Field of Science

Can we target antivirals to the host cell?

Two papers published recently explore the idea of targeting antiviral compounds to the host cell using high-throughput organic synthesis and an in vitro screen. Both groups identify a single novel inhibitor of virus replication and both are host specific.  One paper goes further and identifies the target and also the in vivo potential of this compound. These papers highlight the hopes and pitfalls of targeting the host cell against viruses while shedding light on the basic biology of these viruses.

   We can protect ourselves from the harmful effects of viral infection through the use of vaccination, antivirals or indeed behavioral changes. Yet not all viruses have proved to be amenable to these and some, like measles and mumps, are quite easily combated with mass immunization campaigns while the likes of HIV have shown to be quite sensitive to antiviral compounds.

Respiratory infections are a problem! hastings.gov.uk
   For a majority of viruses though, we have neither effective vaccines nor antivirals. We need these now more than ever. And, even those which we do have vaccines and/or antivirals (Influenza A), resistance is quickly built up over time rendering them clinically useless. How then are we able to generate new, more effective antiviral molecules against a whole range of viral pathogens, especially given the cost of taking a single drug to patients?; we want more bang for our buck with antivirals. One way maybe through the targeting not of a viral gene or protein but a cellular one. The virus life cycle is so intimately connected to that of the host cell that removal of cellular pathways may have a dramatic effect on virus functioning and applications like these have the beauty of possibly targeting both established and emerging pathogens.

   Krumm et al, from the Emory University School of Medicine, Atlanta, U.S.A, demonstrate the feasibility of this approach using viruses from the paramyxo- and the orthomyxoviruses (generally called the 'myxoviruses') as their model of choice. Read the paper here. Using this, they discover a novel compound that demonstrates host-cell specificity while greatly inhibiting virus replication. Even at nanomolar concentrations (potent!).

   This work falls on the back of a series of experiments they reported back in 2008 where they screened a 137,500 compound library against in vitro infection with the related measles, canine distemper and human parainfluenza 3 viruses. This was to determine if they could uncover broad range, potent inhibitors of a large number of viruses. Moving away from the 'one-bug, one-drug' paradigm, this work identified a class of 11 compounds that did just that: they significantly inhibited replication and cytopathic effect of all the three viruses. They use the data from this series of experiments to find a host-targeted compound.

They state that their 'ideal' inhibitor would be:
In search of candidates with a host-directed antiviral profile, we anticipated three distinct features of desirable compounds: a) potent inhibition of virus replication at the screening concentration (3.3 ┬ÁM); b) a primary screening score, representative of the selectivity index (CC50/EC50), close to the cut-off value for hit candidates due to some anticipated host-cell interference ( = 1.9); and c) a broadened viral target spectrum in counter screening assays that extends to other pathogens of the myxovirus families

initial compound A) and B), the synthetically optimised JMN3-003

   A single compound was taken forward and then synthetically optimized (high-throughput synthetic chemistry anybody?) to achieve even better inhibition. No longer do we have to rely on the natural diversity of compounds out there. This new and improved compound (JMN3-003) could inhibit not just myxo-virus replication but also that of clinically relevant RNA (Sindbis virus) and to a lesser extent DNA viruses, such as vaccinia virus. The broad activity of the molecule suggests that it may target a more general aspect of viral replication than others had previously. The activity of this drug was also host-cell specific, suggesting that it did not target a virus structure but rather one found within the host cell, whose activity would change depending on the species assayed.

JMN3-003 antiviral activity

   This molecule had little or no effect on the metabolic activity of the immortalized cell lines used or of other primary cell cultures when administered at >7,000X the active dose, possibly hinting at the safety profile of such a drug. And, under liver cell extract stability tests, the compound showed a desirable extrapolated half-life of around 200 minutes. Host transcription and translation weren't affected by drug administration.To be safe, the authors put forward that this compound may only be preferentially used in acute respiratory infections where the treatment time would be substantially shorter. This group did not carry out any in vivo work so the real safety of this compound cannot really be assessed. See below for how this should be done.


