The host interactome of influenza virus presents new potential targets for antiviral drugs

INTRODUCTION

Influenza viruses belong to the Orthomyxoviridae family, whose members are defined by a segmented, single‐stranded, negative‐sense RNA genome (which is replicated in the nucleus), and an envelope that is derived from the host cell 1. Influenza A viruses have eight genome segments that encode for 10 or 11 viral proteins, depending on the strain. Nine of these proteins are found in the virion. These include the following: the HA, NA, and M2 proteins that are all inserted into the lipid envelope; the matrix (M1) protein that lies beneath the membrane; the NP that coats the viral genome; the polymerase complex (PB1, PB1, and PA) that is associated with the encapsidated genome; and the nuclear export protein (NEP). The remaining viral proteins, NS1 and PB1‐F2, are expressed in infected cells but are not packaged into the virus particle.

Influenza virus initiates infection via attachment of HA to sialic acid‐containing proteins on the host cell membrane (Figure 1). The virus particle then enters the cell by pH‐dependent endocytosis, although there appears to be flexibility in the pathway that is used, with an estimated two thirds using a clathrin‐dependent pathway and the remaining third entering via an undefined pathway that is independent of both clathrin and caveolin 2. Once in the acid environment of the late endosome, the HA undergoes a conformational change and drives fusion of the viral envelope with that of the endosome 3. In addition, the M2 protein, which has ion channel activity 4, pumps H+ ions into the interior of the virion and this dissociates M1 from the viral ribonucleoprotein complexes (vRNPs). The released vRNPs enter the cytoplasm through the fusion pore and are transported into the nucleus via interaction of NP with karyopherin alpha proteins 5, which are part of the nuclear import machinery. Once in the nucleus, the incoming viral polymerase complex initiates genome transcription. In a process known as “cap‐snatching”, the PB2 protein binds to the 5′ methyl cap of cellular pre‐mRNAs, and the PA protein cleaves the pre‐mRNA to produce a capped primer that is used to start transcription 6, 7, 8. The PB1 protein contains the RNA‐dependent RNA polymerase activity, and it is also responsible for the addition of a poly(A) tail via a stuttering mechanism 9. PB1 also catalyzes genome replication, which occurs via a positive sense cRNA intermediate that is an exact copy of the vRNA. In the case of replication, it is still unclear whether initiation is primer‐dependent,—independent or a combination of both depending on anti‐genome or genome synthesis 10, 11. The newly‐synthesized vRNA, NP, and polymerase proteins are complexed together with M1 and NEP in the nucleus and via an interaction with Crm1, NEP exports the new vRNPs back out of the nucleus 12, 13. The assembly pathway for influenza virus is one of the least well‐understood stages of the viral life cycle. We know that the viral glycoproteins, HA and NA, traffic through the endoplasmic reticulum and accumulate at the plasma membrane in specific regions termed lipid raft domains 14. These serve as a virus assembly platform, and the other components of the virion are recruited via mechanisms that have yet to be fully determined. Evidence strongly suggests that HA initiates bud formation, and it has recently been shown that the M2 protein provides membrane scission activity and thereby completes bud formation 15. Finally, the neuraminidase activity of the NA protein is required to release virus from the cell by cleaving sialic acid attachments from HA and other surface glycoproteins 16.

NS1 and PB1‐F2 are accessory proteins that promote virus replication indirectly by either subverting or promoting cellular signaling pathways. NS1 is a multifunctional protein, but its major function is to inhibit activation of the cellular innate immune response 17. In the absence of NS1 expression, influenza virus is rendered non‐pathogenic in an immune‐competent host, and thus it is classified as a viral pathogenicity factor 18. PB1‐F2 is expressed from an alternative ORF in the PB1 gene. This ORF is absent in some viruses 19, which obviously indicates that it is nonessential. However, its presence in pandemic strains or avian viruses correlates with increased lung inflammation and pathology, which contributes to increased virulence and therefore PB1‐F2 constitutes the second pathogenicity factor encoded by influenza virus 20.

In addition to the viral proteins, there are numerous cellular proteins involved at each stage of the influenza virus life cycle. A few of these are mentioned above and have been identified and characterized through detailed studies of individual viral proteins, most often interaction studies. However, these likely only represent a small fraction of required host factors and efforts to broaden our knowledge in this area will provide a much more sophisticated picture of the influenza virus replication cycle. The advent of RNA interference (RNAi) technology now allows one to query the participation of each encoded host protein in a particular function such as virus replication. A number of studies 21, 22, 23, 24, 25 have recently used this approach to unveil the human proteins that are essential for efficient influenza virus replication, and in doing so, they have considerably expanded our view of how influenza virus interacts with its host cell. This review will discuss these findings and their implication for development of new options for influenza therapy.

