Defective viral genomes and severe influenza

Defective influenza virus RNAsThe virulence of a virus – its capacity to cause disease – is determined by both viral and host factors. Even among healthy individuals, infection with a particular virus may have different outcomes ranging from benign to lethal. The study of influenza viruses that cause mild or fatal outcomes reveals that defective viral genomes play a role in determining viral virulence.

Defective viral genomes arise during the replication of many different viruses. They are produced by polymerase errors during copying and are incorporated into virus particles. However, because the genomes contain deletions (pictured – image credit), they are defective and cannot sustain a reproductive cycle. As a consequence, virus stocks containing defective genomes often have low infectivity.

Two influenza H1N1 viruses, one isolated from an individual with mild symptoms (M-IAV) and one from a fatal case (F-IAV) were studied to determine why virulence differed. The F-IAV isolate replicated to higher titers in cell culture, and is more pathogenic in mice, than the M-IAV isolate. Accumulation of defective genomes in both cell cultures and in mice was higher for the M-IAV isolate than for the F-IAV isolate.

Single amino acid changes in subunits of the viral RNA polymerase were identified which, when introduced into the genome of the 2009 H1N1 pandemic virus, led to production, in infected cells, of high or low levels of defective RNA genomes. Viruses that produce high levels of defective genomes were less pathogenic in mice than viruses that produce low levels of defective genomes.

To determine if levels of defective genomes correlate with virulence in humans, a series of isolates was obtained from patients with either mild or severe influenza. In cell culture, isolates from cases of severe influenza produced ten times less defective RNA than isolates from mild cases. However, the mutations in these isolates were different from those previously studied, showing that different amino acid changes can lead to the production of defective RNA.

How does the production of defective viral genomes regulate virulence? One hypothesis is that defective RNAs are good inducers of the innate immune system, leading to an enhanced antiviral state, a reduction in viral replication, and mild disease. Viruses that produce low levels of defective RNAs are poor inducers of the antiviral state; consequently higher viral replication ensues, accompanied by more severe disease. In support of this hypothesis, viruses that produce low levels of defective genomes were found to induce lower amounts of interferon-stimulated genes than viruses that produce high levels of defective genomes. Furthermore, the increased viral replication in mice associated with viruses that produce low levels of defective genomes was found to cause more infiltration of neutrophils, and depletion of macrophages in the mouse lung. These effects are similar to those observed in lethal human influenza virus infections.

Central to thinking about the evolution of viral virulence is to understand the selective pressures involved. In the case of more lethal viruses that produce low levels of defective genomes, the consequence is that the virus is able to replicate to higher titers in the host. In turn, higher virus titers in the lung could facilitate more efficient transmission. As transmission to another host is a major selective force, the increase in influenza virus virulence caused by low defective RNA production makes evolutionary sense. However, as not all influenza virus strains evolve towards greater virulence, there must also be fitness costs associated with this property.

TWiV 450: Ben tenOever and RNA out

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): iTunesRSSemail

Become a patron of TWiV!

Show notes at microbe.tv/twiv

TWiV 430: The persistence of herpesvirus

The TWiX cabal discuss sexual transmission of Zika virus in mice, and how immune escape enables herpes simplex virus escape from latency.

You can find TWiV #430 at microbe.tv/twiv, or listen below.


Click arrow to play
Download TWiV 430 (63 MB .mp3, 104 min)
Subscribe (free): iTunesRSSemail

Become a patron of TWiV!

TWiV 428: Lyse globally, protect locally

The TWiVsters explain how superspreader bacteriophages release intact DNA from infected cells, and the role of astrocytes in protecting the cerebellum from virus infection.

You can find TWiV #428 at microbe.tv/twiv, or listen below.

[powerpress url=”http://traffic.libsyn.com/twiv/TWiV428.mp3″]

Click arrow to play
Download TWiV 428 (65 MB .mp3, 108 min)
Subscribe (free): iTunesRSSemail

Become a patron of TWiV!

TWiV 426: I’m Axl, and I’ll be your cervid today

The sages of TWiV explain how chronic wasting disease of cervids could be caused by spontaneous misfolding of prion protein, and the role of the membrane protein Axl in Zika virus entry into cells.

You can find TWiV #426 at microbe.tv/twiv, or listen below.

[powerpress url=”http://traffic.libsyn.com/twiv/TWiV426.mp3″]

Click arrow to play
Download TWiV 426 (66 MB .mp3, 110 min)
Subscribe (free): iTunesRSSemail

Become a patron of TWiV!

TWiV 412: WO, open the borders and rig the infection

The TWiVome reveal the first eukaryotic genes found in a bacteriophage of Wolbachia, and how DNA tumor virus oncogenes antagonize sensing of cytoplasmic DNA by the cell.

You can find TWiV #412 at microbe.tv/twiv, or listen below.

[powerpress url=”http://traffic.libsyn.com/twiv/TWiV412.mp3″]

Click arrow to play
Download TWiV 412 (73 MB .mp3, 121 min)
Subscribe (free): iTunesRSSemail

Become a patron of TWiV!

