Interview with J. Michael Bishop

A major new feature of the fourth edition of Principles of Virology is the inclusion of 26 video interviews with leading scientists who have made significant contributions to the field of virology. For the chapter on Transformation and Oncogenesis, Vincent spoke with Nobel Laureate J. Michael Biship, of the University of California, San Francisco, about his career and his work on oncogenes.

TWiV 386: The dolphins did it

TWiVDid you know that the evolution of ancient retroviruses, millions of years ago, can be traced by studying their genomes in the chromosomes of contemporary animals? Ted Diehl and Welkin Johnson join the TWiV team to tell us how they did it with mammals. All without a single wet experiment! They also join in the discussion about virus dispersal by hand dryers.

You can find TWiV #386 at microbe.tv/twiv, or listen below.

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TWiV 382: Everyone’s a little bit viral

TWiVOn episode #382 of the science show This Week in Virology, Nels Elde and Ed Chuong join the TWiV team to talk about their observation that regulation of the human interferon response depends on regulatory sequences that were co-opted millions of years ago from endogenous retroviruses.

You can find TWiV #382 at microbe.tv/twiv, or listen below.

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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 360: From Southeastern Michigan

On episode #360 of the science show This Week in Virology, Vincent visits the University of Michigan where he and Kathy speak with Michael, Adam, and Akira about polyomaviruses, virus evolution, and virus assembly, on the occasion of naming the department of Microbiology & Immunology a Milestones in Microbiology site.

You can find TWiV #360 at www.microbe.tv/twiv. Or you can watch the video below.

TWiV 337: Steamer

On episode #337 of the science show This Week in Virology, Vincent meets up with Michael and Steve to discuss their finding of a transmissible tumor in soft-shell clams associated with a retrovirus-like element in the clam genome.

You can find TWiV #337 at www.microbe.tv/twiv.

Retroviral influence on human embryonic development

EmbryogenesisAbout eight percent of human DNA is viral: it consists of retroviral genomes produced by infections that occurred many years ago. These endogenous retroviruses are passed from parent to child in our DNA. Some of these viral genomes are activated for a brief time during human embryogenesis, suggesting that they may play a role in development.

There are over 500,000 endogenous retroviruses in the human genome, about 20 times more than human genes. They were acquired millions of years ago after retroviral infection. In this process, viral RNA is converted to DNA, which then integrates into cell DNA. If the retroviral infection takes place in the germ line, the integrated DNA may be passed on to offspring.

The most recent human retroviral infections leading to germ line integration took place with a subgroup of human endogenous retroviruses called HERVK(HML-2). The human genome contains ~90 copies of these viral genomes, which might have infected human ancestors as recently as 200,000 years ago. HERVs do not produce infectious virus: not only is the viral genome silenced – no mRNAs are produced – but they are littered with lethal mutations that have accumulated over time.

A recent study revealed that HERVK mRNAs are produced during normal human embryogenesis. Viral RNAs were detected beginning at the 8-cell stage, through epiblast cells in preimplantation embryos, until formation of embryonic stem cells (illustrated). At this point the production of HERVK mRNA ceases. Viral capsid protein was detected in blastocysts, and electron microscopy revealed the presence of virus-like particles similar to those found in reconstructed HERVK particles. These results indicate that retroviral proteins and particles are present during human development, up until implantation.

Retroviral particles in blastocysts are accompanied by induction of synthesis of an antiviral protein, IFITM1, that is known to block infection with a variety of viruses, including influenza virus. A HERVK protein known as Rec, produced in blastocysts, binds a variety of cell mRNAs and either increases or decreases their association with ribosomes.

Is there a function for HERVK expression during human embryogenesis? The authors speculate that modulation of the ribosome-binding activities of specific cell mRNAs by the viral Rec protein could influence aspects of early development. As Rec sequences are polymorphic in humans, the effects could even extend to individuals. In addition, HERVK induction of IFITM1 might conceivably protect embryos against infection with other viruses.

The maintenance of open reading frames in HERV genomes, over many years of evolution, suggests a functional role for these elements. Evidence for such function comes from the syncytin proteins, which  are essential for placental development: the genes encoding these proteins originated from HERV glycoproteins. However, not all endogenous retroviruses are beneficial: a number of malignant diseases have been associated with HERV-K expression.

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.

Amyotrophic lateral sclerosis and viruses

Many people have a new awareness of the disease known as amyotrophic lateral sclerosis, or ALS, thanks to the Ice Bucket Challenge initiated by the ALS Association. Fewer might know that retroviruses have been proposed to play a role in the development of the disease.

