Assembly of influenza virus

Our discussion of influenza virus replication has so far brought us to the stage of viral RNA synthesis. Last time we discussed the formation of viral RNAs, an event which takes place in the cell nucleus. Now we’ll consider how these RNAs participate in the assembly of new infectious viral particles, as illustrated in the following figure.


For simplicity, the nucleus is not shown. But remember that the viral RNAs have to be exported from the nucleus to the cytoplasm, where viral assembly occurs. First, the viral mRNAs are translated to produce all the proteins needed to synthesize a new virus particle. The mRNAs encoding the HA and NA glycoproteins are translated by ribosomes that are bound the the endoplasmic reticulum – the membranous organelle that assists in transporting certain proteins to the plasma membrane. As the HA and NA proteins are produced, they are inserted into the membrane of the endoplasmic reticulum as shown. These proteins are then transported to the cell surface via small vesicles that eventually fuse with the plasma membrane. As a result, the HA and NA are inserted in the correct direction in the lipid membrane of the cell. The M2 protein is sent to this location in a similar way.

The (-) strand viral RNAs that will be packaged into new virus particles are produced in the cell nucleus, then exported to the cytoplasm. These RNAs are joined with the viral proteins PA, PB1, PB2, and NP. Viral proteins other than HA, NA, and M2 are produced by translation on free ribosomes, as shown for M1. The latter protein binds to the membrane where HA, NA, and M2 have been inserted. The assembly consisting of viral RNAs and viral proteins – called a ribonucleoprotein complex or RNP –  travels to the site of assembly. The virion then forms by a process called budding, during which the membrane bulges from the cell and is eventually pinched off to form a free particle.

As new virions are produced by budding, they would immediately bind to sialic acid receptors on the cell surface, were it not for the action of the viral NA glycoprotein. This enzyme removes sialic acids from the surface of the cell, so that newly formed virions can be released. This requirement explains how the neuraminidase inhibitors Tamiflu and Relenza function: they prevent cleavage of sialic acid from the cell surface. In the presence of these inhibitors, virions bud from the cell surface, but they remain firmly attached. Therefore Tamiflu and Relenza block infection by preventing the spread of newly synthesized virus particles to other cells.

New influenza antiviral drugs

zac0010977900001The success in treating AIDS with multiple combinations of three antiviral drugs is a model for the successful management of viral resistance. Can we expect that a deep arsenal of anti-influenza virus drugs will be developed?

So far we have  three antiviral drugs against influenza viruses. Most H3N2 strains are resistant to rimantidine, which targets the viral M2 ion channel, although it is still effective against H1N1 strains. There are two neuraminidase (NA) inhibitors: oseltamivir (Tamiflu) and zanamavir (Relenza). The latter is effective against H1N1 and H3N2 strains, but widespread resistance of H1N1 strains to oseltamivir has emerged. Clearly a deeper anti-influenza pipeline is needed.

The news is good: there are a number of promising new NA inhibitors in development. One is CS-8958, a modified version of R-125489 produced by Daiichi Sankyo Co. by adding an acyl chain to the 3-O position. The two drugs were tested in NA inhibition assays against a wide variety of H1N1, H3N2, and influenza B viruses. Importantly, CS-8958 was also found to inhibit NA activity of oseltamivir resistant H1N1, H3N2 and B patient isolates. CS-8958 also significantly improved survival of mice infected with a mouse-adapted H1N1 strain. Interestingly, CS-8958 protected mice when administered up to 10 days before infection. Zanamavir was protective when given to mice no earlier than 1 day before infection. These results may not translate to humans, but in the best of circumstances, CS-8958 might have prophylactic value in treating influenza. This property is useful for controlling outbreaks in communities and institutions.

Daiichi Sankyo Co. started a Phase III trial for adults and a pediatric Phase II/III trial with CS-8958 in November 2008. The double-blind study is meant to confirm that a single inhaled dose of CS-8958 is as effective as 75 mg of oseltamivir administered orally twice daily for 5 consecutive days.

Another promising NA inhibitor is A-322278, a modified version of A315675, a pyrrolidine-based compound developed by Abbott Laboratories. A315675 has been shown to be as active against H1N1 and H3N2 strains as zanamavir and oseltamivir. In the study cited below, the effect of the drug on mortality rates, mean weight loss, mean days of death, and lung viral titers was assessed. A-322278 was shown to protect mice against challenge with both wild type H1N1 virus and the oseltamivir resistant NA mutant H274Y. 

Other influenza antivirals in development include BioCryst Pharmaceutical’s NA inhibitor, Peramivir, an intravenous/intramuscularly administered drug in Phase 3 development; T-705, an oral RNA polymerase inhibitor from Toyama Chemical, in Phase 2, and DAS181, an inhaled drug from Nexbio, which is in Phase 1. DAS181, or Fludase, is unique in that it incorporates a sialidase that removes sialic acids from mucosal membranes, thereby preventing viral attachment via the HA glycoprotein.