Activity changes depending on the cell used. Human PBMCs or primate Veros

   So, we have demonstrated that the compound inhibits virus replication. But just how exactly does it do this? At what stage of the virus replication cycle does it act? Entry? Transcription? replication? assembly? exit? The group addressed these points through the use of assays that measure virus envelope fusion with the cell membrane (virus entry) and virus gene expression and replication (post entry). This work showed that fusion was not inhibited when the drug was added suggesting a post entry step was being targeted. When compared to a known inhibitor of virus polymerase activity, JMN3-003 looked very similar. Other experiments, assessing levels of mRNA and genomes produced from virus replication showed a significant inhibition in one or both processes during measles or influenza virus infection. Some component associated with virus polymerase reactions is being targeted. What it is, sadly this paper did not address, yet further experiments I am sure will uncover the mechanism of action of this compound and maybe through this identify an important host-virus association that has been previously overlooked. Recently, evidence has suggested that these viruses require the activity of host kinases for proper, regulated replication. Could it be these?

Little risk of resistance?


   Their experiments also suggest that it is extremely difficult for these viruses to adapt to the inhibition from JMN3-003. After 90 days of replication on cells treated with the compound, little resistance had emerged. Again, the reasons for this were not addressed. When compared to previous problems of drug resistance seen in Influenza A and HIV-1, this activity looks very promising.

RSV - the biggest cause of bronchiolotis in young children. http://www.empowher.com/
   Interestingly, this approach has been used for another medically important virus (Respiratory syncitial virus, RSV - also a paramyxovirus) and just published recently in the journal PNAS (Bonavia et al 2011). This group used effectively the same strategy as described above, except using a lot more compounds (1.7 million). They picked up a number of molecules that also targeted a post-entry step in virus replication, possibly virus replication ( RdRp activity); it also shows broad spectrum activity against a range of RNA viruses. And again, no resistant viruses emerged in the experiments. 

   This work went a little further than that I mentioned earlier and Bonavia et al were able to pull down the drug target using a type of affinity purification with immobilized samples of the compound (iTRAQ quantitative chemical proteomics). The protein binding most strongly to this may mostly likely be the target. In this case, the two compounds tested both targeted seperated enzymes in the same pathway, the de novo pyrimidine biosynthesis pathway, that is the nucleotides A, T and U (A and U being used extensively by RNA viruses). To assess the safety of targeting this important cellular pathway, the group looked at both in vitro and in vivo rodent models of disease. Rapidly-dividing cells showed high sensitivity to the compound while significant histopathological changes were observed in lymphoid and gut tissues in the cotton rat. This is not good for a potential clinical drug that may be administered to very young babies. 

Pyramidine biosynthesis - a good antiviral target? http://upload.wikimedia.org/

   Sadly, these compounds exhibited no antiviral activity in vivo and rather one increased RSV virus replication. This is possibly down to the potential immunosupressive nature of the target (inhibits rapidly dividing cells - T and B cells) or the ability of circulating pyramidines in the animal's body to rescue the inhibition of their synthesis inside the cell.

   I wonder how the first compound, JMN3-003 would fare in the many animal models of myxovirus infection. And, what would the target be when the chemical pull-down was applied? would it be the same pathway? Both these papers highlight the ability of this approach to identify readily targeted host cell components to reduce virus replication in vitro and potentially overcome the problems of narrow virus targeting and resistance while the second paper goes further and shows us how the results may not be pleasing. The targeting of host factors has obvious implications for patient health and this cotton rat model displays what can go wrong. We should learn from this and expand any potential drug candidates into in vivo modeling quickly and at the start of any experiments, not like what was done with JMN3-003, although that's not to say the same will happen with that compound.