I previously summarized a 2008 paper on ALS in a piece called Retroviruses and amyotrophic lateral sclerosisSera from some ALS patients had previously been shown to contain elevated levels of reverse transcriptase, an enzyme found in retrovirus particles. In the 2008 paper, RNAs encoding this enzyme were reported in the brains of ALS patients, and their origin appears to be the human endogenous retrovirus HERV-K.

The progress made in understanding the relationship of endogenous retroviruses with ALS is summarized in a review published in August of 2014 entitled Retroviruses and amyotrophic lateral sclerosis (the paper is open access). The authors conclude:

A comprehensive study of the expression or reactivation of endogenous retroviral elements in ALS has not yet been undertaken. The literature on HERV-W involvement in ALS is difficult to interpret. Two independent reports, however, have shown increased HERV-K expression in both serum and brain tissue in ALS patients. It remains unknown if HERV-K expression is an epiphenomenon or plays a pathophysiological role in the disease.

I am pleased to participate in the Ice Bucket Challenge to help raise awareness of ALS and raise money to work on the disease.

De-discovering pathogens: Viral contamination strikes again

Spin column

Qiagen spin column at right. The silica layer is white. The spin column is placed in the microcentrifuge tube, left, to remove liquids and elute nucleic acids.

Do you remember the retrovirus XMRV, initially implicated as the cause of chronic fatigue syndrome, and later shown to be a murine virus that contaminated human cells grown in mice? Another virus thought to be associated with human disease has recently been shown to be a contaminant, derived from a piece of laboratory plasticware that is commonly used to purify nucleic acids from clinical samples.

During a search for the causative agent of seronegative hepatitis (disease not caused by hepatitis A, B, C, D, or E virus) in Chinese patients, a novel virus was discovered in sera by next generation sequencing. This virus, provisionally called NIH-CQV, has a single-stranded DNA genome that is a hybrid between parvoviruses and circoviruses. When human sera were screened by polymerase chain reaction (PCR), 63 of 90 patient samples (70%) were positive for the virus, while sera from 45 healthy controls were negative. Furthermore, 84% of patients were positive for IgG antibodies against the virus, and 31% were positive for IgM antibodies (suggesting a recent infection). Among healthy controls, 78% were positive for IgG and all were negative for IgM. The authors concluded that this virus was highly prevalent in some patients with seronegative hepatitis.

A second independent laboratory also identified the same virus (which they called PHV-1) in sera from patients in the United States with non-A-E hepatitis, while a third group identified the virus in diarrheal stool samples from Nigeria.

The first clue that something was amiss was the observation that the novel virus identified in all three laboratories shared 99% nucleotide and amino acid identity. This would not be expected in virus samples from such geographically, temporally, and clinically diverse samples. Another problem was that in the US non-A-E study, all patient sample pools were positive for viral sequences. These observations suggested the possibility of viral contamination.

When nucleic acids were re-purified from the US non-A-E samples using a different method, none of the samples were positive for the novel virus. Presence of the virus was ultimately traced to the use of column-based purification kits manufactured by Qiagen, Inc. Nearly the entire novel viral genome could be detected by deep sequencing in water that was passed through these columns.

The nucleic acid purification columns contaminated with the novel virus were used to purify nucleic acid from patient samples. These columns (pictured), produced by a number of manufacturers, are typically a few inches in length and contain a silica gel membrane that binds nucleic acids. The clinical samples are added to the column, which is then centrifuged briefly to remove liquids (hence the name ‘spin’ columns). The nucleic acid adheres to the silica gel membrane. Contaminants are washed away, and then the nucleic acids are released from the silica by the addition of a buffer.

Why were the Qiagen spin columns contaminated with the parvovirus-circovirus hybrid? A search of the publicly available environmental metagenomic datasets revealed the presence of sequences highly related to PHV-1 (87-99% nucleotide identity). The datasets containing PHV-1 sequences were obtained from sampled seawater off the Pacific coast of North America, and coastal regions of Oregon and Chile. Silica, a component of spin columns, may be produced from diatoms. If the silica in the Qiagen spin columns was produced from diatoms, and if PHV-1 is a virus of ocean-dwelling diatoms, this could explain the source of contamination.

In retrospect it was easy to be fooled into believing that NIH-CQV might be a human pathogen because it was only detected in sick, and not healthy patients. Why antibodies to the virus were detected in samples from sick and healthy patients remains to be explained. However NIH-CQV/PHV-1 is likely not associated with any human illness: when non-Qiagen spin columns were used, PHV-1 was not found in any patient sample.

The lesson to be learned from this story is clear: deep sequencing is a very powerful and sensitive method and must be applied with great care. Every step of the virus discovery process must be carefully controlled, from the water used to the plastic reagents. Most importantly, laboratories involved in pathogen discovery must share their sequence data, something that took place during this study.

Trust science, not scientists.