The fact that some of the new inhibitors are not affected by the amino acid changes that lead to oseltamivir and zanamavir failure is encouraging. If the antivirals fare well in clinical trials, it will be possible to devise combinations of the drugs to better cope with emerging resistance.

Yamashita, M., Tomozawa, T., Kakuta, M., Tokumitsu, A., Nasu, H., & Kubo, S. (2008). CS-8958, a Prodrug of the New Neuraminidase Inhibitor R-125489, Shows Long-Acting Anti-Influenza Virus Activity Antimicrobial Agents and Chemotherapy, 53 (1), 186-192 DOI: 10.1128/AAC.00333-08

Baz, M., Abed, Y., Nehme, B., & Boivin, G. (2008). Activity of the Oral Neuraminidase Inhibitor A-322278 against the Oseltamivir-Resistant H274Y (A/H1N1) Influenza Virus Mutant in Mice Antimicrobial Agents and Chemotherapy, 53 (2), 791-793 DOI: 10.1128/AAC.01276-08

Tamiflu resistance of influenza H1N1 strains

124790416_5dc7e07b8f_mYesterday the New York Times ran an article on the resistance to Tamiflu of current influenza H1N1 strains circulating in the US. I wrote a post about this issue on 22 December 2008, so I’m happy to see the Times following my lead. But there is an issue with the Times article that I’d like to address here. According to the article:

The single mutation that creates Tamiflu resistance appears to be spontaneous, and not a reaction to overuse of the drug.

Drug-resistant viruses are not ‘reactions’ to overuse of the drug. The drug selects, from the diverse viral population in an individual, those viruses that can multiply in its presence. Usually the drug-resistant mutants are already in the host, and outpace other drug-sensitive viruses. Is that what the writer means by ‘spontaneous’? Not in this case. What apparently happened is that the mutation that causes drug-resistance, a change from histidine to tyrosine at position 274 of the viral NA protein, emerged in parts of the world were little Tamiflu is used. There was some other reason why this change was selected for in those populations. The article implies that the his->tyr change accompanied a second amino acid change at position 193 of the HA protein which improved the ability of the virus to infect people. This change did not affect resistance to Tamiflu, but apparently it only persisted when the change at 274 was also present. It so happened that the 274 change also conferred resistance to Tamiflu. Thus, when this virus arrived in parts of the world where Tamiflu is used, the resistance was noted. None of this is made particularly clear from the article.

I also have an issue with the author describing the amino acid changes in the ‘N’ and ‘H’ genes. The correct nomenclature is NA and HA. The author might have been mislead by the strain designation which uses only ‘H’, e.g. H1N1. It’s a small point but I believe that the devil is in the details.

What about the two other anti-influenza drugs? And the other strain currently circulating, H3N2?

Most of the flu in the US now is caused by H1N1 strains. So although the H3N2 strains are sensitive to Tamiflu, it’s not much help.

The Tamiflu-resistant H1N1 strains are sensitive to another drug, Relenza (zanamivir). But that drug must be inhaled and is not appropriate for everyone. However, these H1N1 strains are sensitive to Rimantadine, so its use is a good alternative. Most H3N2 strains are resistant to Rimantadine, which is why it has not been used much in recent years.

Nevertheless, our anti-influenza drug arsenal is much too small. It’s worth recalling the following information from Principles of Virology (ASM Press):

With about 1016 human immunodeficiency virus (HIV) genomes on the planet today, it is highly probable that somewhere there exist HIV genomes that are resistant to every one of the antiviral drugs that we have now or are likely to have in the future.

AIDS is no longer a death sentence because we have a deep arsenal of antiviral drugs that can control the infection. Patients are treated with a combination of three anti-HIV-1 drugs at a time. When resistance inevitably emerges, the patient is switched to another combination of three. The high levels of HIV-1 replication in many hosts, coupled with the large numbers of viral mutants that are produced, ensure that resistance will emerge.

Influenza virus shares similar features as HIV-1: high replication rates in many hosts, and the generation of large numbers of viral mutants. Therefore any antiviral strategy that employs only three drugs  is bound to fail. The difference with influenza, of course, is that an excellent vaccine is available, and should be used whenever possible. The antiviral compounds should only be used in the face of an outbreak when immunization has not been sufficiently comprehensive. However, I suspect that the use of Tamiflu and Relenza is far more prevalent than desired. How many people rush for a prescription at the first signs of a respiratory infection? And how many of those have already been immunized? This was not the intended use for these antiviral compounds.

If we want to seriously use antiviral to treat influenza (which I don’t think is a good idea except in certain cases), we need to have a far deeper arsenal of antiviral drugs.