RNA viruses appear to use oxidative stress generated during infection as a mechanism to control capping their RNA genomes
Oxidative stress influences positive strand RNA virus genome synthesis and capping
RNA viruses are known to induce oxidative stress in cells that they infect. While this has long been thought to be an undesirable side effect of the infection process, a growing body of literature suggests that more is going on than just viruses mucking up the works. Several studies have shown that blocking the oxidative stress response in infected cells and animals can reduce viral infection, indicating that oxidative stress may contribute to effective genome replication. Our study links virus-induced oxidative stress to alteration of viral enzymatic activity during RNA virus genome replication.
Our study, like so many before it, began with a “Hmm…that’s funny…” moment rather than a “Eureka!” Our lab has studied the structure and function of viral RNA capping enzymes for a number of years. We had demonstrated the guanylyltransferase function of the flavivirus NS5 capping enzyme a few years before (Issur et al., 2009), but because NS5 has none of the signature sequence or structural motifs found in other capping enzymes it has proved something of an enigma. We were examining the effects of oxidant induced disulfide bonds on the reaction, and to our surprise treatment with oxidants caused the guanylation signal to increase drastically.
At first we didn’t know what to make of this result, but it then dawned on us that oxidative stress would rise late in infection when positive strand RNA capping was occurring, and the in vitro oxidative activation of the NS5 guanylyltransferase could be mimicking to this process. We then decided to see what would happen when we blocked oxidative stress during infection, and lo and behold we saw a reduction in viral replication and an increase in uncapped viral RNA in infected cells. Although more work needs to be performed, these data suggest that oxidative stress is important for capping and replicating viral genomes.
Viruses have evolved to exploit structures and processes in cells to aid their replication cycle. A common feature of the membranes RNA viruses replicate their genomes on is the presence of a potential, such as the oxidation potential between the ER and the cytoplasm. It stands to reason that viruses, which have usurped so many other aspects of cell physiology to aid their own replication, have found a way to utilize and perturb membrane potentials for their own gain. It seems that while stress may not be good for humans, viruses seem to thrive on it.
Figure: BHA reduces positive strand RNA synthesis and capping. (A) Treatment of cells with BHA alters the ratio of positive to negative strand RNAs in infected cells. BHK cells were infected with KUNV (MOI=0.1) and treated with 200 μM BHA for 48 h. Total RNA was extracted and strand specific qRT-PCR was performed. The ratio of average positive and negative strand Cq values for each sample are shown and average Cq values are in the text. An unpaired t test with equal variance yielded p=0.0129, demonstrating a significant reduction in positive strand KUNV RNA in BHA as compared to DMSO. (n=3) (B) Treatment with BHA increased the abundance of uncapped viral RNA. BHK cells were infected with KUNV as above and treated with DMSO or BHA and total RNA extracted at 48 h post infection. Capped and uncapped RNAs were fractionated by immunoprecipitation from total RNA samples, and KUNV and cellular GAPDH RNAs were detected from the uncapped fraction by qRT-PCR. An unpaired t test with equal variance yielded p=0.048 for uncapped KUNV RNA but insignificant differences for GAPDH, demonstrating a significant increase in uncapped KUNV RNA but not GAPDH RNA with BHA treatment. (n=3).
Introducing the Authors
Dr. Brian Geiss, Dr. Jordan Steel, and Becky Gullberg (Arthropod-Borne and Infectious Diseases Laboratory, Colorado State University).
About the Research
Oxidative stress influences positive strand RNA virus genome synthesis and capping.
Virology, Volume 475, 15 January 2015, Pages 219 – 229
Rebekah C. Gullberg, J. Jordan Steel, Stephanie L. Moon, Elnaz Soltani, Brian J. Geiss