A promising Ebolavirus antiviral compound

ATP EBOV antiviralA small molecule antiviral compound has been shown to protect rhesus monkeys against lethal Ebolavirus disease, even when given up to three days after virus inoculation.

The compound, called GS-5734, is a nucleoside analog. After uptake into cells, GS-5734 is converted to a nucleoside triphosphate (illustrated, bottom panel) which is incorporated by the viral RNA dependent RNA polymerase as it copies the viral genome. However, the nucleoside is chemically different from ATP (illustrated, top) and no further nucleotides can be incorporated into the growing RNA strand. RNA synthesis ceases, blocking production of infectious virus particles.

In cell culture GS-5734 inhibits viral replication at micromolar concentrations, in a variety of human cell types including monocyte-derived macrophages, primary macrophages, endothelial cells, and a liver cell line. The drug inhibits replication of several strains of Zaire ebolavirus, including Kikwit and Makona (from the West African outbreak); Bundibugyo ebolavirus, and Sudan ebolavirus. It also inhibits replication of another filovirus, Marburg virus, as well as viruses of different families, including respiratory syncytial virus, Junin virus, Lassa fever virus, and MERS-coronavirus, but not chikungunya virus, Venezuelan equine encephalitis virus, or HIV-1.

The RNA dependent RNA polymerase of Ebolaviruses has not yet been produced in active form, so the authors determined whether GS-5734 inhibits a related polymerase from respiratory syncytial virus. As predicted, the compound was incorporated into growing RNA chains by the enzyme, and caused premature termination.

Typically tests of antiviral candidates begin in a small animal, and if the results are promising, proceed to nonhuman primates. While a mouse model of Ebolavirus infection is available, the serum from these animals degrades GS-5374. Consequently a rhesus monkey model of infection was used to test the compound.

After intravenous administration of GS-5374, the NTP derived from it was detected in peripheral blood mononuclear cells, testes, epididymis, eyes, and brain within 4 hours. All 12 monkeys inoculated intramuscularly with Zaire ebolavirus died by 9 days post-infection. In contrast, all animals survived after administration of GS-5374 2 or 3 days after virus inoculation. These animals also had reduced virus associated pathology as measured by liver enzymes in the blood and blod clotting. Viral RNA in serum reaches 109 copies per milliliter on days 5 and 7 in untreated animals, and was undetectable in 4 of 6 treated animals.

It is likely that resistant viruses can be obtained by passage in the presence of GS-5734; whether such mutant viruses emerge early in infection, and at high frequency, is an important question that will impact clinical efficacy of the drug. The authors did not detect changes in the viral RNA polymerase gene that might be assoicated with resistance, but further work is needed to address how readily such mutants arise.

These promising results have lead to the initiation of a phase I clinical trial to determine whether GS-5734 is safe to administer to humans, and if the drug reaches sites where Ebolaviruses are known to replicate. However, determining the efficacy of the compound requires treatment of acutely Ebolavirus infected humans, of which there are none. It might be of interest to determine the ability of GS-5734 to clear persistent virus from previously infected individuals.

You can bet that GS-5734 has already been tested for activity against Zika virus.

A protein platform for priming

Priming RNA synthesisThe enzymes that make copies of the DNA or RNA genomes of viruses – nucleic acid polymerases – can be placed into two broad categories depending on whether or not they require a primer, a short piece of DNA or RNA, to get going. The structure of the primer-independent RNA polymerase of hepatitis C virus reveals how a priming platform is built into the enzyme.

The requirement for a primer in the initiation step of nucleic acid synthesis varies among the different classes of polymerases. All DNA polymerases are primer-dependent enzymes, while DNA-dependent RNA polymerases initiate RNA synthesis de novo – without a primer. Some RNA-dependent RNA polymerases can also initiate RNA synthesis without a primer: the enzyme begins by adding the first base complementary to the template RNA (illustrated). Other RNA-dependent RNA polymerases require a primer to initiate synthesis. Examples shown on the illustration include the protein-linked primer of picornaviruses, which consists of the protein VPg covalently attached to two U residues. The primer for influenza virus mRNA synthesis is a capped oligonucleotide 12-14 bases in length that is cleaved from the 5′ end of cellular mRNA.

The structure of the RNA-dependent RNA polymerase of hepatitis C virus reveals how a primer-independent RNA polymerase positions the first nucleotide on the RNA template. This process is illustrated below. With the RNA template (dark green) in the active site of the enzyme (panel A), a short beta-loop (brown) provides a platform on which the first complementary nucleotide (light green) is added to the template. The second nucleotide is then added (panel B), producing a dinucleotide primer for RNA synthesis. At this point nothing further can happen because  the priming platform blocks the exit of the RNA product from the enzyme (panel B). The solution to this problem is that the polymerase undergoes a conformational change that moves the priming platform out of the way and allows the newly synthesized complementary RNA (panel C, light green) to exit as the enzyme moves along the template strand.

 HCV priming of RNA synthesis

The structure of the RNA polymerase of hepatitis C virus reveals that it is not really a primer-independent enzyme: a dinucleotide primer is synthesized by the polymerase using a protein platform in the active site. Such protein platforms also appear to be involved in the priming of RNA synthesis by other flaviviruses (dengue and West Nile viruses), influenza virus (genome RNA synthesis is primer independent), reovirus, and bacteriophage phi6. Perhaps all viral RNA-dependent RNA polymerases are dependent on such priming platforms to initiate RNA synthesis.

TWiV 311: Bulldogs go viral

On episode #311 of the science show This Week in Virology, Vincent visits the University of Georgia where he speaks with Zhen Fu and Biao He about their work on rabies virus and paramyxoviruses.

You can find TWiV #311, audio and video versions, at www.microbe.tv/twiv.

TWiV 100: TWiV catches a big fish

Hosts: Vincent Racaniello, Alan Dove, Rich Condit, and David Baltimore

Vincent, Alan, and Rich celebrate the 100th episode of the podcast This Week in Virology by talking about viruses with Nobel Laureate David Baltimore.

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Alan – TimeTree
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The neurons that shaped civilization
Vincent – Ahead of the Curve: David Baltimore’s Life in Science by Shane Crotty

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TWiV 60: Making viral RNA

TWiV_AA_200Hosts: Vincent Racaniello and Dickson Despommier

Vincent and Dick continue Virology 101 with a discussion of how RNA viruses produce mRNA and replicate their genomes.

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Dick The Double Helix by James D. Watson
Vincent
Worms and Germs Blog

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