ResearchBlogging.org

Bonavia, A., Franti, M., Pusateri Keaney, E., Kuhen, K., Seepersaud, M., Radetich, B., Shao, J., Honda, A., Dewhurst, J., Balabanis, K., Monroe, J., Wolff, K., Osborne, C., Lanieri, L., Hoffmaster, K., Amin, J., Markovits, J., Broome, M., Skuba, E., Cornella-Taracido, I., Joberty, G., Bouwmeester, T., Hamann, L., Tallarico, J., Tommasi, R., Compton, T., & Bushell, S. (2011). Organic Synthesis Toward Small-Molecule Probes and Drugs Special Feature: Identification of broad-spectrum antiviral compounds and assessment of the druggability of their target for efficacy against respiratory syncytial virus (RSV) Proceedings of the National Academy of Sciences, 108 (17), 6739-6744 DOI: 10.1073/pnas.1017142108

Krumm, S., Ndungu, J., Yoon, J., Dochow, M., Sun, A., Natchus, M., Snyder, J., & Plemper, R. (2011). Potent Host-Directed Small-Molecule Inhibitors of Myxovirus RNA-Dependent RNA-Polymerases PLoS ONE, 6 (5) DOI: 10.1371/journal.pone.0020069

Yoon, J., Chawla, D., Paal, T., Ndungu, M., Du, Y., Kurtkaya, S., Sun, A., Snyder, J., & Plemper, R. (2008). High-Throughput Screening--Based Identification of Paramyxovirus Inhibitors Journal of Biomolecular Screening, 13 (7), 591-608 DOI: 10.1177/1087057108321089

Investigating how ebola kills - one receptor at a time

  
   The emergence of a deadly Ebola virus (EBOV) strain into human populations has been our constant worry for over 40 years now. And news of the most recent case where a 12-year-old girl was killed near a major trade hub in Uganda earlier this month only serves to remind us of the devastating impact it can have. Infection with the Zaire strain of EBOV, the one that caused the death of the girl, initially causes a rapid onset of fever and headaches culminating in internal and extrernal bleeding, vomiting and diarrhea and eventually, death. No specific therapy or vaccines are available.

   While Ebola virus Zaire is known to cause up to 90% mortality the exact mechanisms of how it causes disease in humans are not understood. Kondratowicz et al, publishing recently in the journal PNAS, add to our knowledge of EBOV pathogenesis and biology by identifying the receptor molecule (TIM-1) on the surface of our cells that the virus uses to infect humans. 



What is a virus receptor?



EBOV glycoprotein (GP) synthesis and functions. http://www.bio.davidson.edu/
   Viruses are parasites of cells; that is, in order to survive they need to enter our cells and replicate. One major barrier to this, however, is the cell's plasma membrane: an outer covering made up of fats and proteins that protects the cell from the harsh outside environment. On the other hand, viruses have evolved diverse strategies to bypass this barrier and EBOV is not any different (reviewed in TWiV here). Ebola glycoproteins (GP) 1 and 2 lie on the outside of the virus particle and mediate both attachment to the cell via a receptor molecule AND fusion of the virus membrane with the cell membrane releasing the infectious virus genome into the host cytoplasm. This process also appears to involve a peculiar process known as macropinocytosis.


   Viruses do not infect every cell within the human body - they are pretty selective, having adapted their replication cycle to a particular host over hundreds or thousands of years. What then dictates this selectivity? and what role does this host cell choice have on the replication of the virus? The answer lies upon the particular receptor moleculas the viruses use.

EBOV GP structure. http://www.als.lbl.gov/


What about the ebola receptor?


   A number of cell surface molecules have been identified that play a role in the complex process of EBOV entry, all mainly only increasing its efficiency. Although none have been shown to specifically interact and bind to the virus GP 1/2, these may play more of a general role in enhancing entry. How then are we meant to identify what molecules act as specific receptors? More specifically, what does a virus receptor behave like? if we saw one, what would it look like?


   To prove that a particular molecule is a receptor for a specific virus, it should fulfil a set of predictions. Primarily, it must:



  • Physically interact with virus proteins (GP 1/2) on it's surface
  • Expression of it must significantly enhance infection (shoudn't be found on any cells it can't infect) and if it cannot infect a particular cell, does forced expression restore infectability?
  • Removal or blocking of receptor molecule will prevent infection of cell previously susceptable

How did the group do this? 


