TWiV 485: Fishing with defective flies

The TWiV posse considers viral insulin-like peptides encoded in fish genomes, and insect antiviral immunity by production of viral DNA from defective genomes of RNA viruses.

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TWiV 477: Raiders of the lost Arc

The TWiVodrome explains how a gag-like protein from a retrotransposon forms virus-like particles that carry mRNA within vesicles across the synapse.

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A transmissible cancer of soft-shell clams

Mya_arenariaA leukemia-like cancer is killing soft-shell clams along the east coast of North America. The cancer is transmitted between animals in the ocean, and appears to have originated in a single clam as recently as 40 years ago.

Hemic neoplasm is a disease of marine bivalves that is characterized by proliferation of morphologically and functionally aberrant hemocytes, the cells that circulate in the circulatory fluid of mollusks. A newly identified LTR retrotransposon called Steamer correlates with neoplastic disease in clams. LTR retrotransposons are DNA sequences in the genome that are thought to be precursors to retroviruses. The normal clam genome contains 10-20 copies of Steamer, compared with 150-300 copies in neoplastic hemocytes.

Integration of retroviruses into the genome is a known mechanism for disrupting cellular growth control, leading to uncontrolled proliferation and ultimately development of cancer. Whether Steamer causes neoplasia, by integrating near an oncogene, can be determined based on where this retroelement has inserted in the clam genome.

Analysis of 12 Steamer integration sites revealed that 7 were present in neoplastic samples from clams collected at sites in New York, Maine, and Prince Edward Island, Canada. Examination of nuclear and mitochondrial DNA revealed that cancerous hemocytes collected from clams at all three sites contain similar mutations that are not present in normal tissues. These observations indicate that these neoplasms  are nearly genetically identical and could not have arisen from the hosts. The cancer probably originated in a single clam and was then transmitted to other animals.

Cells from different vertebrates are usually rejected by the host immune system, which recognizes foreign cells by the major histocompatibility (MHC) system. However, mollusks do not have MHC, which may explain why tumor cells can be transmitted among clams. Two other transmissible tumors have been described: the canine venereal tumor, which is sexually passed among dogs, and Tasmanian devil facial tumor disease, transmitted by biting. In both cases MHC molecules are low in tumor cells, explaining why they are not rejected by the recipient animal.

How cancer could spread among clams hundreds of miles apart is not known. Clams are filter feeders, which may lead to uptake of neoplastic cells released into the water by diseased animals. Movement of clams by humans might also have played a role in dissemination of disease along the northeastern US seaboard.

There are no known contagious human cancers, and it is unlikely that the steamer clam neoplasia could be transmitted to humans. It is not known how extensively hemic neoplasia will spread among soft-shell clams, or whether the disease could spread to other bivalves.

Unexpected viral DNA in RNA virus-infected cells

vesicular stomatitis virusMany years ago a claim was made that cells infected with respiratory syncytial virus contained infectious DNA copies of the viral genome. When this paper was published, retroviral reverse transcriptase had been discovered, which explained how DNA copies of retroviral RNA genomes were made in infected cells. Although the respiratory syncytial viral genome is RNA, it does not encode a reverse transcriptase, and how a DNA copy of this genome could be made in infected cells was unknown. The observation was initially met with great fanfare, and was suggested to account for why some RNA virus infections persist, and even to explain autoimmune diseases. However the findings were never duplicated, and the authors fell into scientific obscurity. Now it appears that they might not have been entirely wrong.

It is now quite clear that DNA copies of viral RNA are made in infected cells. A DNA copy of lymphocytic choriomeningitis virus (LCMV) RNA has been detected in mouse cells, and this DNA can be integrated into cellular DNA. LCMV DNA was only detected in cells that produce a retrovirus, implicating reverse transcriptase in this process. DNAs of various RNA viruses, including bornaviruses and filoviruses, have been found integrated into the genome of many animals. An explanation for the genesis of these DNA copies is provided by studies of cells infected with vesicular stomatitis virus.

Vesicular stomatitis virus (pictured) is an enveloped virus with a genome of (-) strand RNA. In infected cells the (-) strand RNA is copied by the viral RNA polymerase to form 5 mRNAs which encode the viral proteins. Infection of various human cell lines resulted in the production of DNA complementary to VSV RNA. The DNAs are single stranded and appear to be produced from the viral mRNAs, not the viral genome.

How might VSV DNA be produced in virus infected cells? About 20% of the human genome consists of a mobile genetic element called LINE-1 (Long Interspersed Nuclear Element). These elements, also called retrotransposons, encode a reverse transcriptase. This enzyme converts LINE-1 mRNA into DNA, which can then integrate elsewhere in the cell genome – hence the name mobile genetic element. LINE-1 encoded reverse transcriptase can also produce DNA copies of cellular mRNAs and other mobile elements that do not encode their own reverse transcriptase. Thanks to LINEs, our genomes are littered with mobile genetic elements.

