virology blog

About viruses and viral disease

protease

TWiV 267: Snow in the headlights

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On episode #267 of the science show This Week in Virology, Vincent, Alan, Rich and Kathy review a protease essential for influenza pathogenesis in mice, and directionality of rhinovirus RNA exit from the capsid.

You can find TWiV #267 at www.microbe.tv/twiv.

TWiP 32: Evasive trypanosomes and schistosomes

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t cruzi and nfkbHosts: Vincent Racaniello and Dickson Despommier

Vincent and Dickson discuss immune evasion by the cruzain protease of T. cruzi, and novel tetraspanin antigens of S. japonicum.

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Click the arrow above to play, or right-click to download TWiP #32 (65 MB .mp3, 90 minutes).

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A new target for hepatitis C virus

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When infection with hepatitis C virus goes from acute to chronic, severe liver disease may occur which requires organ transplantation. Nearly 200 million people are chronically infected with HCV, necessitating approaches to preventing and treating infections. No HCV vaccine is available, and current antiviral therapy consists of administration of interferon plus ribavirin, a combination that is effective about half the time and is associated with undesirable side effects. New antiviral compounds that target a viral protease and RNA polymerase are currently in clinical trials may eventually reach the market. But our experience with HIV-1 has shown that combinations of three drugs are the most effective for derailing the emergences of drug resistant viruses. The third target for HCV could be NS5A, a viral protein without a known function.

To identify new inhibitors of HCV, a chemical library of one million compounds was screened for the ability to inhibit viral replication in cell culture. The active compound were then subjected to a second screen to eliminate inhibitors of known viral enzymes: the viral protease, RNA polymerase, and helicase. One of the remaining inhibitors was further refined chemically until a very potent derivative was obtained. This molecule, called BMS-790052, has a 50% inhibitory concentration in the picomolar range, and inhibits all the viral genotypes tested. It is the most powerful inhibitor of HCV discovered.

The compound was tested for safety and bioavailability in various animal species. After oral administration, the compound was found in plasma and liver, despite a molecular mass of over 700 daltons. Six different levels of the compound were tested in HCV infected individuals. No adverse effects were reported, and the highest amount administered reduced viral levels in the blood 2,000 fold after one day. These results are promising, but larger trials will now be needed to further confirm the safety and efficacy of the drug.

What is the target of BMS-790052? Two lines of evidence suggest that the compound inhibits the viral protein NS5A. The drug appears to bind NS5A, and viruses resistant to the drug have amino acid changes in this protein. Although NS5A is known to be required for viral replication, its precise function is not known. Because NS5A does not have an easily assayable enzymatic function, it has not previously been a target of drug discovery. The identification of a compound that inhibits NS5A function is an important step forward in HCV drug development. The general approach used to discover BMS-790052 should be useful in identifying inhibitors of other viral proteins that do not have well defined and measurable activities.

I discussed this paper on Futures in Biotech episode #60. If you would like to listen only to the conversation about BMS-790052, download this mp3 file, or listen to the discussion below.

[audio:https://www.virology.ws/fib60.mp3 | titles=FIB 60]

Gao M, Nettles RE, Belema M, Snyder LB, Nguyen VN, Fridell RA, Serrano-Wu MH, Langley DR, Sun JH, O’Boyle DR 2nd, Lemm JA, Wang C, Knipe JO, Chien C, Colonno RJ, Grasela DM, Meanwell NA, & Hamann LG (2010). Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature, 465 (7294), 96-100 PMID: 20410884

Influenza HA cleavage is required for infectivity

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The influenza virus hemagglutinin (HA) is the viral protein that attaches to cell receptors. The HA also plays an important role in the release of the viral RNA into the cell, by causing fusion of viral and cellular membranes. HA must be cleaved by cellular proteases to be active as a fusion protein.

The HA on the influenza virion is a trimer: it is made up of three copies of the HA polypeptide. The cleavage site for cell proteases on the HA protein is located near the viral membrane.

influenza-ha-cleavage

In the diagram, the globular head of the HA protein, which attaches to cell receptors, is at the top, and the viral membrane is at the bottom. For clarity, only one HA cleavage site is labeled. The uncleaved form of the protein is called HA0; after cleavage by a cellular enzyme, two proteins are produced, called HA1 (blue) and HA2 (red). The two subunits remain together at the surface of the virus particle. The new amino(N)-terminal end of HA2 that is produced by cleavage contains a sequence of hydrophobic amino acids called a fusion peptide. During entry of influenza virus into cells, the fusion peptide inserts into the endosomal membrane and causes fusion of the viral and cell membranes. Consequently, the influenza viral RNAs can enter the cytoplasm. The fusion process is described in a previous post.

If the HA protein is not cleaved to form HA1 and HA2, fusion cannot occur. Therefore influenza viruses with uncleaved HA are not infectious. Cleavage of the viral HA occurs after newly synthesized virions are released from cells. Influenza viruses replicate efficiently in eggs because of the presence of a protease in allantoic fluid that can cleave HA. However, replication of many influenza virus strains in cell cultures requires addition of the appropriate protease (often trypsin) to the medium.

In humans, influenza virus replication is restricted to the respiratory tract, because that is the only location where the protease that cleaves HA is produced. However, the HA protein of highly pathogenic H5 and H7 avian influenza virus strains can be cleaved by proteases that are produced in many different tissues. As a result, these viruses can replicate in many organs of the bird, including the spleen, liver, lungs, kidneys, and brain. This property may explain the ability of avian H5N1 influenza virus strains to replicate outside of the human respiratory tract.

Like the HA proteins of highly pathogenic H5 and H7 viruses, the HA of the 1918 influenza virus strain can also be cleaved by ubiquitous cellular proteases. Consequently, the virus can replicate in cell cultures in the absence of added trypsin.

The H5 and H7 HA proteins have multiple basic amino acid residues at the HA1-HA2 cleavage site which allows cleavage by widely expressed proteases. But the 1918 H1 HA does not have this feature. Nor does the 1918 N1 help recruit proteases that cleave the HA, a mechanism that allows the A/WSN/33 influenza virus strain to multiply in cells without trypsin. An understanding of how the 1918 H1 HA protein can be cleaved by ubiquitous proteases is essential for understanding the high pathogenicity of this strain.

Chaipan, C., Kobasa, D., Bertram, S., Glowacka, I., Steffen, I., Solomon Tsegaye, T., Takeda, M., Bugge, T., Kim, S., Park, Y., Marzi, A., & Pohlmann, S. (2009). Proteolytic Activation of the 1918 Influenza Virus Hemagglutinin Journal of Virology, 83 (7), 3200-3211 DOI: 10.1128/JVI.02205-08