Asparagine Is a Critical Limiting Metabolite for Vaccinia Virus Protein Synthesis during Glutamine Deprivation

Abstract

Viruses actively interact with host metabolism because viral replication relies on host cells to provide nutrients and energy. Vaccinia virus (VACV; the prototype poxvirus) prefers glutamine to glucose for efficient replication to the extent that VACV replication is hindered in glutamine-free medium. Remarkably, our data show that VACV replication can be fully rescued from glutamine depletion by asparagine supplementation. By global metabolic profiling, as well as genetic and chemical manipulation of the asparagine supply, we provide evidence demonstrating that the production of asparagine, which exclusively requires glutamine for biosynthesis, accounts for VACV’s preference of glutamine to glucose rather than glutamine’s superiority over glucose in feeding the tricarboxylic acid (TCA) cycle. Furthermore, we show that sufficient asparagine supply is required for efficient VACV protein synthesis. Our study highlights that the asparagine supply, the regulation of which has been evolutionarily tailored in mammalian cells, presents a critical barrier to VACV replication due to a high asparagine content of viral proteins and a rapid demand of viral protein synthesis. The identification of asparagine availability as a critical limiting factor for efficient VACV replication suggests a new direction of antiviral strategy development.IMPORTANCEViruses rely on their infected host cells to provide nutrients and energy for replication. Vaccinia virus, the prototypic member of the poxviruses, which comprise many significant human and animal pathogens, prefers glutamine to glucose for efficient replication. Here, we show that the preference is not because glutamine is superior to glucose as the carbon source to fuel the tricarboxylic acid cycle for vaccinia virus replication. Rather interestingly, the preference is because the asparagine supply for efficient viral protein synthesis becomes limited in the absence of glutamine, which is necessary for asparagine biosynthesis. We provide further genetic and chemical evidence to demonstrate that asparagine availability plays a critical role in efficient vaccinia virus replication. This discovery identifies a weakness of vaccinia virus and suggests a possible direction to intervene in poxvirus infection.

Identification of Vaccinia Virus Inhibitors and Cellular Functions Necessary for Efficient Viral Replication by Screening Bioactives and FDA-Approved Drugs

“…It was released into the cell culture medium immediately after synthesis, which allowed it to be detected in real-time without the need to lyse the cells. Therefore, we used a reporter VACV, vLGluc, which was engineered to express Gluc in the WR strain of VACV under the control of a well-characterized viral late F17 promoter [ 20 ]. The luciferase expressed from this virus was found predominantly (99.8%) in the cell-culture medium.…”

“…BS-C-1 cells (ATCC CCL-26) were purchased from ATCC and grown in Eagle’s Minimum Essential Medium (EMEM) supplemented as described above. The recombinant VACV—Western Reserve virus expressing Gluc under viral late promoter was described elsewhere, and it was a gift from Dr. Bernard Moss [ 20 ]. Wildtype and recombinant viruses were amplified in BS-C-1 cells, and they were cushion- and gradient-purified prior to use, as described elsewhere [ 20 ].…”

Glutamine reliance in cell metabolism

“…These studies suggest that most glutamine-dependent protein translation activities can still proceed under asparagine supplementation in a glutamine-deprived environment, although the exact mechanism is still unknown. Furthermore, studies performed in endothelial cells, Kaposi's sarcoma-associated herpesvirus (KSHV)transformed cancer cells and several normal fibroblast or epithelial cell lines reported a similar effect of asparagine on supporting cell survival and protein translation after glutamine deprivation 44,49,50 . Interestingly, high intracellular asparagine levels have recently been identified to be essential for breast cancer metastasis 51 .…”

Asparagine deprivation causes a reversible inhibition of Human Cytomegalovirus acute virus replication

“…However, glutamine is also required for the de novo synthesis of asparagine by ASNS ( Figure 6A ). A recent study demonstrated that addition of asparagine could rescue vaccinia virus replication following glutamine deprivation (21). Therefore, we investigated whether loss of asparagine synthesis contributes to inhibition of HCMV replication following glutamine deprivation.…”

“…Despite ASNS knockdown, global proteins translation levels were maintained Furthermore, given the block in virus replication occurs relatively early in infection and knockdown cells could still fully support replication of HSV-1 and IAV, suggests the loss of replication is not simply due to loss of protein production, but rather indicates asparagine levels feed into a signalling pathway that influences replication of HCMV. While asparagine depletion had little effect on HSV-1 or IAV replication, a recent report demonstrated that, like HCMV, Vaccinia virus (VACV) is also highly dependent on asparagine levels and knockdown of ASNS resulted in attenuation of virus replication (21). Interestingly, despite the similar phenotype, there appears to be differences in the effects of asparagine depletion on the two viruses.…”