On episode #332 of the science show This Week in Virology, Vincent visits Vanderbilt University and meets up with Seth, Jim, and Mark to talk about their work on a virus of Wolbachia, anti-viral antibodies, and coronaviruses.

You can find TWiV #332 at www.twiv.tv.

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.


On episode #331 of the science show This Week in Virology, the TWiV team discusses the possible association of the respiratory pathogen enterovirus D68 with neurological disease.

You can find TWiV #331 at www.twiv.tv.

HeLa RNA is everywhere

1 April 2015

Immortal LifeThe first immortal human cell line ever produced, HeLa, originated from a cervical adenocarcinoma taken from Henrietta Lacks. The cell line grew so well that it was used in many laboratories and soon was found to contaminate other cell lines. Now HeLa RNA has made its way into human sequence databases.

Although the cause of Henrietta Lacks’ cervical tumor was not known in her lifetime, we now understand that it was triggered by infection with human papillomavirus (HPV) type 18. When this virus infects the cervical epithelium, the viral DNA may integrate into the host genome, causing the cells to become transformed and eventually malignant. HeLa cells are known to contain integrated HPV18 DNA.

There are many different types of cancer, each caused by errors in DNA. The Cancer Genome Atlas (TCGA) is a database for collecting the DNA sequences of diverse cancers from many different individuals. It was established to help understand what mutations cause various types of cancer. As viruses are known to be responsible for about 20% of human cancers, searching this database for viral sequences can advance our understanding of their role in this disease. For example, almost every genome from patients with cervical cancer contains HPV DNA.

A recent search of the TCGA for viral sequences revealed that, in addition to cervical cancer, HPV18 sequences were found in many other cancers, including colon, head and neck, kidney, liver, lung, ovary, rectum, and stomach. The HPV18 sequences in non-cervical cancers resembled the viral sequence found in HeLa cells, both in integration site and single nucleotide variations. In other words, the HPV18 in these cancers closely matches that of the viral genome integrated into HeLa cells, and their presence is likely due to contamination.

Further analysis revealed that the contaminated samples originated from only two genome sequencing centers, the University of North Carolina Lineberger Comprehensive Cancer Center, and the Michael Smith Genome Sciences Centre of the British Columbia Cancer Agency. All the contamination took place in 2011 and 2012, and was limited to 18 (6%) of the sequencing machines.

The contamination with HeLa nucleic acid was observed only in datasets derived from sequencing of RNA, not DNA. I asked the senior author Jim Pipas how he thought this contamination might have taken place:

I can think of two possibilities. One is that the RNA isolated from the tumor was somehow contaminated with HeLa sequences. The other is that HeLa cell RNA was sequenced on the same machine as the tumors and the contamination is from the sequencing machine itself.

It is well known that nucleic acids can become contaminated during their manipulation in the laboratory. The use of sensitive techniques such as PCR and deep sequencing reveal such contamination when it previously went unnoticed. High profile examples of nucleic acid contamination include the retrovirus XMRV associated with chronic fatigue syndrome, and a virus believed to cause hepatitis (a contaminant from laboratory plasticware).

As virus discoverer Eric Delwart noted on TWiV 86, ‘DNA is a real problem. It’s everywhere’. Apparently so is HeLa cell RNA.

1 comment

On episode #330 of the science show This Week in Virology, the TWiVers explain how a protein platform assists the hepatitis C virus RNA polymerase to begin the task of making viral genomes.

You can find TWiV #330 at www.twiv.tv.

Priming RNA synthesisThe enzymes that make copies of the DNA or RNA genomes of viruses – nucleic acid polymerases – can be placed into two broad categories depending on whether or not they require a primer, a short piece of DNA or RNA, to get going. The structure of the primer-independent RNA polymerase of hepatitis C virus reveals how a priming platform is built into the enzyme.