A new function for oncoproteins of DNA tumor viruses

oncoproteinsOncogenes of DNA tumor viruses encode proteins that cause cells to divide incessantly, eventually leading to formation of a tumor. These oncoproteins have now been found to antagonize the innate immune response of the cell (link to paper).

Most cells encountered by viruses are not dividing, and hence do not efficiently support viral DNA synthesis. The genomes of adenoviruses, polyomaviruses, and papillomaviruses encode proteins that cause cells to divide. This effect allows for efficient viral replication, because a dividing cell is producing the machinery for DNA synthesis. Under certain conditions, infections by these viruses do not kill cells, yet they continue to divide due to the presence of viral oncoproteins. Such incessant division gives the cells new properties – they are called transformed cells – and they may eventually become a tumor.

These so-called viral oncoproteins include large T antigen (of SV40, a polyomavirus); E6 and E7 (papillomavirus), and E1A (adenovirus). These viral proteins kick cells into mitosis by inactivating cell proteins (such as Rb, pictured) that are normally involved in regulating cell growth. The cells divide, and in the process produce proteins involved in DNA replication, which are then used for viral replication. These oncoproteins accidentally cause tumors: the replication of none of these viruses is dependent on transformation or tumor formation.

Cells transformed with T, E6/E7, or E1A proteins are commonly used in laboratories because they are immortal. An example is the famous HeLa cell line, transformed by human papillomavirus type 18 (which originally infected Henrietta Lacks and caused the cervical tumor that killed her). Another commonly used transformed cell line is 293 (human embryonic kidney cells transformed by adenovirus E1A). It’s been known for some time that when DNA is introduced into normal (that is, not transformed) cells, they respond with an innate response: interferons are produced. In contrast, when DNA is introduced into the cytoplasm of a transformed cell, there is no interferon response.

To understand why HeLa and HEK 293 cell lines did not respond to cytoplasmic DNA, the authors silenced the viral oncogenes by disrupting them with CRISPR/Cas9. The altered cells produced interferon in response to cytoplasmic DNA. Furthermore, they produced new transformed lines by introducing genes encoding E6, E7, E1A, or T into normal mouse embryonic fibroblasts. These new transformed cells failed to respond to cytoplasmic DNA.

Cytoplasmic DNA is detected in cells by an enzyme called cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase) together with an adaptor protein known as STING (stimulator of interferon genes). When cytoplasmic DNA is detected by this system, the antiviral interferons are produced. The viral oncoproteins were found to directly bind STING, but not cGAS. A five amino acid sequence within E1A and E7 proteins was identified that is responsible for overcoming the interferon response to cytoplasmic DNA. When this sequence was altered, interaction of the oncoprotein with cGAS was reduced, and antagonism of interferon production in response to cytoplasmic DNA was blocked.

These findings provide a new function for the oncoproteins from three DNA tumor viruses: antagonism of the interferon response to cytoplasmic DNA. Normally DNA is present in the cell nucleus, and when it is detected in the cytoplasm, this is a signal that a virus infection is underway. The cytoplasmic DNA is sensed by the cGAS-STING system, leading to interferon production and elimination of infection. A herpesvirus protein has been identified that binds to STING and blocks interferon responses to cytoplasmic DNA. Clearly antagonism of the cGAS-STING DNA sensing system is of benefit to DNA viruses.

An interesting question is what selection pressure drove the evolution of viral oncogenes. One hypothesis, described above, is that they are needed to induce a cellular environment that supports viral DNA synthesis. The other idea, favored by the authors of this new work, is that oncogenes arose as antagonists of innate immune signaling. But I can’t imagine these DNA viruses without oncogenes, because they would not be able to replicate very efficiently. Could both functions have been simultaneously selected for? Why not – the same five amino acid sequence that binds cGAS also binds cellular proteins (such as Rb), disrupting their function and leading to uncontrolled cell growth!

TWiV 392: Zika virus!

Four virologists discuss our current understanding of Zika virus biology, pathogenesis, transmission, and prevention, in this special live episode recorded at the American Society for Microbiology in Washington, DC.

You can find TWiV #392 at microbe.tv/twiv, or listen/watch below.

[powerpress url=”http://traffic.libsyn.com/twiv/TWiV392.mp3″]

Click arrow to play
Download TWiV 392 (62 MB .mp3, 85 min)
Subscribe (free): iTunesRSSemailGoogle Play Music

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.

[powerpress url=”http://traffic.libsyn.com/twiv/TWiV382.mp3″]

Click arrow to play
Download TWiV 382 (82 MB .mp3, 114 min)
Subscribe (free): iTunesRSSemail

TWiV 353: STING and the antiviral police

On episode #353 of the science show This Week in Virology, the TWiVniacs discuss twenty-eight years of poliovirus shedding by an immunodeficient patient, and packaging of the innate cytoplasmic signaling molecule cyclic GMP-AMP in virus particles.

You can find TWiV #353 at www.microbe.tv/twiv.