   To search for the receptor molecule that EBOV uses, Kondratowicz et al sought to correlate EBOV infection with the expression of the receptor molecule. Using a non-related virus (Vesicular Stromatitis virus) that was engineered to express EBOV glycoproteins as well as a green fluorescent protein (GFP), they infected a panel of 54 tumour cell lines. By counting the number of green cells (i.e those that EBOV could enter) they could see which cells were most easily infected and hence expressed the required receptor molecule. 

Percent transduction (GFP expression) of the VSV-EBOV-GP virus (A), Non-EBOV-VSV (B) in a range of cell lines. Correlated with TIM-1 expression date (C).

TIM-1 on the cell surface
   The group then searched through gene expression data corresponding to the cell lines and attempted to pull out a set of candidate receptors common to all susceptible cells, i.e. those that generated a lot of green cells. One gene that they found to be significantly correlated with infection was TIM-1, a type one transmembrane protein expressed on dividing kidney cells and some immune cells. They do note that TIM-1 is not found on all the susceptible cells, suggesting a role for other unknown factors in EBOV entry. Alas the hunt may continue.


   Kondratowicz et al further provides ample evidence that TIM-1 plays a significant role in EBOV glycoprotein mediated entry: he team showed that if you knockdown expression of TIM-1 on the surface of infectable cells with siRNAs, no longer does EBOV infect the cells; they also showed that expressing TIM-1 in cells not previously able to be infected, or ones that weren't all that good at it, allowed the entry of EBOV. Soluble TIM-1 is able to outcompete cell surface TIM-1 for binding to EBOV GP thus causing an inhibition of entry. TIM-1 EBOV GP binding is also demonstrated, fulfilling the requirement of direct interaction between the two. They go on to identify the expression of TIM-1 on the epithelial cells lining the human respiratory tract, a fact important as the means that EBOV initially infects us. Interestingly, the receptor is also found in conjunctiva around the eye. And, finally, infection with viable 'live' EBOV (not the VSV + EBOV glycoproteins) was blocked by administration of an anti-TIM-1 antibody. 

   We may safely conclude that TIM-1 is at least one of the receptors used by EBOV to infect human cells. These results shed light on the possible early events of EBOV infection in humans: aerosolised virus particles entering via the respiratory tract bind to an infect the epithelial cells lining it. From here, further cells are infected, like immune cells, which may allow spread throughout the body. Interestingly, TIM-1 was also found on the conjunctiva indicating a role for hand/aerosol-eye contact mediated infection. 

Death from ebola may soon be a thing of the past. http://www.documentingreality.com/

   This discovery however, does not end the story of the EBOV receptor. Not all cells that can be infected express TIM-1. What receptors are being used in this case? we just don't know yet. What we do know is that we may be on to a worthwile antiviral against ebola. For if we bock virus access to TIM-1 (like what was done in with the antibodies in this experiment) we may be able to severely inhibit EBOV infection and disease.



ResearchBlogging.org
Kondratowicz AS, Lennemann NJ, Sinn PL, Davey RA, Hunt CL, Moller-Tank S, Meyerholz DK, Rennert P, Mullins RF, Brindley M, Sandersfeld LM, Quinn K, Weller M, McCray PB Jr, Chiorini J, & Maury W (2011). From the Cover: T-cell immunoglobulin and mucin domain 1 (TIM-1) is a receptor for Zaire Ebolavirus and Lake Victoria Marburgvirus. Proceedings of the National Academy of Sciences of the United States of America, 108 (20), 8426-31 PMID: 21536871

The ecology of virus emergence - the role of rodents and biodiversity

   The inter-species transmission of viruses and other pathogens (see data below) poses a serious threat to public health, the global economy as well our environment and biodiversity. Just look at Ebola; SARS and Hendra viruses. With the numbers of emerging viruses increasing year on year, how best are we to deal with this incoming threat? As they say, the most effective bioterrorist is nature herself; so how can we stop her?

