Characterization of dengue virus resistance to brequinar in cell culture.
Brequinar is an inhibitor of dihydroorotate dehydrogenase, an enzyme that is required for de novo pyrimidine biosynthesis. Here we report that brequinar has activity against a broad spectrum of viruses. The compound not only inhibits flaviviruses (dengue virus, West Nile virus, yellow fever virus, and Powassan virus) but also suppresses a plus-strand RNA alphavirus (Western equine encephalitis virus) and a negative-strand RNA rhabdovirus (vesicular stomatitis virus). Using dengue virus serotype 2 (DENV-2) as a model, we found that brequinar suppressed the viral infection cycle mainly at the step of RNA synthesis. Supplementing the culture medium with pyrimidines (cytidine or uridine) but not purines (adenine or guanine) could be used to reverse the inhibitory effect of the compound. Continuous culturing of DENV-2 in the presence of brequinar generated viruses that were partially resistant to the inhibitor. Sequencing of the resistant viruses revealed two amino acid mutations: one mutation (M260V) located at a helix in the domain II of the viral envelope protein and another mutation (E802Q) located at the priming loop of the nonstructural protein 5 (NS5) polymerase domain. Functional analysis of the mutations suggests that the NS5 mutation exerts resistance through enhancement of polymerase activity. The envelope protein mutation reduced the efficiency of virion assembly/release; however, the mutant virus became less sensitive to brequinar inhibition at the step of virion assembly/release. Taken together, the results indicate that (i) brequinar blocks DENV RNA synthesis through depletion of intracellular pyrimidine pools and (ii) the compound may also exert its antiviral activity through inhibition of virion assembly/release.
Qing
Min
M
Novartis Institute for Tropical Diseases, 10 Biopolis Rd., Chromos Building, Singapore 138670, Singapore.
Zou
Gang
G
Wang
Qing-Yin
QY
Xu
Hao Ying
HY
Dong
Hongping
H
Yuan
Zhiming
Z
Shi
Pei-Yong
PY
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D001713:Biphenyl Compounds / Q000494:pharmacology*; D002522:Chlorocebus aethiops; D003716:Dengue Virus / Q000187:drug effects* / Q000235:genetics; D024882:Drug Resistance, Viral; D011743:Pyrimidines / Q000494:pharmacology; D012367:RNA, Viral / Q000235:genetics; D014709:Vero Cells; D011798:Quinaldines
D000998:Antiviral Agents; D001713:Biphenyl Compounds; D011743:Pyrimidines; D012367:RNA, Viral; C046943:brequinar; D011798:Quinaldines; C030986:pyrimidine