CURRENT OPTIONS FOR INFLUENZA THERAPY

There are four drugs currently approved for the treatment or prevention of influenza and all of these act on viral proteins (Table 1). Amantadine and rimantadine both target the M2 protein and are specific for influenza A virus. By inhibiting M2 ion channel activity, they block acidification of the incoming virus particle and therefore prevent the release of the viral genome 26. However, because of widespread resistance to these drugs in current circulating viruses, the Centers for Disease Control (CDC) has recommended that they not be used during the 2010/2011 influenza season in the USA. The drugs that are recommended for clinical use are oseltamivir and zanamivir, which target the neuraminidase activity of the NA protein and are active against both influenza A and B viruses 27. NA inhibition prevents release of the newly‐formed virus particle at the last step of the replication cycle. Although oseltamivir is an oral drug, zanamivir is administered via inhalation. Another NA inhibitor, peramivir, obtained approval from the FDA for emergency use during the 2009 pandemic, but this approval has since expired 28. Peramivir can be administered intravenously and is therefore of particular use for critically ill patients.

One of the major issues with drugs that target viral proteins is that resistance is very likely to develop. This is particularly true of RNA viruses that have a more error‐prone polymerase and for influenza virus, the segmented nature of the genome allows for mutations to be transferred to new virus strains during reassortment. Mutations in the M2 protein that confer resistance to the adamantanes are found in nearly 100% of H3N2 influenza A viruses, as well as the pandemic H1N1 influenza A viruses that are currently circulating (CDC surveillance). In contrast, the seasonal H1N1 viruses that were circulating prior to the H1N1 pandemic remain sensitive to the adamantanes. The pattern is reversed when looking at resistance to oseltamivir, where the H3N2 viruses and pandemic H1N1 viruses are sensitive whereas the seasonal H1N1 viruses are 100% resistant. Oseltamivir resistance arose over a relatively short timescale (three seasons), which was unexpected as it was believed that oseltamivir resistant viruses carried a fitness deficit 29. However, evidence suggests that compensatory mutations, which arose before the drug resistance mutation, rescued the fitness and therefore allowed for worldwide spread of these viruses 30. Note that so far mutations that confer oseltamivir resistance confer little or no resistance to zanamivir. The co‐circulation of viruses that are resistant to either of the two classes of antiviral drugs obviously brings with it the concern that a multi‐drug resistant virus may emerge. We may have been granted a reprieve from this situation in that the prevalence of seasonal H1N1 viruses has dropped significantly since the appearance of the pandemic H1N1 virus, and therefore the oseltamivir‐resistance mutation is no longer in circulation. Nevertheless, if H1N1 viruses have a propensity for developing oseltamivir resistance and this is acquired by the pandemic H1N1 virus in addition to its existing adamantane resistance, we would have few therapeutic options at our disposal. Some examples of multi‐drug resistant viruses have been isolated from patients undergoing therapy, but so far, they have not been seen in general circulation 31, 32. Examination of these resistant H1N1 viruses in animal models for influenza virus has shown that they do not appear to be attenuated either in terms of pathogenicity or transmission 33, 34. This current status fuels the argument that new options for influenza antiviral therapy are sorely needed, and moreover, that we must start exploring different targets, including non‐viral ones.

HOST FACTORS AS POTENTIAL ANTIVIRAL DRUG TARGETS

Almost all approved antiviral drugs on the market are highly specific for a particular virus or family of viruses by virtue of the fact that they target viral proteins. Understandably, this strategy is used because of the need to establish selectivity and to not cause undue harm to the host. The downside is that resistance is far more likely to develop if the target is virus‐encoded and also that there are a limited number of viral proteins that possess properties amenable to developing pharmaceutically acceptable inhibitors (“druggable”). As described above, all approved influenza antiviral drugs are directed at either the M2 or NA proteins. For small RNA viruses in particular, the number of host functions they rely on is likely to far outnumber the viral functions, so by identifying these required host factors, we will immediately increase the number of potential drug targets.

DAS181 or Fludase is a developmental therapeutic candidate for influenza and is unique in that it targets a cellular component. DAS181 is a recombinant protein with sialidase activity and acts by removing sialic acid from proteins in the airway and thus prevents influenza virus attachment 35. Maraviroc, which was approved in 2007 as an antiretroviral drug, is another example of a host‐directed antiviral. Maraviroc targets one of the HIV‐1 co‐receptors, C‐C chemokine receptor type 5 (CCR5) 36, and thereby prevents virus entry. Support for the value of pursuing CCR5 as a drug target came from individuals who have a deletion in their CCR5 gene leading to loss of function and who are more resistant to HIV‐1 infection 37. Admittedly, maraviroc has also taught us that antiviral drugs directed at cellular targets are not immune to resistance, as maraviroc‐resistance has been observed both in the laboratory and in patients. Interestingly, resistance seen in vitro results from the virus adapting to use the drug‐bound form of CCR5, while in patients, the drug selects for the growth of viruses that use the alternative receptor, C‐X‐C chemokine receptor type 4, which are therefore insensitive to a CCR5‐inhibitor 38. This only emphasizes the close interplay between virus and host cell and how the role of any required cellular protein will have to be carefully dissected in order to validate it as a drug target. One should also distinguish between chronic and acute infections, as the length of drug treatment for acute infections such as influenza will usually be limited to about a week. Therefore, inhibition of a cellular function will be temporary, and perhaps, there is greater opportunity to explore the potential for host‐directed antiviral therapies in this setting, rather than with a chronic infection.

