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

ABSTRACT

Many viral infections cause host shutoff, a state in which host protein synthesis is globally inhibited. Emerging evidence from vaccinia and influenza A virus infections indicates that subsets of cellular proteins are resistant to host shutoff and continue to be synthesized. Remarkably, the proteins of oxidative phosphorylation, the cellular-energy-generating machinery, are selectively synthesized in both cases. Identifying mechanisms that drive selective protein synthesis should facilitate understanding both viral replication and fundamental cell biology.

INTRODUCTION

All viruses rely entirely on cellular ribosomes for translation of viral proteins because they lack their own genes encoding mRNA translational machinery. Through various tactics, viruses evolved mechanisms to utilize or manipulate the translational machineries of the cells that they infect. One such mechanism, induced by many viruses, is the global suppression of host protein synthesis known as “host shutoff” (1, 2). Host shutoff targets all major steps of protein synthesis: causing inhibition of mRNA transcription, interference with mRNA processing, destabilization of mRNAs, and suppression of almost every step of mRNA translation (initiation, elongation, and termination; Fig. 1) (1).

FIG 1.

Virus-induced host shutoff and selective synthesis of oxidative phosphorylation (OXPHOS) proteins during VACV and IAV infections. The steps that can be targeted during viral infections to induce host shutoff are noted in red. Certain genes may resist host shutoff at these steps for selective protein synthesis. The oxidative phosphorylation mRNAs that resist mRNA decay during IAV infection or enhance their translation efficiency during VACV infection are noted in green. Continuous synthesis of oxidative phosphorylation proteins contributes to maintaining or increasing mitochondrial energy production function during host shutoff.

To trigger a host shutoff, each type of virus has devised particular mechanisms. For example, vaccinia virus (VACV), the prototypic member of poxvirus with a large (∼200-kbp) DNA genome, depletes host mRNAs soon after entering cells by inducing mRNA decay and inhibiting transcription (3–6). The former is initiated through the activity of two virus-encoded decapping enzymes, D9 and D10, which remove the 7-methylguanosine cap (m⁷G) at the 5′ ends of mRNAs, exposing the mRNAs for rapid degradation (4, 5). This suppresses cellular mRNA translation and diverts cellular resources to viral replication (7). Influenza A virus (IAV), an RNA virus, also induces a host shutoff mainly by reducing the abundance of cellular mRNAs (8). Alternatively, poliovirus, a picornavirus, deactivates cellular factors critical for host mRNA translation (9, 10), although host mRNA depletion may also play a role. For further reading on the subject of host shutoff, we refer you to several excellent review papers on the topic (1, 11, 12). In the backdrop of the global host shutoff, various mechanisms permit efficient production of viral proteins, e.g., an overwhelmingly high level of viral mRNAs, and alternative translation modes; also, many published reports discuss the specifics of how viral proteins are efficiently synthesized (2, 13).

SELECTIVE SYNTHESIS OF CELLULAR PROTEINS DURING VIRALLY INDUCED HOST SHUTOFF

Host shutoff gives infecting viruses clear advantages: the cellular translation machinery and protein building blocks (for example, ribosomes and amino acids) can be reallocated for viral protein synthesis, and a considerable fraction of cellular energy (ATP) can be devoted to viral replication rather than to cellular protein synthesis (especially mRNA translation, which consumes approximately 30% to 40% of usable energy) (14, 15). In addition, shutoff blunts the host's antiviral immune responses (16, 17), many of which require nascent protein synthesis. Despite the advantages that viruses gain by these tactics, viruses do require certain cellular functions for replication, so they have evolved ways to circumvent the obstacles inherent in host shutoff. It is conceivable that subsets of cellular proteins bypass the global inhibition and are selectively synthesized to circumvent shutoff.

To fulfill the viral replication requirement, a subset of cellular proteins may be selectively synthesized by production of more of the necessary nascent transcripts, by increasing their translation efficiency, or by rendering them resistant to virus-induced mRNA decay (Fig. 1). For some genes, transcriptional induction is essential but may take several hours to implement, e.g., genes related to antiviral or stress responses are not expressed under normal conditions and so likely must be induced. A more immediate and timely response is translational upregulation or maintenance of the levels of existing mRNAs. Induction or maintenance of mRNA levels of some specific genes has been observed during host shutoff caused by a number of viral infections (18–20). For example, lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) induces host shutoff because a virus-encoded SOX endonuclease degrades host mRNAs. The infection also induces transcription of interleukin-6 (IL-6), and IL-6 is resistant to SOX-induced RNA degradation (18, 19). In the case of herpes simplex virus, a virally encoded endoribonuclease degrades most cellular mRNAs, but it spares a small number of cellular mRNAs (20). In such cases, an mRNA that is resistant to degradation might be continually used as the template for protein synthesis.

If we can understand the extent and mechanisms of selective host protein synthesis during host shutoff, then we should not only gain insight into fundamental aspects of viral replication but also discover novel interventional strategies for fighting viral diseases. To this end, numerous studies examined the mechanisms that viruses use to shunt host expression to favor viral replication, but few focused on the extents and underlying mechanisms of selective synthesis of cellular proteins. One obstacle barring investigation into this area has been a lack of an unbiased, global analysis of mRNA translation during host shutoff. This type of high-resolution profile would aid in identifying genes that are selectively synthesized in the context of a particular viral infection and, in doing so, facilitate mechanistic investigations.