Increasing numbers of emerging infectious diseases. Jones et al (2008)

First of all, we can identify a number of possible routes that may be followed if we are to successfully combat or at least limit human infection with other animal viruses. These would include:

  • Identifying what viruses may make (or are in the process of making) the jump into humans, i.e. what viruses are out there?

  • What are the molecular mechanisms behind inter-species transmission and adaptation?

  • Can we identify temporal and geographical (or cultural) 'hotspots' that correlate with increased risk of virus emergence?

  • How can we develop potential vaccines/antivirals to further protect vulnerable local/global populations?

And, perhaps most importantly - and the most difficult aspect:

  • How are we going to fund this and what are the most cost-effective measures of doing this, i.e can we identify the best places to protect ourselves and place our resources there?
  
Hantavirus - a deadly group of re-emerging viruses. http://virology-online.com/
Deer mouse
   
Orrock et al, publishing recently in the journal American Naturalist, give their contribution to this complex virus protection scheme by identifying key ecological regulators of the potential emergence of a fatal human virus from its rodent species reservoir. Knowledge of this may allow us to pin-point potential danger areas in terms of countries/regions or seasons (by applying their principles to other viruses and ecosystems), which would increase the risk of human infection. By placing emphasis on these areas we could develop a safer, more cost effective strategy to protect at-risk populations.



  
   The model system the group used was that of the rodent-borne hantvirus, Sin Nombre virus (SNV) infecting its host, the deer mouse, Peromyscus maniculatus on the Californian Channel islands. This virus was only relatively recently found to be present among Channel islands deer mice. Hantaviruses are a group of trisegmented negative sense RNA viruses that naturally infect rodent species around the world. Two groups are recognised: one found throughout the new world and the other, throughout the old. When a human is infected by one of the new world viruses (Sin nombre virus, for example), they may develop what is known as hantavirus cardiopulmonary syndrome (HCPS), a life-threatening disease (with up to 50% mortality) caused by leakage of fluids into the lungs. Humans get infected through coming into contact with infected rodents through their aerosolised urine, feces or saliva. In the U.S, the deer mouse (Peromyscus maniculatus) is the primary reservoir of this viruses. Environmental determinants of rodent density are thought to play a significant role in the risk of rodent-human disease through increasing the chances of human/deer mouse contact.


   

Specifically, they obtained SNV-specific antibody data relating to infected mice across all islands, giving them an accurate estimate as to the prevalence of SNV in these mouse populations. The group also compiled data corresponding to a number of environmental factors of these islands, including: area, perimeter, elevation, annual precipitation ( a good correlate with island productivity) and finally the number of deer mouse predators found across the islands. These numbers allowed Orrock et al to determine which ecological factor correlated well with SNV prevalence individually or in combination with others; data that would allow for the prediction of at-risk areas across the islands.


   A number of factors were identified, for example: SNV prevalence correlated well with annual precipitation on the islands as well as island area, which I guess may be expected given that these factors influence the food sources that the mice eat as well as potential space to leave and breed.



   Predator richness negatively correlates with SNV prevalence, suggesting that if we were to artificially remove top predator species from this ecosystem, rodent population density would increase, leading to more and more SNV-infected mice with a greater chance of infecting both themselves and humans. The authors state that the protection of both biodiversity and individual predators within ecosystems would serve to protect human populations from rodent-human virus transmission through the better regulation of host density. Also, environmental increases in primary productivity within the isalnd may also increase the risk of emergence.



   So, Orrock et al have demonstrated the key role of a number of ecological regulators of virus prevalence using a unique island-rodent virus model system. They specifically focused on a potentially fatal virus that can infect humans and hence their work has medical significance within the island system. They have also identified possible situations (bigger islands/more rainfall/low predator richness) that favor an increase in SNV prevalence although, they have not determined exactly why each occurs. This adds to the debate on the relevance of biodiversity to general protection from zoonotic disease.


   This work supplies important evidence for the potential prediction of 'at-risk' regions - not just within the Californian Channel islands - that pose a threat of virus emergence. Seasons with increased rainfall that increase primary production and hence possibly increase rodent densitie. Predictions such as these have recently been used within China and South Korea to identify areas for targeted control of hantavirus infections.





