There aren’t enough human organs to meet the needs for transplantation, so we have turned to pigs. Unfortunately pig cells contain porcine endogenous retroviruses, PERVS, which could infect the transplant recipient, leading to tumor formation. But why worry? Just use CRISPR to purge the PERVs.

The genomes of many species on Earth are littered with endogenous retroviruses. These are DNA copies of retroviral genomes from previous infections that are integrated into germ line DNA and passed from parent to offspring. About 8% of the human genome consists of ERVs. The pig genome is no different – it contains PERVs (an acronym made to play with). The genome of an immortalized pig cell line called PK15 contains 62 PERVs. Human cells become infected with porcine retroviruses when they are co-cultured with PK15 cells.

The presence of PERVS is an obvious problem for using pig organs for transplantation into humans – a process called xenotransplantation. The retroviruses produced by pig cells might infect human cells, leading to problems such as immunosuppression and tumor formation. No PERV has ever been shown to be transmitted to a human, but the possibility remains, especially with   the transplantation of increasing numbers of pig organs into humans.

The development of CRISPR/Cas9 gene editing technology made it possible to remove PERVs from pigs, potentially easing the fears of xenotransplantation. This technology was first used to remove all 62 copies of PERVS from the PK15 cell line. But having PERV-free pig cells doesn’t help humans in need of pig organs – for that you need pigs.

To make pigs without PERVs, CRISPR/Cas9 was used to remove the PERVs from primary (that is, not immortal) pig cells in culture. Next, the nuclei of these PERV-less cells was used to replace the nucleus of a pig egg cell. After implantation into a female, these cells gave rise to piglets lacking PERVs.

In theory such PERV-less piglets can be used to supply organs for human transplantation, eliminating the worrying about infecting humans with pig retroviruses. But first we have to make sure that the PERV-free pigs, and their organs, are healthy. The more we study ERVs, the more we learn that they supply important functions for the host. For example, the protein syncytin, needed to form the placenta, is a retroviral gene, and the regulatory sequences of interferon genes come from retroviruses. There are likely to be many more examples of essential functions provided by ERVs. It would not be a good idea to have transplanted pig organs fail because they lack an essential PERV!

Sharon Isern and Scott Michael return to TWiV for a Zika virus update, including their work on viral evolution and spread, and whether pre-existing immunity to dengue virus enhances pathogenesis.

Click arrow to play
Download TWiV 454 (65 MB .mp3, 108 min)
Subscribe (free): iTunes, RSS, email

Become a patron of TWiV!

Show notes at microbe.tv/twiv

The short answer to the question posed in the title of this blog is: we don’t know.

Why would we even consider that a prior dengue virus infection would increase the severity of a Zika virus infection? The first time you are infected with dengue virus, you are likely to have a mild disease involving fever and joint pain, from which you recover and develop immunity to the virus. However, there are four serotypes dengue virus, and infection with one serotype does not provide protection against infection with the other three. If you are later infected with a different dengue virus serotype, you may even experience more severe dengue disease involving hemorrhagic fever and shock syndrome.

The exacerbation of dengue virus disease has been documented in people. Upon infection with a different serotype, antibodies are produced against the previous dengue virus encountered. These antibodies bind the new dengue virus but cannot block infection. Dengue virus then enters and replicates in cells that it does not normally infect, such as macrophages. Entry occurs when Fc receptors on the cell surface bind antibody that is attached to virus particles (illustrated). The result is higher levels of virus replication and more severe disease. This phenomenon is called antibody-dependent enhancement, or ADE.

When Zika virus emerged in epidemic form, it was associated with microcephaly and Guillain-Barré syndrome, diseases that had not been previously known to be caused by infection with this virus. As Zika virus and dengue virus are closely related, because ADE was known to occur with dengue virus, and both viruses often co-circulated, it was proposed that antibodies to dengue virus might exacerbate Zika virus disease.

It has been clearly shown by several groups that antibodes to dengue virus can enhance Zika virus infection of cells in culture. Specifically, adding dengue virus antibodies to Zika virus allows it to infect cells that bear receptors for antibodies – called Fc receptors. Without Fc receptors, the Zika virus plus dengue antibodies cannot infect these cells. ADE in cultured cells has been reported by a number of groups; the first was discussed here when it appeared on bioRxiv.

