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  • Although the reasons why viral RNA does not bind


    Although the reasons why viral RNA does not bind to and activate PKR in WNV-infected cells are not known, some of the known characteristics of flavivirus infections provide possible clues. The viral (R,S)-Anatabine protein forms dimers that associate with ER membranes and viral RNA (Lindenbach et al., 2007) and these interactions may prevent PKR from binding to the genomic RNA. Also, the WNV genomic 3′SL RNA was previously reported to bind to several cellular proteins that are thought to be required for efficient initiation of viral minus strand RNA synthesis (Brinton, 2002, Davis et al., 2007) and the interactions with these proteins may prevent the interaction of this SL with PKR. The WNV genomic RNA contains a 5′ cap, an RNA modification reported to prevent PKR activation by cellular RNAs (Nallagatla et al., 2007). Interactions between the 5′SL and translation factors may mask this structure from detection by PKR. Alternatively, the reported interaction between NS5 and 5′ nts of the viral genome (Dong et al., 2008) may prevent the RNA structures in this region from interacting with PKR. Flaviviruses replicate in the cytoplasm and induce extensive ER membrane proliferation and rearrangement. Perinuclear vesicles that are formed by invaginations of the rough ER membrane contain the double-stranded viral RNA replication intermediates and once formed facilitate an exponential amplification of genome RNA (Gillespie et al., 2010, Mackenzie, 2005, Welsch et al., 2009). At later times in the infection cycle, the sequestering of replicating dsRNA in vesicles, the close proximity of viral RNA replication sites to sites of genome RNA translation and packaging and the membrane association of the capsid proteins may all participate in the evasion of WNV dsRNA regions from detection by PKR. However, it is not known how the viral RNAs are hidden from PKR during the early stages of the infection cycle when viral genome and antisense RNA replication occurs symmetrically at low levels (Lindenbach et al., 2007). Possibly the viral RNA levels are not high enough to outcompete Nck efficiently. Viral RNA tertiary interactions may play a role in preventing interaction with PKR. Also, GU wobble pairs in RNAs were previously shown to inhibit PKR activation in vitro (Nallagatla and Bevilacqua, 2008). Not only the number but also the clustering of GU pairs is required to sufficiently alter a dsRNA structure so that it is unable to activate PKR (Nallagatla and Bevilacqua, 2008). More than 500 GU wobble base pairs (bp) were predicted to form in a whole genome fold of the WNV Eg101 RNA (Ann Palmenberg, unpublished data). In the context of the whole genomic RNA, 13 of the 22 GU pairs formed by the small 3′sfRNA sequence are long distance interactions between nts in this region and nts located near the 5′ end of the viral RNA and most of these GU pairs are clustered. However, the “free” 3′sfRNA is predicted to form only 12 GU pairs and these are not clustered. The decrease in GU pairs would be expected to increase the ability of the “free” 3′sfRNA to activate PKR. The location of the small 3′sfRNAs that accumulate in infected cells is not known. They may also be “hidden” from PKR though association with multiple cell proteins and/or be sequestered in a cell compartment.
    Materials and methods
    Acknowledgments This work was supported by Public Health Service research grant AI048088 to M.A.B. from the National Institute of Allergy and Infectious Diseases, National Institutes of Health. We thank Graeme Conn for providing purified PKR protein and for technical advice and Bronislava Stockman for technical assistance.
    Introduction Garlic supplementation is thought to provide preventive or therapeutic effects against various diseases that are caused by oxidative stress [1]. Several clinical studies reported that the intake of aged garlic extract reduces the risk of cardiovascular disease [2,3]. The sulfur-containing amino acids S-1-propenylcysteine (S1PC) and S-allylcysteine (SAC) previously isolated from aged garlic extracts have been demonstrated to exhibit high oral bioavailability [[4], [5], [6]]. Additionally, SAC has been shown to increase stress resistance and to reduce the accumulation of reactive oxygen species (ROS) in Caenorhabditis elegans-based oxidative stress models. The transcription factor SKN-1, an ortholog of nuclear factor erythroid 2 like 2 (Nfe2l2/Nrf2) in mammals, has been demonstrated to play an integral role in mediating the antioxidant response [7]. The molecular mechanism underlying SKN-1 activation by SAC or other sulfur-containing compounds, however, remain unclear.