The requirement for a primer in the initiation step of nucleic acid synthesis varies among the different classes of polymerases. All DNA polymerases are primer-dependent enzymes, while DNA-dependent RNA polymerases initiate RNA synthesis de novo – without a primer. Some RNA-dependent RNA polymerases can also initiate RNA synthesis without a primer: the enzyme begins by adding the first base complementary to the template RNA (illustrated). Other RNA-dependent RNA polymerases require a primer to initiate synthesis. Examples shown on the illustration include the protein-linked primer of picornaviruses, which consists of the protein VPg covalently attached to two U residues. The primer for influenza virus mRNA synthesis is a capped oligonucleotide 12-14 bases in length that is cleaved from the 5′ end of cellular mRNA.

The structure of the RNA-dependent RNA polymerase of hepatitis C virus reveals how a primer-independent RNA polymerase positions the first nucleotide on the RNA template. This process is illustrated below. With the RNA template (dark green) in the active site of the enzyme (panel A), a short beta-loop (brown) provides a platform on which the first complementary nucleotide (light green) is added to the template. The second nucleotide is then added (panel B), producing a dinucleotide primer for RNA synthesis. At this point nothing further can happen because  the priming platform blocks the exit of the RNA product from the enzyme (panel B). The solution to this problem is that the polymerase undergoes a conformational change that moves the priming platform out of the way and allows the newly synthesized complementary RNA (panel C, light green) to exit as the enzyme moves along the template strand.

 HCV priming of RNA synthesis

The structure of the RNA polymerase of hepatitis C virus reveals that it is not really a primer-independent enzyme: a dinucleotide primer is synthesized by the polymerase using a protein platform in the active site. Such protein platforms also appear to be involved in the priming of RNA synthesis by other flaviviruses (dengue and West Nile viruses), influenza virus (genome RNA synthesis is primer independent), reovirus, and bacteriophage phi6. Perhaps all viral RNA-dependent RNA polymerases are dependent on such priming platforms to initiate RNA synthesis.


On episode #329 of the science show This Week in Virology, the TWiV team reviews identification of immune biomarkers in CFS/ME patients, and how a cell nuclease controls the innate immune response to vaccinia virus infection.

You can find TWiV #329 at www.twiv.tv.

Sabin type 2 poliovirusViruses can be broadly classified according to whether or not the particle is enveloped – surrounded by a membrane taken from the host cell – or naked. Some naked viruses apparently are more modest than we believed.

Members of the family Picornaviridae, which include Hepatitis A virus, poliovirus, and Coxsackieviruses, have non-enveloped particles that consist of a protein shell surrounding the viral RNA genome (poliovirus is illustrated). Examples of viruses that are enveloped include dengue virus, influenza virus, and measles virus.

Recently it was discovered that hepatitis A virus (HAV) particles are released from cells in membrane vesicles containing 1-4 virus particles. These membranous structures resemble exosomes, which are also released from uninfected cells and play roles in various biological processes. Enveloped hepatitis A virus particles are present in the blood of infected humans. However virus in the feces, which is transmitted to other hosts, is not enveloped.

Viral envelopes typically contain viral glycoproteins, such as the HA protein of influenza viruses, which serve important functions during replication, such as attachment to cell receptors. Envelope glycoproteins are also the target of antibodies that block viral infection. The presence of an envelope makes HAV resistant to neutralization with antibodies, because the membrane contains no viral proteins that can be blocked by antibodies.

Two other non-enveloped picornaviruses, Coxsackievirus B and poliovirus, are also released from cells within membrane vesicles. These virus particles are in vesicles derived from the autophagy pathway, which captures and recycles cytoplasmic contents by ejecting them from the cell.

What is the function of the membrane acquired by these naked viruses? Perhaps immune evasion: the presence of the cell membrane makes HAV and Coxsackievirus B virus particles resistant to neutralization with antibody. The ability to deliver multiple virus particles to a single cell might help to overcome genetic defects in the viral genome that are a consequence of the high mutation rates of these viruses.

An interesting problem is how these cloaked viruses enter cells, because there is no evidence that the membranes contain any viral proteins that could interact with a cell receptor. Nevertheless, entry of enveloped HAV and poliovirus into cells requires the known viral receptor. Perhaps the vesicles are taken into the cell by endocytosis, where viral particles are released from the vesicles, and then bind receptors to initiate escape of the genome.