The important question is whether antibodies to dengue virus enhance Zika virus disease in animals, and there the results are mixed. In one experiment, mice were injected with serum from people who had recovered from dengue virus infection, followed by challenge with Zika virus. These sera, which cause ADE of Zika virus in cultured cells, led to increased fever, viral loads, and death of mice.

These finding were not replicated in two independent studies conducted in rhesus macaques (paper one, paper two). In these experiments, the macaques were first infected with dengue virus, and shown to mount an antibody response to that virus. Over one year later the animals were infected with Zika virus (the long time interval was used because in humans dengue ADE is observed mainly with second infections 12 months or more after a primary infection). Both groups concluded that prior dengue virus immunity did not lead to more severe Zika virus disease.

Which animals are giving us the right answer, mice or monkeys? It should be noted that the mouse study utilized an immunodeficient strain lacking a key component of innate immunity. As the authors of paper one concluded, it’s probably not a good idea to use immune deficient mice to understand the pathogenesis of Zika virus infection of people.

When it comes to viral pathogenesis, we know that mice lie; but we also realize that monkeys exaggerate. Therefore we should be cautious in concluding from the studies on nonhuman primates that dengue virus antibodies do not enhance Zika virus pathogenesis.

The answer to the question of whether dengue antibodies cause Zika virus ADE will no doubt come from carefully designed epidemiological studies to determine if Zika virus pathogenesis differs depending on whether the host has been previously infected with dengue virus. Such studies have not yet been done*.

You might wonder about the significance of dengue virus antibodies enhancing infection of cells in culture with Zika virus. An answer is provided by the authors of paper one:

In vitro ADE assays using laboratory cell lines are notoriously promiscuoius and demonstrate no correlation with disease risk. For example, DENV-immune sera will enhance even the homotypic serotype responsible for a past infection in the serum is diluted to sub-neutralizing concentrations.

The conundrum of whether ADE is a contributor to Zika virus pathogeneis is an example of putting the cart before the horse. For dengue virus, we obtained clear evidence of ADE in people before experiments were done in animals. For Zika virus, we don’t have the epidemiological evidence in humans, and therefore interpreting the animals results are problematic.

*Update 8/12/17: A study has been published on Zika viremia and cytokine levels in patients previously infected with dengue virus. The authors find no evidence of ADE in patients with acute Zika virus infection who had previously been exposed to dengue virus. However the study might not have been sufficiently powered to detect ADE.

From the Vector-Borne Viruses Symposium in Hamilton, Montana, Dickson and Vincent speak with Diane Griffin about her career and her work on understanding viral infections of the central nervous system.

Click arrow to play
Download TWiV 453 (39 MB .mp3, 64 min)
Subscribe (free): iTunes, RSS, email

Become a patron of TWiV!

Show notes at microbe.tv/twiv

Lynda Coughlan joins the weekly virtual bus companions for a discussion of a host defense peptide from frogs that destroys influenza virus, and mouse models for acute and chronic hepacivirus infection.

Click arrow to play
Download TWiV 452 (68 MB .mp3, 113 min)
Subscribe (free): iTunes, RSS, email

Become a patron of TWiV!

Show notes at microbe.tv/twiv

Frogs don’t get flu (as far as I know) but their skin contains a peptide that inhibits the replication of influenza virus (link to paper).

Frog skin contains host defense peptides (HDPs), part of the innate immune defenses of many species. They were first found in amphibians by Michael Zasloff, who, as part of his research, performed surgery on frogs and then returned them to an aquarium – which was not sterile. He wondered why the frogs always healed without signs of infection, which lead him to discover the antimicrobial peptides, called magainins, in frog skin. HDPs had been first discovered years earlier in the silk moth.

Amphibian HDPs are active against bacteria, fungi, viruses, and protozoa. To discover HDPs that inhibit influenza virus, 32 HDPs from skin secretions of the Indian frog Hydrophylax bahuvistara were screened by mixing them with virus followed by a plaque assay. One peptide was found to potently inhibit influenza virus replication without cell toxicity. It was called urumin, after the whip sword known as urumi.

Urumin inhibits infectivity of influenza H1N1 viruses far better than H3N2 viruses. The reason is that the peptide targets the viral H1 hemagglutinin, one of two glycoproteins in the viral envelope. Furthermore, the peptide appears to interact with the conserved stalk region of the HA glycoprotein, and not with the globular head.

