A huge host contribution to virus mutation rates

HIV-1 mutation rateThe high mutation rate of RNA viruses enables them to evolve in the face of different selection pressures, such as entering a new host or countering host defenses. It has always been thought that the sources of such mutations are the enzymes that copy viral RNA genomes: they make random errors which they cannot correct. Now it appears that a cell enzyme makes an even greater contribution the mutation rate of an RNA virus.

Deep sequencing was used to determine the mutation rate of HIV-1 in the blood of AIDS patients by searching for premature stop codons in open reading frames of viral RNA. Because stop codons terminate protein synthesis, they do not allow production of infectious viruses. Therefore they can be used to calculate the mutation rate in the absence of selection. The mutation rate calculated in this way, 0.000093 mutations per base per cell, was slightly higher than previously calculated from studies in cell culture.

When HIV-1 infects a cell, the enzyme reverse transcriptase converts its RNA genome to DNA, which then integrates into the host cell genome. Identification of stop codons in integrated viral DNA should provide an even better estimate of the mutation rate of reverse transcriptase, because mutations that block the production of infectious virus have not yet been removed by selection. The mutation rate calculated by this approach was 0.0041 mutations per base per cell, or one mutation every 250 bases. This mutation rate is 44 times higher than the value calculated from viral RNA in patient plasma (illustrated).

Sequencing of integrated viral DNA from many patients revealed that the vast majority of mutations leading to insertion of stop codons – 98% – were the consequence of editing by the cellular enzyme APOBEC3G. This enzyme is a deaminase that changes dC to dU in the first strand of viral DNA synthesized by reverse transcriptase. APOBEC3G constitutes an intrinsic defense against HIV-1 infection, because extensive mutation of the viral DNA reduces viral infectivity. Indeed, most integrated HIV proviruses are not infectious as a consequence of APOBEC3G-induced mutations. That infection proceeds at all is due to incorporation of the viral protein vif in the virus particles. Vif binds APOBEC3G, leading to its degradation in cells.

The mutation rate of integrated HIV-1 DNA calculated by this method is much higher than that of other RNA viruses. This high mutation rate is driven by the cellular enzyme, APOBEC3G. At least half of the mutations observed in plasma viral RNAs are also contributed by this enzyme.

It has always been thought that error-prone viral RNA polymerases are largely responsible for the high mutation rates of RNA viruses. The results of this study add a new driver of viral variation, a cellular enzyme. APOBEC enzymes are known to introduce mutations in the genomes of other viruses, including hepatitis B virus, papillomaviruses, and herpesviruses. Furthermore, the cellular adenosine deaminase enzyme can edit the genomes of RNA viruses such as measles virus, parainfluenza virus, and respiratory syncytial virus. Cellular enzymes may therefore play a much greater role in the generation of viral diversity than previously imagined.

TWiV 320: Retroviruses and cranberries

On episode #320 of the science show This Week in Virology, Vincent speaks with John Coffin about his career studying retroviruses, including working with Howard Temin, endogenous retroviruses, XMRV, chronic fatigue syndrome and prostate cancer, HIV/AIDS, and his interest in growing cranberries.

You can find TWiV #320 at www.microbe.tv/twiv.

TWiV 301: Marine viruses and insect defense

On episode #301 of the science show This Week in Virology, Vincent travels to the International Congress of Virology in Montreal and speaks with Carla Saleh and Curtis Suttle about their work on RNA interference and antiviral defense in fruit flies, and viruses in the sea, the greatest biodiversity on Earth.

You can find TWiV #301 at www.microbe.tv/twiv.

TWiV 293: Virology Down Under

On episode #293 of the science show This Week in VirologyVincent visits Melbourne, Australia and speaks with Melissa, Alex, Gilda, and Paul about their work on HIV infection of the central nervous system, West Nile virus, microbicides for HIV, and the Koala retrovirus.

You can find TWiV #293 at www.microbe.tv/twiv.

TWiV 288: ebircsnart esreveR

On episode #288 of the science show This Week in Virology, the Twivsters discuss how reverse transcriptase encoded in the human genome might produce DNA copies of RNA viruses in infected cells.

You can find TWiV #288 at www.microbe.tv/twiv.

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.

TWiV 163: What Rous wrought

fungal christmas treeHosts: Vincent Racaniello, Dickson DespommierRich Condit, and Alan Dove

Vincent, Dickson, Rich, and Alan review the 100 year old finding by Peyton Rous of a transmissible sarcoma of chickens, a discovery that ushered in the era of tumor virology.

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TWiV 100: TWiV catches a big fish

Hosts: Vincent Racaniello, Alan Dove, Rich Condit, and David Baltimore

Vincent, Alan, and Rich celebrate the 100th episode of the podcast This Week in Virology by talking about viruses with Nobel Laureate David Baltimore.

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Virology lecture #7: Reverse transcription and integration


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Visit the virology W3310 home page for a complete list of course resources.

TWiV 66: Reverse transcription

Hosts: Vincent Racaniello and Dickson Despommier

Vincent and Dickson continue virology 101 with a discussion of information flow from RNA to DNA, a process known as reverse transcription, which occurs in cells infected with retroviruses, hepatitis B virus, cauliflower mosaic virus, foamy viruses, and even in uninfected cells.

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Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv or leave voicemail at Skype: twivpodcast. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.