ResearchBlogging.org
Orrock, J., Allan, B., & Drost, C. (2011). Biogeographic and Ecological Regulation of Disease: Prevalence of Sin Nombre Virus in Island Mice Is Related to Island Area, Precipitation, and Predator Richness The American Naturalist, 177 (5), 691-697 DOI: 10.1086/659632

Interferons, interferons, interferons - what exactly DO they do?

   The interferon (IFN) signalling pathway acts as a primary defense against all viruses through the induction of expression of hundreds of genes following infection; the exact functions of each are, at best, poorly understood. In order to gain a better insight into the antiviral mechanism of the induced genes, Schoggins, et al. (2011) performed a sensitive high-throughput screen of the effects of each one on infection with a range of RNA viruses.



Structure of the IFN-alpha protein. http://www.wikipedia.com/

Abstract:

The type I interferon response protects cells against invading viral pathogens. The cellular factors that mediate this defence are the products of interferon-stimulated genes (ISGs). Although hundreds of ISGs have been identified since their discovery more than 25 years ago1, 2, 3, only a few have been characterized with respect to antiviral activity. For most ISG products, little is known about their antiviral potential, their target specificity and their mechanisms of action. Using an overexpression screening approach, here we show that different viruses are targeted by unique sets of ISGs. We find that each viral species is susceptible to multiple antiviral genes, which together encompass a range of inhibitory activities. To conduct the screen, more than 380 human ISGs were tested for their ability to inhibit the replication of several important human and animal viruses, including hepatitis C virus, yellow fever virus, West Nile virus, chikungunya virus, Venezuelan equine encephalitis virus and human immunodeficiency virus type-1. Broadly acting effectors included IRF1, C6orf150 (also known as MB21D1), HPSE, RIG-I (also known as DDX58), MDA5 (also known as IFIH1) and IFITM3, whereas more targeted antiviral specificity was observed with DDX60, IFI44L, IFI6, IFITM2, MAP3K14, MOV10, NAMPT (also known as PBEF1), OASL, RTP4, TREX1 and UNC84B (also known as SUN2). Combined expression of pairs of ISGs showed additive antiviral effects similar to those of moderate type I interferon doses. Mechanistic studies uncovered a common theme of translational inhibition for numerous effectors. Several ISGs, including ADAR, FAM46C, LY6E and MCOLN2, enhanced the replication of certain viruses, highlighting another layer of complexity in the highly pleiotropic type I interferon system.
  
   The interferons are a multifunctional family of around 20 cell-signaling proteins that are secreted from cells following viral infection (as reviewed here). Our cells expend a lot of energy attempting to detect infection and following this, they express high concentrations of IFN proteins. Following secretion, they bind to receptors - and activate - nearby cells alerting them to the viral assault. 

   Through a complex signaling network a range of genes are actively expressed across the genome that alter the cell in such a way that it becomes harder for viruses to infect them. A rapid antiviral defence system is set-up within the host's tissues and organs. As shown in mice lacking STAT1 a key IFN signal mediator, this IFN signaling network is required to limit viral replication and disease yet also bide time for the development of an adaptive immune response. IFNs are extremely important in our fight against viruses. Only a handful of these IFN-stimulated genes (ISGs) have been characterised while hundreds still sit untouched.

 
What did they do?
 
   Using previously published gene expression data the group chose 389 ISGs for characterisation. To determine what function these ISGs have on virus replication, Schoggins, et al. developed an intracellular assay in which a retroviral vector expressing high levels of both the individual ISG alongside a red
fluorescent protein was used to infect IFN pathway-deficient cells in vitro and  then 48 - 72 hours following ISG expression, these exact cells were again infected with a range of RNA viruses expressing a green-fluorescent protein (see figure below). Viruses used included: hepatitis C virus, HIV, yellow fever virus, west Nile virus, Venezuelan equine encephalitis virus and chikungunya virus. 