It is also possible that a particular host function is required for the replication of multiple viruses, which opens the door to the development of an antiviral drug with broad‐spectrum activity. This would be particularly useful for the treatment of diseases caused by emerging or neglected viruses for which there is little incentive for specific drug development by the pharmaceutical industry.

GENOME‐WIDE RNAI SCREENS UNVEIL THE HOST FACTORS REQUIRED BY INFLUENZA VIRUS

Genome‐wide RNAi screens are proving to be a vital tool in our efforts to gather more information on host‐pathogen interactions because they provide a global view of all host factors that are required by a particular pathogen. For influenza virus, five studies have used this approach to define the cellular network that is critical for efficient replication of the virus. In addition to learning how these host factors facilitate virus replication, it is hoped that we can use this information to pursue new targets for antiviral therapy.

SELECTION OF NEW TARGETS FOR THERAPEUTIC INTERVENTION

Faced with this vast amount of new data, how does one progress to the identification of host factors that would make suitable targets for anti‐influenza virus drugs? Beyond anything else, it is important to gather more supporting information that the host factor plays a critical role in influenza virus replication. The precise mechanism of action will have to be elucidated, and it must be determined whether the required function is something that is amenable to inhibition by a small molecule. In some cases, small molecule inhibitors of the host factors already exist and can be used as validation for the requirement of that cellular function. For example, König et al. 24 identified the calcium/calmodulin‐dependent kinase, CAMK2B, in their screen, and they showed that a specific inhibitor of this kinase, KN93, could inhibit influenza virus replication in a dose‐dependent manner. Similarly, Karlas et al. 23 showed that TG003, an inhibitor of CDC‐like kinase 1 (CLK1), can reduce influenza virus growth. When interpreting these results, one has to ensure that the inhibitor is specific for the intended target, as it is often the case that small molecules can inhibit multiple related proteins to varying extents. Apart from chemical inhibitors, it is also possible to test whether enzymatic activity is required by expressing dominant negative forms of the enzyme and determining whether this reduces influenza virus replication. Knock‐out mice, when available, are particularly beneficial in terms of examining whether the loss of the cellular factor has an impact on influenza virus replication in vivo and if this translates into reduced pathogenicity. This was demonstrated for one of the hits identified in the screens, where p27‐deficient mice were shown to have lower viral titers in their lungs compared with wild‐type mice 23. In addition, knock‐out mice provide evidence that the cellular factor is not required for viability. Although this is a comforting finding, it is unclear whether it is entirely necessary in order to support a case for a host factor as a drug target. With drug‐mediated inhibition, especially if treating an acute disease such as influenza, the loss of cellular function is temporary and it may be quite possible for the host to withstand this without deleterious consequences.

Of the 85 cellular factors that were identified in two or more of the influenza virus screens, 50 are considered to have druggable properties (Table 4), according to the Integrated Druggable Genome Database available from Sophic (http://www.sophicalliance.com/). Of these, 21 have been confirmed to be required for replication of wild‐type influenza virus (underlined in Table 4), so could serve as top candidates for further exploration.

Further insight into the role of the cellular protein in the influenza virus life cycle is crucial in order to comprehend the consequences of manipulating its function. For this, we will rely on integrating the data from the RNAi screens with those studies that use alternative approaches to address virus‐host interactions. For example, protein interaction studies such as the yeast‐two‐hybrid assay employed by Shapira et al. 25 and gene expression data that may indicate whether these pathways are regulated during influenza virus infection. It is expected that those host factors for which we obtain multiple lines of evidence for an involvement in the influenza virus life cycle will naturally attract more research interest, and that in turn will foster their development as potential drug targets.

CONCLUSION

In summary, the use of RNAi screens has expanded our knowledge of the number of cellular proteins potentially involved in the replication of influenza virus by several fold. The fact that multiple such studies were published over a short time span has provided us the opportunity to assess the usefulness of the approach and more importantly to learn how one should interpret such data. Clearly, if we restrict ourselves to analyzing the overlapping hits at the level of gene name, we will limit the pool of required host factors to a few key members. However, if a cross‐comparison is performed at the level of gene function, the multiple studies provide support for the participation of distinct cellular pathways or protein complexes, some of which are not seen if analyzing the studies on an individual basis. Overall, these studies provide strong evidence that the vATPase and COPI complexes, the ribosomal, mRNA splicing and nuclear trafficking machinery, and kinase‐regulated signaling are all required for efficient replication of influenza A virus. Of these, the COPI complex is the best example of a new cellular function that was uncovered by the use of RNAi screening and this is sure to spur further research to understand how it facilitates influenza virus replication. The screens also present new avenues to explore in terms of potential targets for antiviral drugs. Some of these cellular factors may be specifically required by influenza virus, whereas others may also play a role in other virus infections, which presents the opportunity for development of a host‐directed drug with broad‐spectrum activity.

CONFLICT OF INTEREST

The authors have declared that there is no conflict of interest.