A Mouse Model System for Zika Virus Infection

IFN signaling

Type I IFN receptor binding and signal transduction.

By Gertrud U. Rey

Zika virus (ZIKV) infection causes microcephaly in newborns and is causally associated with Guillian–Barré syndrome in adults. To date, there are no drugs available to prevent or treat ZIKV infection. ZIKV vaccine research is challenging because adult immunocompetent mice are resistant to ZIKV infection and disease.

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TWiV 484: Float like a mimivirus STING like a bat

The TWiVumvirate discuss the giant Tupanvirus, with the longest tail in the known virosphere, and dampened STING dependent interferon activation in bats.

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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.


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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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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.

TWiV 336: Brought to you by the letters H, N, P, and Eye

On episode #336 of the science show This Week in Virology, the TWiVsters explore mutations in the interferon pathway associated with severe influenza in a child, outbreaks of avian influenza in North American poultry farms, Ebolavirus infection of the eye weeks after recovery, and Ebolavirus stability on surfaces and in fluids.

You can find TWiV #336 at www.microbe.tv/twiv.

Interfering with interferon

During a discussion about blogging on the Coast to Coast Bio Podcast, it was suggested that science professors should spend more time writing about their research – by explaining what problems they are trying to solve, how they approach them, and why they are interesting. My goal here at virology blog is mainly to teach virology. But explaining what we do in my virology laboratory can be an effective instructional tool.

About five years ago I became very interested in the innate immune response to viral infections. The innate response is considered the first line of immune defense because it is active even before infection begins. Many viral infections are halted by the innate immune system, which responds very quickly – within minutes to hours after infection.

The key to innate defenses are cellular proteins – toll-like receptors and cytoplasmic molecules – that can detect viruses and turn on pathways that lead to the synthesis of the antiviral interferons (IFN).

In 2005, I attended a virology meeting in Italy where I learned about two cellular proteins that can sense the presence of viral nucleic acids. I found it absolutely amazing that these cytoplasmic proteins – called MDA-5 and RIG-I – can detect viral RNA as a foreign molecule. When these proteins sense viral RNA, they interact with a mitochondrial protein called IPS-1, which then initiates a signal transduction cascade.

When I returned from that meeting I immediately began experiments to understand how our favorite viruses – poliovirus, rhinovirus, and encephalomyocarditis virus (EMCV) – interact with the innate defense system. To our surprise, we found that RIG-I, MDA-5, and IPS-1 are all degraded in cells infected with these viruses. The following figure shows sensing of viral RNAs (green) by these proteins, and the pathways that lead to IFN synthesis. The red arrowheads show which proteins are cleaved in virus-infected cells.


What does this mean? By eliminating these cell proteins, viruses could prevent the innate immune system from functioning properly – they could block synthesis of IFN. But we don’t have any evidence to support this hypothesis. RIG-I, MDA-5, and IPS-1 disappear quite late in infection, perhaps too late to impact IFN production.

A few days after our paper on the cleavage of RIG-I was published, another group reported similar findings. They found that in cells infected with EMCV, RIG-I is cleaved. In mouse cells lacking the RIG-I protein, normal amounts of IFN is produced after EMCV infection. They postulated that the presence of MDA-5 allowed viral infection to be sensed by the innate immune system. Next, they infected MDA-5-deficient cells with EMCV. In this case, no IFN was produced. This result made sense, they reasoned, because not only was MDA-5 absent, but RIG-I was cleaved by viral infection. To support this hypothesis, they infected cells lacking MDA-5 with EMCV, then added back new RIG-I protein to these cells. The addition of RIG-I lead to the synthesis of IFN, supporting their idea that both MDA-5 and RIG-I are important for sensing EMCV infection.

These results are still not conclusive – they do not prove that cleavage of MDA-5 and RIG-I antagonize the innate immune response. To prove this point, it is necessary to produce altered forms of the proteins that cannot be degraded in virus-infected cells. If cleavage of these proteins benefits viral replication, then synthesis of cleavage-resistant forms should lead to production of higher levels of IFN and reduced viral yields in infected cells.

I’m not sure if the results of these experiments would be worth the 6-9 months required for completion. We already know dozens of ways that viruses antagonize the innate immune system. I don’t believe that adding a few more mechanisms will substantially advance our knowledge of how viruses block immune responses. More importantly, I don’t think that these experiments are ‘fundable’. That is, it would be hard to convince the NIH to provide the money to do the experiments. There are other more interesting experiments that I’d like to do. But we’ll save a description of those for another time.

Drahos J, & Racaniello VR (2009). Cleavage of IPS-1 in cells infected with human rhinovirus. Journal of Virology PMID: 19740998

Barral, P., Sarkar, D., Fisher, P., & Racaniello, V. (2009). RIG-I is cleaved during picornavirus infection Virology, 391 (2), 171-176 DOI: 10.1016/j.virol.2009.06.045

Barral, P., Morrison, J., Drahos, J., Gupta, P., Sarkar, D., Fisher, P., & Racaniello, V. (2007). MDA-5 Is Cleaved in Poliovirus-Infected Cells Journal of Virology, 81 (8), 3677-3684 DOI: 10.1128/JVI.01360-06

Papon L, Oteiza A, Imaizumi T, Kato H, Brocchi E, Lawson TG, Akira S, & Mechti N (2009). The viral RNA recognition sensor RIG-I is degraded during encephalomyocarditis virus (EMCV) infection. Virology PMID: 19733381

A viral protease interferes with interferon

Viruses are known to evade the early, or innate, host defenses by interfering with the sensing of infection, production of IFN, and synthesis and activity of ISGs. Today we’ll examine the evidence that the function of one or more ISGs is blocked in poliovirus-infected cells.