A) ISG/RFP-expressing retrovirus, B) experimental outline
   
Example results

  Using basic Fluorescent Activated Cell Sorting (FACs) they were able to sort the cells based upon what colour they were, for example: red and the ISG inhibited the virus; green and it did not. More importantly they were able to specifically quantify the levels of green and red to assess the exact levels of virus replication in the cell population. The strongest inhibitors were extensively validated. On top of being expressed individually, some ISGs were expressed in combination to determine whether their antiviral effects were additive. The group were finally able to pinpoint where in the virus replication cycle inhibition took place: entry, transcription, translation, replication or exit.





 The major findings included:

  • Most ISGs inhibit virus replication
   As would be expected for an antiviral response, and one which has been shown to already inhibit a wide range of viruses, the majority of the tested ISGs inhibited replication to some degree across a range of viruses looked at. This is a good thing. This means your assay is doing what is supposed to do.
  • There are two types of antiviral genes: modest and strong inhibitors
   Owing to how the IFN system has evolved, we may categorise the ISGs into two antiviral classes: modest and strong inhibitors. The modest ones act specifically, targeting limited aspects of the virus replication cycle; the stronger ones function as a positive feedback, increasing the expression of key IFN signaling genes
  • Inhibition is additive - more ISGs equals more inhibition
   Upon the activation of the IFN pathway, hundreds of genes are upregulated resulting in an antiviral state within the cell. The defence system is not set up so that protection lies in the hands of a single gene/protein but in the hands of many - it is a truly cooperative process. In this system, following the expression of different combinations of ISGs together within a single cell, the author's noted that inhibition of viral replication increased. 
  • Translational inhibition is the most common antiviral mechanism
   The group asked, using a number of assays, at what point do these ISG's inhibit hepatitis C virus replication - is it: entry or translation of HCV mRNA/genome. They found that, in this case, it was not entry but translation. Although, this effect is probably particular to positive-sense RNA viruses. 
  • Some ISGs enhanced virus infection

   Interestingly, they found that following expression of a number of ISG's, virus replication increased. Something of a surprise for an antiviral pathway. The mechanisms of why/how these genes did this was not addressed but we can assume that in a real-life infection, the other 380-odd inhibitory genes also upregulated would cancel these out. 


HCV-induced hepatocellular carcinoma. WIll this IFN research aid in potential HCV antivirals? http://www.stanford.edu


Some caveats exist, however:

  • Over-expression systems may not reflect in vivo situation
   The results from this work are extremely interesting, important and useful yet the way it was done may not accurately represent what happens when you are infected. Although, I feel that this was not what was set out to be determined by the investigators, which I think were stimulated by the need of novel antiviral candidates. This research will of course aid in that field through the identification of potentially life-saving targets. 
  • Only a relatively small number of diverse viruses were screened
   The viruses tested in this system included only a relatively small sample of virus diversity - mainly focusing upon small positive sense RNA viruses (HCV etc.), and one retrovirus: HIV-1. We saw that a number of these ISG's inhibited these viruses but does this reflect what may happen with other viruses? What effect would they have on large DNA viruses, negative-sense RNA viruses or double-stranded RNA viruses? While the fact that the majority of the genes inhibited replication probably wouldn't change, the specifics most likely would. A number of these genes would specifically target pathways and systems that are preferentially used by these small positive sense RNA viruses while some would target those used by other viruses.This experiment would only 'see' those affecting these small positive sense RNA viruses. Although, this may not be a bad thing. These viruses are major causes of morbidity and mortality in both human and other animal populations worldwide.

  • Doesn't take into consideration virus IFN modulation

   Every virus probably has different ways of inhibited the IFN response themselves before they are eliminated from the host following ISG expression. This system would not pick up of the majority of these modulatory mechanisms as they occur prior to the expression of ISGs. So, again, as I mentioned before, this work does not reflect what would happen during infection in vivo and nor does it really matter.



ResearchBlogging.org
Schoggins, J., Wilson, S., Panis, M., Murphy, M., Jones, C., Bieniasz, P., & Rice, C. (2011). A diverse range of gene products are effectors of the type I interferon antiviral response Nature DOI: 10.1038/nature09907