-dependent RNA polymerases from double-stranded RNA viruses.

Probably one of the first proteinaceous enzymes was an RNA-dependent RNA polymerase (RDRP). Although there are several conserved motifs present in the RDRPs of most positive and double-stranded RNA (dsRNA) viruses, the RDRPs of the dsRNA viruses show no detectable sequence similarity outside the conserved motifs. There is now, however, a group of dsRNA viruses of lower eucaryotes whose RDRPs are detectably similar. The origin of this sequence similarity appears to be common descent from one or more noninfectious viruses of a progenitor cell, an origin that predates the differentiation of protozoans and fungi. The cause of this preservation of sequence appears to be constraints placed on the RDRP by the life-style of these viruses--the maintenance of a stable, persistent, noninfectious state.     

Infidelity of DNA synthesis by reverse transcriptase

The fidelity of purified DNA polymerase from avian myeloblastosis virus in precisely copying polynucleotide templates was determined. With poly (dA-dT) · poly (dA-dT) as a template, one molecule of the incorrect basepaired nucleotide (dCTP) is incorporated for every 6000 nucleotides polymerized. When copying the ribo strand of poly (rA) · poly (dT) the error rate is approximately one in 600. It is suggested that the enzyme makes similar errors $\text{in}\text{vivo}$ and thus could be mutagenic.     

Translesion synthesis by the UmuC family of DNA polymerases.

Translesion synthesis is an important cellular mechanism to overcome replication blockage by DNA damage. To copy damaged DNA templates during replication, specialized DNA polymerases are required. Translesion synthesis can be error-free or error-prone. From E. coli to humans, error-prone translesion synthesis constitutes a major mechanism of DNA damage-induced mutagenesis. As a response to DNA damage during replication, translesion synthesis contributes to cell survival and induced mutagenesis. During 1999-2000, the UmuC superfamily had emerged, which consists of the following prototypic members: the E. coli UmuC, the E. coli DinB, the yeast Rad30, the human RAD30B, and the yeast Rev1. The corresponding biochemical activities are DNA polymerases V, IV, eta, iota, and dCMP transferase, respectively. Recent studies of the UmuC superfamily are summarized and evidence is presented suggesting that this family of DNA polymerases is involved in translesion DNA synthesis.     

Ribonucleotide discrimination by translesion synthesis DNA polymerases

The well-being of all living organisms relies on the accurate duplication of their genomes. This is usually achieved by highly elaborate replicase complexes which ensure that this task is accomplished timely and efficiently. However, cells often must resort to the help of various additional “specialized” DNA polymerases that gain access to genomic DNA when replication fork progression is hindered. One such specialized polymerase family consists of the so-called “translesion synthesis” (TLS) polymerases; enzymes that have evolved to replicate damaged DNA. To fulfill their main cellular mission, TLS polymerases often must sacrifice precision when selecting nucleotide substrates. Low base-substitution fidelity is a well-documented inherent property of these enzymes. However, incorrect nucleotide substrates are not only those which do not comply with Watson–Crick base complementarity, but also those whose sugar moiety is incorrect. Does relaxed base-selectivity automatically mean that the TLS polymerases are unable to efficiently discriminate between ribonucleoside triphosphates and deoxyribonucleoside triphosphates that differ by only a single atom? Which strategies do TLS polymerases employ to select suitable nucleotide substrates? In this review, we will collate and summarize data accumulated over the past decade from biochemical and structural studies, which aim to answer these questions.     

Protection from RNA and DNA viruses by IL-32

Several studies have documented a proinflammatory role for IL-32, which induces IL-1α, IL-1β, IL-6, TNF, and chemokines via NF-κB, p38MAPK, and AP-1. However, IL-32 also participates in the responses to infection with viruses such as HIV-1 and influenza. In this study, we explored these antiviral properties of IL-32. Vital staining assays demonstrated that low concentrations (5-10 ng/ml) of rIL-32γ protected epithelial WISH cells from vesicular stomatitis virus-induced cell death. By lactate dehydrogenase assays, treatment with IL-32γ resulted in a 3- to 4-fold decrease in viral load. Specific silencing of IL-32 revealed that the antiviral responses triggered by the synthetic analogs of ssRNA viruses (polyuridine) and dsRNA viruses (polyinosinic-polycytidylic acid) were significantly weaker (2- to 3-fold more virus) in WISH cells in the absence of IL-32. Importantly, we discovered that the polyinosinic-polycytidylic acid-induced increase in production of IFN-α in human PBMC was nearly completely abolished when IL-32 was silenced. Moreover, we observed that IL-32 antagonizes the DNA virus HSV-2 in epithelial Vero cells as well as in human umbilical cord endothelial cells, as production of HSV-2 increased 8-fold upon silencing of IL-32 (p < 0.001). Mechanistically, we found that IL-32 used the PKR-eIF-2α as well as the MxA antiviral pathways. Unexpectedly, a considerable part of the antiviral properties of IL-32 was not dependent on IFNs; specific blockade of IFN activity reduced the antiviral properties of IL-32 only moderately. In conclusion, these data suggest a central role for IL-32 in the immune response to RNA and DNA viruses, which may be exploitable for clinical use in the future.