Currently two different antiviral drugs, oseltamivir and relenza, are used to control influenza virus infection. Viruses resistant to these drugs were still inhibited by urumin, indicating that should urumin ever be licensed, it would be useful in the event that oseltamivir and relenza resistant viruses became more common.

Examination of urumin treated virus particles by electron microscopy revealed that they are disrupted by the peptide. How urumin breaks influenza virus particles is not known. However, the HDP nisin destroys bacteria by first binding to a bacterial membrane component, then moving into the membrane. After binding to HA, urumin might in a simlar way disrupt the membrane of influenza virus particles.

Urumin also reduced disease, death, and the amount of virus in the lung in mice intranasally infected with influenza virus.

These observations suggest that urumin is worthy of additional study as an influenza virus inhibitor. HDPs are attractive antimicrobial compounds because resistance to their mechanisms of action is lower than for other types of inhibitors. However, enthusiasm for urumin is dampened because, despite extensive study, no HDP has yet been approved by the US Food and Drug Administration for use in humans. The obstacles to therapeutic success of HDPs have not been identified.

Could ancient host proteins contribute to the replication of a modern virus? The answer is, not very well (link to paper).

Viruses are obligate intracellular parasites, which means that they have to get inside of a host cell to produce more viruses. The genomes of all viruses, even the biggest ones, do not encode anywhere near the number of proteins that are needed to replicate. The cell provides thousands of proteins that are involved in energy production, membrane synthesis, protein synthesis, transport, and so much more.

The difficulty in studying ancient proteins is that none of them exist. But we can make good guesses about what very old proteins might look like, by examining modern proteins, seeing how they vary among organisms, and calculating how they might look like billions of years ago. The field of predicting what ancient proteins might look like is quite active.

Investigators have predicted what ancient versions of a cell protein called thioredoxin might have looked like. They have synthesized such ‘ancient’ thioredoxins and shown that they are stable and active. Thioredoxins are found in nearly all organisms, where they act as antioxidants.

Ancient thioredoxins that have been synthesized include those from the last common ancestors of bacteria; of archaea; and of archaea and eukaryotes (all around 4 billion years old); the last common anestor of cyanobacterial, deinococcus, and thermus groups (about 2.5 billion years old);  the last common ancestor of gamma-proteobacteria; of eukaryotes; and of fungi and animals (around 1.5 billion years old).

These ancient thioredoxins work in a modern E. coli. This bacterium has two thioredoxin genes, and if they are both deleted, growth occurs, but very slowly. If genes encoding ancient thioredoxins are introduced into these mutated bacteria, they can compensate for the growth deficiency. The older thioredoxins (4 billion years) compensate less well than ones that are closer in time (1.5 billion years).

It’s amazing that an ancient protein can work in a modern E. coli. But could ancient thioredoxins support viral growth?

Thioredoxin from E. coli is an essential part of the DNA polymerase complex of the bacteriophage T7 (pictured – image credit). This virus does not form plaques on E. coli lacking the two thioredoxin genes. The only ancient thioredoxin gene that allows phage T7 plaque formation is from the last common ancestor of cyanobacterial, deinococcus, and thermus groups, which is about 2.5 billion years old and has 57% amino acid identity with the E. coli enzyme. But the effienciency of plaque formation was very poor – about 100 million times worse than on regular  E. coli. None of the older thioredoxins worked.

Why would an ancient thioredoxin work for E. coli but not for bacteriophage T7? Over billions of years, thioredoxin evolves but it must still be able to carry out its function for E. coli. The viruses that infected bacteria 4 billion years ago were very different from contemporary viruses, and so the ancient thioredoxin does not work for modern viruses. Today’s thioredoxin could change so that it would not support T7 replication – as long as the enzyme still works for E. coli.

The authors of this work view it as a proof of principle: that virus growth is not supported by an ancient version of a modern protein required for virus replication. They would like to apply this approach to produce plants that are resistant to viruses, which have serious effects on global agricultural productivity.

I think the work is amazing not only because an ancient protein can be made, but it supports growth of the host and not that of a virus. It might therefore be possible to reconstruct the host-virus arms race, starting from ancient proteins. In this race, the gene encoding an essential cell protein can evolve so that it no longer supports virus replication. Next, the viral genome changes to adapt to the altered cell protein. And so the game goes back and forth.