OXIDATIVE PHOSPHORYLATION PROTEINS AS TARGETS FOR SELECTIVE SYNTHESIS DURING VACV- AND IAV-INDUCED HOST SHUTOFF

Over the past decade, advances in next-generation sequencing techniques promoted the development of many innovative methodologies. RNA sequencing (RNA-Seq) and ribosome profiling (Ribo-Seq), which, respectively, measure genome-wide mRNA levels and mRNAs that are being actively translated, are particularly relevant to the topic discussed here (21, 22). The relative translation efficiency of individual mRNAs can also be assessed by analysis of the ratios of Ribo-Seq reads to RNA-Seq reads (22, 23). The analysis simultaneously measures cellular and viral transcriptional and translational landscapes and can dissect gene regulation, at multiple steps from transcription to translation, with unprecedented sensitivity, resolution, and scale. This provides powerful tools for a bird's-eye view of gene expression during a host shutoff and can identify selectively synthesized proteins.

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). Extraordinarily, both studies found subsets of genes that continued to be expressed during the shutoff, and, notably, those genes controlling oxidative phosphorylation were found to be enriched.

Oxidative phosphorylation is a cellular housekeeping function that generates biologically usable energy through a series of reactions involving protein complexes located in the inner membrane of mitochondria (25). 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). Since viruses cannot generate energy, they depend entirely on host cell metabolism, so continuous production of oxidative phosphorylation proteins is conceivably one way to maintain or increase the production of energy during a host shutoff.

Although both VACV and IAV target oxidative phosphorylation proteins for selective synthesis, they do so through distinct mechanisms. 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). Thus, levels of oxidative phosphorylation proteins are maintained over the course of IAV infection, but it is not clear whether the energy production capability is maintained or increased. Both studies showed that the transcripts of oxidative phosphorylation genes are shorter than those of other cellular mRNAs. The study in VACV-infected cells also indicated that oxidative phosphorylation mRNAs have a short 5′ untranslated region (5′ UTR) that is likely partially responsible for the elevated translational efficiency. Still, the details of how these mRNAs acquire their translational advantage or why such features confer resistance to mRNA decay are largely elusive.

Such studies not only bring to light unanswered questions but also point to new opportunities. More than 100 oxidative phosphorylation genes are encoded in both the mitochondrial and nuclear genomes. They are transcribed and located in distinct cellular compartments (27). Even more intriguing, the mRNAs are translated either in mitochondria (for mRNAs derived from mitochondrial genome) or the cytoplasm (for mRNAs derived from nuclear genome) using completely distinct translation machineries (28, 29). How do these genes resist accelerated mRNA degradation or elevate translation efficiency in different cellular compartments? Answering this question might be a key to understanding the relationship between nuclear translation and mitochondrial translation.

Mounting evidence indicates that mitochondria constitute the hub of cellular innate immune responses and thus are targets of many antiviral functions (30–33). For example, VACV, as well as other poxviruses, can evade or block many cellular antiviral innate immune responses (34, 35). It is an enigma that a viral infection can inhibit the antiviral, innate immune function of mitochondria while at the same time enhancing their production of energy. Perhaps they achieve this by selectively regulating particular spatial and temporal functions, which might be driven in part by selective protein synthesis. It will be interesting to find whether oxidative phosphorylation is similarly protected during host shutoff caused by other viruses.

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. Likewise, a small number of other proteins, mostly functioning in cellular responses to infection or stress, are also selectively synthesized.

CONCLUSIONS AND PERSPECTIVES

Although many viruses cause host shutoff, most past efforts to understand the phenomenon were devoted to determining how a viral infection shuts off cellular protein synthesis while at the same time shunting the system to selectively produce viral proteins (1, 2). The topic of selective synthesis of cellular proteins is an underinvestigated area with valuable scientific and translational implications. Studies aimed at identifying selectively synthesized proteins at various steps of gene expression have used the powerful tool of comparing total mRNA to ribosome-protected mRNAs. Complementary approaches—for example, mass-spectrometry identification of nascent proteins during a shutoff—are expected to provide another robust analysis.

We expect that future studies will uncover new examples of the phenomenon. The next critical investigation will ask how particular cellular proteins are selectively synthesized, despite the general trend of host shutoff. Conceivably, selective synthesis during viral infection is mechanistically controlled by cis sequence elements of host mRNAs, which guide mRNA processing and translation (e.g., the internal ribosome entry site [IRES], upstream open reading frames [uORFs], or structural features). Transregulatory protein factors, along with certain cellular signaling pathways, likely also play important roles. These mechanisms that viruses tap into are likely also used in various cellular stress responses, which are also characterized by global shutoff of protein synthesis and selective protein synthesis (36).

For future studies, key questions remain. During shutoff, how are cellular resources devoted to synthesis of viral and selective cellular proteins? Did they evolve to utilize some common strategies coordinately? Does each use a distinct mechanism(s) to achieve efficient protein synthesis? The findings from investigations into these questions will not only advance the understanding of viral replication strategies but also shed light on fundamental cellular transcription and translation mechanisms. The latter is in the center of all life processes, and these studies may ultimately lead to new broad-spectrum therapeutics for treatment of viral and other human diseases.