When IFN is added to the medium of cultured cells, hundreds of ISGs are produced, establishing an antiviral state. Many viruses are unable to replicate in cells treated in this way. An example is the inhibition of replication of encephalomyocarditis virus (EMCV), a picornavirus, by IFN shown in this figure:


In this experiment, IFN was added to the medium of HeLa cells for 16 hours, and then the cells were infected with EMCV. At the indicated times after infection, the medium was removed, and EMCV titer was determined by plaque assay. In mock-treated cells, EMCV produces about 100 plaque-forming units (pfu) per cell*, while IFN treatment of cells blocks virus production.

If the same experiment is done using poliovirus, the outcome is quite different. As shown in the figure below, poliovirus replication is reduced by IFN treatment of cells, but a good amount of virus is nevertheless produced.


This result suggests that the genome of poliovirus encodes one or more proteins that antagonize the activity of ISGs. Likely candidates for the ISG-busting activity are the two proteases encoded in the viral genome. These proteases, called 2Apro and 3Cpro, process the viral protein and also are known to inactivate cellular proteins. Consistent with this idea, poliovirus with a one amino acid change in 2Apro was found to be completely sensitive to IFN.

This observation indicates that 2Apro blocks the antiviral activity of one or more ISGs.

The EMCV genome encodes a 2A protein that does not have the ability to cleave other proteins. This observation may explain the exquisite sensitivity of EMCV to IFN. Can poliovirus 2Apro turn EMCV into an IFN-resistant virus? To test this hypothesis, the gene encoding 2A was inserted into the genome of EMCV. The resulting virus, EMCV-2A, was able to replicate in cells treated with IFN.

Which ISGs are targeted by poliovirus 2Apro? That is not an easy question to answer, as there are nearly 1000 known ISGs. We’re working on it.

(*The plaque assay is one of several methods for determining virus titer. We’ll discuss this assay in an upcoming post).

Morrison, J., & Racaniello, V. (2009). Proteinase 2Apro Is Essential for Enterovirus Replication in Type I Interferon-Treated Cells Journal of Virology, 83 (9), 4412-4422 DOI: 10.1128/JVI.02177-08

How influenza virus inhibits early antiviral responses

PrintThe fact that viruses routinely and frequently cause disease shows that our defense mechanisms are imperfect. This occurs in large part because nearly every viral genome encodes one or more countermeasures to modulate host defenses. Influenza virus is no exception. One of the viral proteins, called NS1, is particularly adept at impairing the synthesis of interferons (IFN) by cells.

The influenza NS1 protein, which is encoded by viral RNA 8, inhibits the innate and adaptive immune responses by multiple mechanisms. The protein blocks expression of type I IFN and inflammatory cytokines, and interferes with T-cell activation. Viral mutants with a truncated NS1 protein cause less severe disease in mice, pigs, horses, and macaques. Such viral mutants induce higher levels of IFN synthesis, and better T-cell activation, than wild type virus. It has been suggested that viruses with truncated NS1 proteins might be good candidates for infectious, attenuated vaccines against influenza.

The main sensor of influenza virus infection is the cytoplasmic protein known as RIG-I (illustrated). This protein resides in the cytoplasm and senses the presence of viral RNA – either double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA) with a 5′-phosphate. When these RNAs bind RIG-I, a signaling cascade is initiated which culminates in the production of IFN. The IFNs in turn activate the synthesis of nearly a thousand cellular proteins which have antiviral properties.

In order to function in the pathway leading to IFN induction, RIG-I must be ubiquinated near its amino terminus, on what is known as the CARD domain. Attachment of ubiquitin to RIG-I is accomplished by a cellular enzyme called TRIM25. In cells infected with influenza virus, RIG-I is not ubiquinated, and therefore IFN is not produced. The NS1 protein of influenza virus specifically inhibits ubiqutination of RIG-I by TRIM25. It does so by binding to TRIM25 and preventing it from forming multimers. A specific amino acid sequence in NS1, threonine-leucine-glutamic acid-glutamic acid, is involved in binding to TRIM25. A virus in which the two glutamic acid residues are converted to alanine is defective in blocking TRIM25-mediated ubiquitination of RIG-I. Consequently, in cells infected with this mutant virus, IFN is produced and viral replication in suppressed. As expected, the virus is much less virulent in mice than wild type virus.

Other TRIM proteins (the name stands for TRIpartite Motif, a reference to the fact that the proteins have RING, B-box zinc finger, and coiled-coil domains) are also involved in antiviral defense. TRIM19 inhibits the replication of many DNA and RNA viruses, while TRIM5α blocks replication of HIV. TRIM25 is the first protein of this family whose function has been shown to be inhibited by a viral protein, influenza virus NS1. Given the effectiveness of the innate immune response, it is safe to say that viruses exist only because they encode antagonists of this defense system. It seems likely that viral modulators of TRIM19 and TRIM5α await discovery.

Haye, K., Bourmakina, S., Moran, T., Garcia-Sastre, A., & Fernandez-Sesma, A. (2009). The NS1 protein of a human influenza virus inhibits type I interferon production and the induction of antiviral responses in primary human dendritic and respiratory epithelial cells Journal of Virology DOI: 10.1128/JVI.02323-08

Gack, M., Albrecht, R., Urano, T., Inn, K., Huang, I., Carnero, E., Farzan, M., Inoue, S., Jung, J., & García-Sastre, A. (2009). Influenza A Virus NS1 Targets the Ubiquitin Ligase TRIM25 to Evade Recognition by the Host Viral RNA Sensor RIG-I Cell Host & Microbe, 5 (5), 439-449 DOI: 10.1016/j.chom.2009.04.006