Ribosome Profiling Reveals Translational Upregulation of Cellular Oxidative Phosphorylation mRNAs during Vaccinia Virus-Induced Host Shutoff

Abstract

Vaccinia virus infection causes a host shutoff that is marked by global inhibition of host protein synthesis. Though the host shutoff may facilitate reallocation of cellular resources for viral replication and evasion of host antiviral immune responses, it poses a challenge for continuous synthesis of cellular proteins that are important for viral replication. It is, however, unclear whether and how certain cellular proteins may be selectively synthesized during the vaccinia virus-induced host shutoff. Using simultaneous RNA sequencing and ribosome profiling, two techniques quantifying genome-wide levels of mRNA and active protein translation, respectively, we analyzed the responses of host cells to vaccinia virus infection at both the transcriptional and translational levels. The analyses showed that cellular mRNA depletion played a dominant role in the shutoff of host protein synthesis. Though the cellular mRNAs were significantly reduced, the relative translation efficiency of a subset of cellular mRNAs increased, particularly those involved in oxidative phosphorylation that are responsible for cellular energy production. Further experiments demonstrated that the protein levels and activities of oxidative phosphorylation increased during vaccinia virus infection, while inhibition of the cellular oxidative phosphorylation function significantly suppressed vaccinia virus replication. Moreover, the short 5= untranslated region of the oxidative phosphorylation mRNAs contributed to the translational upregulation. These results provide evidence of a mechanism that couples translational control and energy metabolism, two processes that all viruses depend on host cells to provide, to support vaccinia virus replication during a host shutoff.IMPORTANCE Many viral infections cause global host protein synthesis shutoff. While host protein synthesis shutoff benefits the virus by relocating cellular resources to viral replication, it also poses a challenge to the maintenance of cellular functions necessary for viral replication if continuous protein synthesis is required. Here we measured the host mRNA translation rate during a vaccinia virus-induced host shutoff by analyzing total and actively translating mRNAs in a genome-wide manner. This study revealed that oxidative phosphorylation mRNAs were translationally upregulated during vaccinia virus-induced host protein synthesis shutoff. Oxidative phosphorylation is the major cellular energy-producing pathway, and we further showed that maintenance of its function is important for vaccinia virus replication. This study highlights the fact that vaccinia virus infection can enhance cellular energy production through translational upregulation in the context of an overall host protein synthesis shutoff to meet energy expenditure.KEYWORDS oxidative phosphorylation, RNA-seq, ribosome profiling, translational regulation, host shutoff, poxvirus, vaccinia virus

Global analysis of polysome-associated mRNA in vesicular stomatitis virus infected cells

“…Analysis of cellular mRNAs less-sensitive to this global shut-down of translation identifies several host proteins that promote viral replication. Similar analyses revealed the abundance of viral mRNA contributes to the host-cell shut-off for other viruses including coronaviruses, influenza and vaccinia [47][48][49].…”

Going against the Tide: Selective Cellular Protein Synthesis during Virally Induced Host Shutoff

“…Two recent studies, one from our group and one from Stern-Ginossar's group, respectively, employed simultaneous RNA-Seq and Ribo-Seq to provide systematic views of VACV-and IAV-induced host shutoff (8,24). The studies found that, during both viral infections, host shutoff is driven by mRNA depletion (likely triggered by virus-encoded proteins that rapidly degrade host cell mRNAs).…”

“…When oxidative phosphorylation is impaired by chemical compounds in VACV-or IAV-infected cells, viral replication is significantly reduced (8,24), demonstrating that oxidative phosphorylation is important in their life cycles. In addition to providing energy for viral replication, increasing or maintaining ATP levels may also prevent viral protein aggregation due to large amount of viral protein production during replication (26).…”

“…In VACV infection, the levels of the majority of oxidative phosphorylation mRNAs are reduced; however, the translational efficiency of these mRNAs increases significantly, resulting in a greater abundance of some oxidative phosphorylation proteins. The capacity to generate ATP also increases in VACV-infected cells (24), and elevated RNA translational efficiency that increases protein production likely rapidly boosts energy production. In another route to a similar outcome, IAV-infected cells mainly maintain levels of oxidative phosphorylation proteins by selectively depleting cellular mRNAs, such that those controlling oxidative phosphorylation escape the degradation process (8).…”

“…Ribosomes are also selectively synthesized in host shutoff induced by both VACV and IAV infections (8,24), at least at certain times during infection. Like the requirement for energy, viral replication requires synthesis of viral proteins, so protection of ribosome function might be expected during a viral shutoff.…”