Date Published: August 6, 2018
Publisher: Public Library of Science
Author(s): Irena Corbic Ramljak, Julia Stanger, Antonio Real-Hohn, Dominik Dreier, Laurin Wimmer, Monika Redlberger-Fritz, Wolfgang Fischl, Karin Klingel, Marko D. Mihovilovic, Dieter Blaas, Heinrich Kowalski, George A. Belov.
In nearly all picornaviruses the precursor of the smallest capsid protein VP4 undergoes co-translational N-terminal myristoylation by host cell N-myristoyltransferases (NMTs). Curtailing this modification by mutation of the myristoylation signal in poliovirus has been shown to result in severe assembly defects and very little, if any, progeny virus production. Avoiding possible pleiotropic effects of such mutations, we here used pharmacological abrogation of myristoylation with the NMT inhibitor DDD85646, a pyrazole sulfonamide originally developed against trypanosomal NMT. Infection of HeLa cells with coxsackievirus B3 in the presence of this drug decreased VP0 acylation at least 100-fold, resulting in a defect both early and late in virus morphogenesis, which diminishes the yield of viral progeny by about 90%. Virus particles still produced consisted mainly of provirions containing RNA and uncleaved VP0 and, to a substantially lesser extent, of mature virions with cleaved VP0. This indicates an important role of myristoylation in the viral maturation cleavage. By electron microscopy, these RNA-filled particles were indistinguishable from virus produced under control conditions. Nevertheless, their specific infectivity decreased by about five hundred fold. Since host cell-attachment was not markedly impaired, their defect must lie in the inability to transfer their genomic RNA into the cytosol, likely at the level of endosomal pore formation. Strikingly, neither parechoviruses nor kobuviruses are affected by DDD85646, which appears to correlate with their native capsid containing only unprocessed VP0. Individual knockout of the genes encoding the two human NMT isozymes in haploid HAP1 cells further demonstrated the pivotal role for HsNMT1, with little contribution by HsNMT2, in the virus replication cycle. Our results also indicate that inhibition of NMT can possibly be exploited for controlling the infection by a wide spectrum of picornaviruses.
The family Picornaviridae encompasses 35 genera of small non-enveloped RNA viruses that include numerous animal and human pathogens (www.picornaviridae.com). They are causative agents of diseases with huge impact on health care and economy ranging from the relatively harmless common cold typically caused by rhinoviruses to flaccid paralysis as live-threatening complication from poliovirus infection. Vaccines are available only against few members and currently no antiviral drug has been approved for clinical use [1–3].
To overcome the limitations of previous myr-deficient PV1 mutant studies, we here used gene knockout and pharmacological inhibition of cellular N-myristoyltransferases (NMTs) to explore the effect of radically diminished N-terminal myristoylation on the replication of coxsackievirus B3 (CVB3) and other representatives of the genus Enteroviruses and to evaluate the usefulness of NMT as a potential drug target. For the purpose of comparison we focused on CVB3, as its myristoyl group linked to VP4 displays interactions at the inner side of the capsid almost superimposable to PV1 . A recent study exploring the translation initiation in CVB3 incidentally discovered an essential role for the myristoylated Gly 2 in infectivity, where its mutation to Arg resulted in abnormal polyprotein processing in vitro, but other possible defects have not been analyzed .