The authors have shown that they can select mutant bacteriophage T7 isolates that replicate in the present of an ancient thioredoxin. This result suggests that it might be possible to reconstruct host-virus arms races beginning with an ancestral host protein. If we can make an ancient protein, could we also make an ancient virus? Why not?

Ben tenOever joins the TWiVoli to discuss the evolution of RNA interference and his lab’s finding that RNAse III nucleases, needed for the maturation of cellular RNAs, are an ancient antiviral RNA recognition platform in all domains of life.

Click arrow to play
Download TWiV 450 (58 MB .mp3, 96 min)
Subscribe (free): iTunes, RSS, email

Become a patron of TWiV!

Show notes at microbe.tv/twiv

I just love it when long standing mysteries in virology are suddenly solved, typically by the use of new technologies. In this story, the long standing mystery was why poxvirus mRNAs have a stretch of poly(A) in their 5′-noncoding regions. The answer is that it allows the ribosome to preferentially translate these viral mRNAs over those of the host (link to paper).

Ribosomes, the sites of protein synthesis, are very large assemblies of RNAs and proteins. A ribosome-associated protein called RACK1 is the major player in this story. This protein was previously found to be important for translation of viral RNAs by internal ribosome entry, a process in which ribosomes bypass the 5′-cap structure on mRNAs.

Cells lacking the gene encoding RACK1 seem to be just fine – they grow normally. This result would suggest that RACK1 is not needed for translation of mRNAs via the 5′-cap structure. But the translation of mRNAs of vaccinia virus, a poxvirus, is blocked in cells lacking RACK1. More specifically, the viral mRNAs produced later in infection cannot be translated.

This effect of RACK1 on vaccinia virus mRNA translation prompted a closer look at the protein in virus infected cells – which revealed that it is phosphorylated by a viral protein kinase. Phosphorylation of RACK1 makes the ribosomes preferentially translate vaccina virus mRNAs – an effect that is completely dependent upon the poly(A) in the 5′-untranslated region! Transfer of this poly(A) sequence to a non-viral mRNA enhanced its translation in cells in which RACK1 is phosphorylated.

The amino acid sequence of RACK1 that is phosphorylated is within a loop that contacts the 18S ribosomal RNA subunit. An examination of RACK1 proteins from different species reveals that in plants, unlike in mammals, this loop contains a stretch of negatively charged amino acids. Because a phosphate is negatively charged, it made perfect sense to see if the plant loop sequence, when transferred to mammalian RACK1, could stimulate translation of poly(A) containing mRNAs. It did!

In other words, phosphorylation by a vaccinia virus protein kinase makes RACK1 plant-like! Which is why the authors called their paper ‘Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase”. Exactly what RACK1 protein is doing in plants is a burning question, as is how RACK1 recognizes mRNAs containing a poly(A) leader.

Phosphorylation of RACK1 by the vaccinia virus kinase explains why viral mRNAs with poly(A) leader sequences are preferentially translated in infected cells (late viral mRNAs have this unusual leader). The presence of poly(A) in the 5′-ends of late vaccinia virus mRNAs is considered to be a consequence of polymerase slippage – but it is clearly not a mistake. At one time, long ago, a polymerase might have slipped for the first time, but the resulting poly(A) in the mRNA conveyed a selective advantage that remained. Today I would no longer call the addition of poly(A) to vaccinia virus mRNAs an error!

Why did it take so long to figure out the role of poly(A) at the 5’-end of vaccinia virus mRNAs? The right technology was not available – the key ones were the ability to knock out specific genes in cells, to knock down mRNA levels with RNA interference, and to identify the sites of RACK1 phosphorylation by mass spectrometry. These are not only recently developed techniques, but they have become widely accessible. But don’t forget the use of long-ago isolated vaccinia virus mutants of the protein kinase that were an essential part of this story.

Rich Condit, a poxvirologist, joined Nels Elde (also a poxvirologist, though he dabbles in other systems) and me to discuss this very cool paper on episode #21 of TWiEVO, This Week in Evolution. Rich had some very good insight on this paper; plus he bristled twice during the episode. You’ll have to listen to find out why.

Image credit.