To determine if LINE-1 could make VSV in infected cells, the authors took advantage of their observation that not all human cells produce VSV DNA after infection. Introduction of LINE-1 DNA into one of these cell lines enabled it to produce DNA copies of VSV mRNAs. This result shows that LINE-1 can make VSV DNA in infected cells. Whether LINE-1 actually accomplishes this process in unmodified, VSV infected cells remains to be proven.

The authors also show that viral DNA is produced in cells infected with two other RNA-containing viruses, echovirus and respiratory syncytial virus. The latter of course is the virus used to make the claim nearly 40 years ago that viral DNA is present in infected cells. Whether that viral DNA is infectious, however, is not known. No complete copies of the VSV RNA genome have been observed in infected cells, only copies of viral mRNAs.

It seems likely that in cells infected with RNA viruses, reverse transcriptase encoded by LINE-1 could produce DNA copies of viral RNAs. After entering the nucleus these viral DNAs could become integrated into cellular DNA. If a germline cell were infected, the integrated viral DNA would be passed on to subsequent generations, explaining the presence of DNA copies of various RNA viruses in animal genomes.

A key question is whether the production of DNA copies of viral RNAs is an accident, or benefits the virus or host. There is yet no answer to this question. The authors suggest that DNA copies of viral RNA might contribute to innate immune sensing of viral infection. VSV replication is not altered by the presence or absence of viral DNA, but there could be a role for viral DNA during infection of a host animal.

Integration of arenavirus DNA into the cell genome

retrotransposonIs that title correct? Arenaviruses have an RNA genome which is not known to be copied into DNA at any stage of the replication cycle. How could a DNA copy of this virus be produced and be inserted into the host genome?

The RNA genome of retroviruses is converted to a DNA form during viral replication by the viral enzyme reverse transcriptase. The viral DNA then integrates into the host’s genome, becoming a permanent part of the cell. These events have no counterparts during replication of arenaviruses. The RNA genomes of these viruses are copied via an RNA intermediate, entirely in the cytoplasm of the cell. Nevertheless, the authors of a recent study found a DNA copy of the RNA genome of lymphocytic choriomeningitis virus (LCMV), a commonly studied arenavirus, integrated into host cell DNA.

This unusual story began in 1979 with the report that DNA complementary to the RNA genome of LCMV can be detected in about 1 in 103 to 104 infected cells. The authors speculated that retroviruses were involved but did not provide mechanistic evidence. Eighteen years later, the authors subcloned these cells to produce cell lines containing LCMV DNA. They then used polymerase chain reaction to isolate LCMV DNA from the cloned cells, including the cellular sequence flanking the viral genome. Nucleotide sequence analysis revealed that both cell lines contain a copy of the viral glycoprotein (GP) gene joined to cellular sequences encoding an IAP (intracisternal A type) retrotransposon. The authors identified LCMV integration both in cultured cells and infected mice.

Retrotransposons are sequences related to retroviruses that are found in the genome of many organisms. The gene content and arrangement are similar to retroviruses, but they lack an extracellular phase: they do not encode an env gene. Retrotransposons may be retroviral progenitors, or degenerate forms of these viruses. They are amazingly abundant: 42% of the human genome is made up of retrotransposons.

Based on their experimental results, the authors proposed that the following events occurred in a cell infected with LCVM. During copying of the retrotransposon RNA into a cDNA copy by reverse transcriptase, the enzyme switched templates and began copying the LCMV GP RNA. This hybrid DNA – retrotransposon linked to LCVM sequences –  then integrated into the host genome.

The results reported in this paper have enormous implications for evolution and for human gene therapy. Because retrotransposons transpose most efficiently in the thymus and testicles, the recombination events described could lead to transmission of RNA virus genes in the germline. Therefore the contribution of non-retroviral RNA viruses to evolution of the mammalian genome might be greater than previously believed. In addition, it is possible that RNA virus vectors used for gene therapy could integrate into the human genome via the mechanism described in this study. Such integration could lead to undesired mutation such as activation of oncogenes. Therefore the ability of specific RNA virus vectors to integrate into the human genome should be carefully tested before the vectors are approved for use in humans.

Rolf M. Zinkernagel, Paul Klenerman, Hans Hengartner (1997). A non-retroviral RNA virus persists in DNA form Nature, 390 (6657), 298-301 DOI: 10.1038/36876

M. B. Geuking, J. Weber, M. Dewannieux, E. Gorelik, T. Heidmann, H. Hengartner, R. M. Zinkernagel, L. Hangartner (2009). Recombination of Retrotransposon and Exogenous RNA Virus Results in Nonretroviral cDNA Integration Science, 323 (5912), 393-396 DOI: 10.1126/science.1167375