Opinion
Emerging antiviral targets for influenza A virus

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The potential threat of a pandemic caused by H5N1 influenza A viruses has stimulated increased research on developing new antivirals against influenza A viruses. Current antivirals are directed against the M2 protein (named adamantanes) and the neuraminidase (named zanamivir and oseltamivir). However, both seasonal and H5N1 influenza A viruses have developed resistance to adamantanes and oseltamivir. Accordingly, new antivirals directed at the M2 and neuraminidase proteins, and against the hemagglutinin protein, are being developed. In addition, elucidation of the structural basis for several crucial functions of other viral proteins (specifically the non-structural NS1A protein, the nucleoprotein and the viral polymerase) has identified novel targets for the development of new antivirals. Here, we describe how functional and structural studies led to the discovery of these novel targets and also how structural information is facilitating the rational design of new drugs against previously identified targets.

Introduction

Influenza A and B viruses are enveloped RNA viruses, of which the genomes are in the form of eight segments [1] (Box 1). These viruses cause a highly contagious respiratory disease in humans, resulting in ∼36 000 deaths and >100 000 hospitalizations in the USA annually (www.cdc.gov/flu). Influenza A viruses, but not influenza B viruses, are responsible for the periodic widespread epidemics, or pandemics, that have caused high mortality rates [2]. The most devastating pandemic occurred in 1918, which caused an estimated 40 million deaths worldwide [3]. Less devastating pandemics occurred in 1957 and 1968. H5N1 influenza A viruses (commonly known as ‘avian flu’), which are highly pathogenic in humans, are candidates for the next pandemic influenza virus 4, 5 (Box 2). H5N1 viruses were first transmitted from chickens to humans in 1997 in Hong Kong, killing 6 out of 18 infected people 6, 7. After all the chickens in the poultry markets in Hong Kong were culled, H5N1 viruses continued to circulate in poultry markets in China, but transmission to humans was not documented until 2003 when fatal transmissions to humans resumed [8]. The human mortality rate has been high (∼65%), resulting in >240 deaths. H5N1 viruses in avian species have now spread from Asia to Europe and Africa (www.who.int/csr/disease/avian_influenza/en/index.html). Fortunately, H5N1 viruses have not yet acquired the ability for efficient human-to-human transmission.

The primary means for controlling influenza virus epidemics has been vaccination with inactivated or live-attenuated virus [2]. The hemagglutinin (HA) of the virus in the vaccine elicits an immune response that neutralizes virus infectivity. The antigenic structures of the HAs of both influenza A and B viruses undergo antigenic drift, which results from the selection of mutant viruses that evade antibodies directed against the predominant antigenic type of the HA circulating in the human population. Mutant viruses are readily generated because the viral RNA polymerase has no proof-reading function. Because of antigenic drift, the vaccine has to be reformulated each year.

Pandemic influenza A viruses can apparently arise by two different mechanisms. In one mechanism, influenza A viruses undergo a process called antigenic shift, the reassortment of genomic RNA segments between human and avian influenza A virus strains, resulting in a new (potentially pandemic) virus encoding a novel avian-type HA that is immunologically distinct [2]. The influenza A viruses containing the H2 and H3 HA subtypes that caused pandemics in 1957 and 1968, respectively, resulted from the reassortment of avian and human genomic RNA segments. Influenza B viruses cannot undergo antigenic shift because they infect only humans. A second mechanism for generating pandemic influenza A viruses has been proposed based on the finding that all eight genomic RNAs of the 1918 pandemic virus contain avian-like sequences [9]. This finding suggested that all eight genomic RNAs were derived from an avian virus and that such a progenitor virus then underwent multiple mutations in the process of adapting to the mammalian host. H5N1 viruses, which contain only avian genes, seem to be undergoing this route for acquiring pandemic capability in humans 4, 5. As is the case for other avian HAs, humans have no immunological protection against the H5 HA subtype.

Antivirals have been used for both prophylactic and therapeutic treatments during seasonal epidemics [10]. For example, prophylactic treatment has been used for individuals at high risk who have not been vaccinated, such as the elderly in nursing homes and hospital personnel who could spread infection to patients. The most effective therapeutic treatment of patients requires early diagnosis because antivirals are most effective in alleviating symptoms if given within the first 30 hours after infection. If a H5N1 pandemic occurs, effective control of such a pandemic will require the use of antiviral drugs because it is not likely that sufficient amounts of an effective vaccine will be available, particularly in the early phase of a fast-spreading pandemic 11, 12, 13. Antivirals can be stockpiled and, if used appropriately, should limit the spread of pandemic influenza virus. Importantly, the strategies that have been proposed for the use of antivirals to stem a H5N1 pandemic 11, 12, 13 could lead to more common and effective uses of antivirals during annual epidemics.

Current antivirals are directed against the M2 protein (these antivirals are known as adamantanes) and neuraminidase (NA; antivirals known as zanamivir and oseltamivir) 14, 15, 16, 17, 18. However, both seasonal and H5N1 influenza A viruses have developed resistance to adamantanes and oseltamivir 19, 20, 21, 22, 23, 24, 25, 26. Consequently, new antivirals directed at these two viral proteins, and against HA, are being developed. In addition, novel targets for the development of new antivirals have been identified in the non-structural NS1A protein, the nucleoprotein (NP) and the viral polymerase. Here, we describe how functional and structural studies led to the discovery of these novel targets, and also how structural information is facilitating the rational design of new drugs against previously identified targets.

Section snippets

M2 protein

Adamantanes (amantadine and rimantadine, marketed under the trade names Symmetrel and Flumadine, respectively) are directed against the M2 protein, a 97-amino-acid protein that is assembled into a tetramer that spans the viral membrane 14, 15 (Figure 1). The tetramer possesses low pH-activated (H+)-ion-channel activity that is crucial for uncoating of influenza A virions, which enter cells via endosomes 14, 15. The M2 protein channel promotes the influx of H+ ions into the interior of the

NS1A protein

The NS1 protein from influenza A (NS1A) is a small (230–237 residue), dimeric, multi-functional protein that has a crucial role in the ability of the virus to evade the antiviral response of the infected cell 40, 41. The protein comprises two domains: (i) the N-terminal (residues 1–73) RNA-binding domain (RBD), and (ii) a C-terminal (residues 74–230/237) effector domain (ED) 40, 41. The NS1A RBD binds to double-stranded RNA (dsRNA) in a non-sequence-specific manner 42, 43, 44, 45. The primary,

Conclusions

The potential threat of a pandemic caused by H5N1 influenza A viruses has stimulated increased research on developing new antivirals directed against several viral proteins. An additional incentive has been the emergence of influenza A viruses, both seasonal and pathogenic H5N1 viruses, that are resistant to some of the current antivirals that are directed against the M2 and NA proteins. Accordingly, new antivirals against these two viral proteins are being developed relying largely on

Disclosure statement

The authors are funded by a grant from the National Institutes of Health USA to develop antivirals against the dsRNA- and CPSF30-binding surfaces in the NS1A protein. Applications for patents directed at targeting these two NS1A surfaces for the development of antivirals have been submitted.

Acknowledgements

Research in our laboratories is supported by grants from the National Institutes of Health USA (www.nih.gov).

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