Should HAV, poliovirus, and Coxsackievirus B be reclassified as enveloped viruses? Probably not, in part because the membranes surrounding these virus particles are not needed for infectivity. In contrast, removal of the membrane from influenza virus, dengue virus, or measles virus destroys their infectivity. Enveloped viruses acquire a membrane after the internal components have been assembled, whether they are helical or icosahedral nucleocapsids. In contrast, HAV, poliovirus, and Coxsackievirus B become fully infectious particles before they acquire an envelope.

Another argument against calling picornaviruses enveloped is that viral membranes contain viral glycoproteins that allow attachment to cell receptors and release of the viral genome into the cell. There is no evidence that the membranes of picornaviruses contain viral proteins.

The acquisition of a membrane may have taken place later in the evolution of picornaviruses, to allow more efficient infection or evasion of host responses. Alternatively, the membrane may simply be a by-product acquired when these viruses exit the cell by a non-lytic mechanism.

While the finding of membranes around picornavirus particles is intriguing, I am not yet convinced that these viruses should be considered to be enveloped. I would like to know if other non-enveloped viruses are similarly released from cells in membranous cloaks, and the function of this addition for viral replication in the host.


On episode #328 of the science show This Week in Virology, the TWiVocateurs discuss how the RNA polymerase of enteroviruses binds a component of the splicing machinery and inhibits mRNA processing.

You can find TWiV #328 at www.twiv.tv.

Areas with CWDChronic wasting disease (CWD) is a prion disease of cervids (deer, elk, moose). It was first detected in Wyoming and Colorado, and has since spread rapidly throughout North America (illustrated; image credit). Because prions that cause bovine spongiform encephalopathy (BSE, mad cow disease) are known to infect humans, there is concern that CWD might also cross the species barrier and cause a novel spongiform encephalopathy. Recent experimental results suggest that CWD prions are not likely to directly infect humans.

The prion protein PrPC is encoded by the prnp gene, which is essential for the pathogenesis of transmissible spongiform encephalopathies (TSEs). Transgenic mice have been used to understand the species barrier to prion transmission. When mice are inoculated with human prions, few animals develop disease and the incubation periods are over 500 days. When the mouse prnp gene is replaced with the human gene, the mice become uniformly susceptible to infection with human prions and the incubation period is shorter. The species barrier to prion transmission is therefore associated with differences in the prion protein sequence between host and target species.

Mice have been used to understand whether CWD prions might be transmitted to humans. Mice are not efficiently infected with CWD prions unless they are made transgenic for the cervid prnp gene. Four different research groups have found that  mice transgenic for the human prnp gene are not infected by CWD prions. These findings suggest that CWD prions are not likely to be transmitted directly to humans. However, changing four amino acids in human prnp to the cervid sequence allows efficient infection of transgenic mice with cervid prions.

Another concern is that prions of chronic wasting disease could be transmitted to cows grazing in pastures contaminated by cervids. Prions can be detected in deer saliva and feces, and contamination of grass could pass the agent on to cows. In the laboratory, brain homogenates from infected deer can transmit the disease to cows. Therefore it is possible that cervid prions could enter the human food chain through cows.

A further worry is that BSE prions shed by cows in pastures might infect cervids, which would then become a reservoir of the agent. BSE prions do not infect mice that are transgenic for the cervid prnp gene. However, intracerebral inoculation of deer with BSE prions causes neurological disease,  and the prions from these animals can infect mice that are transgenic for the cervid prnp gene. Therefore caution must be used when using transgenic mice to predict the abilities of prions to cross species barriers.

Although the risk of human infection with CWD prions appears to be low, hunters should not shoot or consume an elk or deer that is acting abnormally or appears to be sick, to avoid the brain and spinal cord when field dressing game, and not to consume brain, spinal cord, eyes, spleen, or lymph nodes. No case of transmission of chronic wasting disease prions to deer hunters has yet been reported.