The more than 200 closely spaced annotated open reading frames, extensive transcriptional read-through, and numerous unpredicted RNA start sites have made the analysis of vaccinia virus gene expression challenging. Genome-wide ribosome profiling provided an unprecedented assessment of poxvirus gene expression. By 4 h after infection, approximately 80% of the ribosomeassociated mRNA was viral. Ribosome-associated mRNAs were detected for most annotated early genes at 2 h and for most intermediate and late genes at 4 and 8 h. Cluster analysis identified a subset of early mRNAs that continued to be translated at the later times. At 2 h, there was excellent correlation between the abundance of individual mRNAs and the numbers of associated ribosomes, indicating that expression was primarily transcriptionally regulated. However, extensive transcriptional readthrough invalidated similar correlations at later times. The mRNAs with the highest density of ribosomes had host response, DNA replication, and transcription roles at early times and were virion components at late times. Translation inhibitors were used to map initiation sites at single-nucleotide resolution at the start of most annotated open reading frames although in some cases a downstream methionine was used instead. Additional putative translational initiation sites with AUG or alternative codons occurred mostly within open reading frames, and fewer occurred in untranslated leader sequences, antisense strands, and intergenic regions. However, most open reading frames associated with these additional translation initiation sites were short, raising questions regarding their biological roles. The data were used to construct a high-resolution genome-wide map of the vaccinia virus translatome. Poxviruses comprise a large family that infect vertebrates and invertebrates, including species that are highly pathogenic for humans as well as for wild and domesticated animals (1, 2). Nevertheless, attenuated poxviruses are being employed as recombinant vectors for vaccines and potential cancer therapeutics. The cytoplasmic site of replication, the large linear double-stranded DNA genome with covalently closed ends, and the encoding of most, if not all, proteins required for DNA and RNA synthesis are distinguishing features of poxviruses. Studies with poxviruses have led to fundamental and far-reaching discoveries, such as the structure and biosynthesis of the 5= cap (3, 4) and the 3= poly(A) sequence (5, 6) on mRNA and numerous proteins that counteract host defenses (7,8). Vaccinia virus (VACV), the prototype poxvirus that was used as the vaccine to eradicate smallpox, has a 200-kbp genome with more than 200 annotated open reading frames (ORFs) that are selectively expressed at early, intermediate, or late times of infection due to stage-specific viral promoters and transcription factors that work in concert with the multisubunit viral DNA-dependent RNA polymerase. Recent genome-wide transcription analyses (high-throughput sequencing of RNA tran-

The nuclear export of the influenza A virus ribonucleoprotein (vRNP) is crucial for virus replication. As a major component of the vRNP, nucleoprotein (NP) alone can also be shuttled out of the nucleus by interacting with chromosome region maintenance 1 (CRM1) and is therefore hypothesized to promote the nuclear export of the vRNP. In the present study, three novel nuclear export signals (NESs) of the NP-NES1, NES2, and NES3-were identified as being responsible for mediating its nuclear export. The nuclear export of NES3 was CRM1 dependent, whereas that of NES1 or NES2 was CRM1 independent. Inactivation of these NESs led to an overall nuclear accumulation of NP. Mutation of all three NP-NESs significantly impaired viral replication. Based on structures of influenza virus NP oligomers, these three hydrophobic NESs are found present on the surface of oligomeric NPs. Functional studies indicated that oligomerization is also required for nuclear export of NP. Together, these results suggest that the nuclear export of NP is important for virus replication and relies on its NESs and oligomerization.T he influenza A virus genome consists of eight negative-sense single-stranded RNA segments (vRNA) (17). Each vRNA segment is associated with multiple copies of the viral nucleoprotein (NP) and three polymerase subunits (PA, PB1, and PB2), forming the viral ribonucleoprotein (vRNP) complex. During an early stage of infection, the vRNPs are released into the cytoplasm from virions following fusion of the viral membrane and endosomal membrane (41). Subsequently, the incoming vRNPs are transported into the nucleus, where viral genome replication and transcription occur (41).One of the determinants for vRNP nuclear import is NP (8, 28, 44, 49, 52, 53), the major protein in the vRNP structure. Thus far, two nuclear localization signals (NLSs) and a nuclear accumulation signal (NAS) have been identified in NP. The stronger NLS is an unconventional signal located in the N-terminal basic region (between residues 3 and 13) of NP (28, 44), and the weaker signal, a classical bipartite NLS, is located between residues 198 and 216 (49). The NAS-spanning residues (327 to 345) were identified through analyses of mutants that lacked both of the NLSs but still exhibited partial nuclear distribution (9).At a late stage of virus infection, the vRNPs exit the nucleus to assemble and bud from the apical plasma membrane of polarized cells (3). The trafficking of the vRNPs into and out of the nucleus is a tightly regulated process (7). The nuclear export of progeny vRNPs is mediated by the CRM1 cellular export receptor (12,23,29,48). Nuclear export protein (NEP; formerly referred to as the NS2 protein), which possesses a nuclear export signal (NES), and the matrix protein (M1) of influenza A virus are deemed responsible for directing export of the vRNPs (29, 31). This process is regulated by the Raf/MEK/ERK pathway (32), which is stimulated by the membrane association of influenza virus hemagglutinin (HA) (25). Interestingly, exogenously expresse...

The poly(A) leader at the 5’-untranslated region (5’-UTR) is an unusually striking feature of all poxvirus mRNAs transcribed after viral DNA replication (post-replicative mRNAs). These poly(A) leaders are non-templated and of heterogeneous lengths; and their function during poxvirus infection remains a long-standing question. Here, we discovered that a 5’-poly(A) leader conferred a selective translational advantage to mRNA in poxvirus-infected cells. A constitutive and uninterrupted 5’-poly(A) leader with 12 residues was optimal. Because the most frequent lengths of the 5’-poly(A) leaders are 8–12 residues, the result suggests that the poly(A) leader has been evolutionarily optimized to boost poxvirus protein production. A 5’-poly(A) leader also could increase protein production in the bacteriophage T7 promoter-based expression system of vaccinia virus, the prototypic member of poxviruses. Interestingly, although vaccinia virus post-replicative mRNAs do have 5’- methylated guanosine caps and can use cap-dependent translation, in vaccinia virus-infected cells, mRNA with a 5’-poly(A) leader could also be efficiently translated in cells with impaired cap-dependent translation. However, the translation was not mediated through an internal ribosome entry site (IRES). These results point to a fundamental mechanism poxvirus uses to efficiently translate its post-replicative mRNAs.

The influenza A virus matrix 1 protein (M1) shuttles between the cytoplasm and the nucleus during the viral life cycle and plays an important role in the replication, assembly, and budding of viruses. Here, a leucine-rich nuclear export signal (NES) was identified specifically for the nuclear export of the M1 protein. The predicted NES, designated the Flu-A-M1 NES, is highly conserved among all sequences from the influenza A virus subtype, but no similar NES motifs are found in the M1 sequences of influenza B or C viruses. The biological function of the Flu-A-M1 NES was demonstrated by its ability to translocate an enhanced green fluorescent protein (EGFP)-NES fusion protein from the nucleus to the cytoplasm in transfected cells, compared to the even nuclear and cytoplasmic distribution of EGFP. The translocation of EGFP-NES from the nucleus to the cytoplasm was not inhibited by leptomycin B. NES mutations in M1 caused a nuclear retention of the protein and an increased nuclear accumulation of NEP during transfection. Indeed, as shown by rescued recombinant viruses, the mutation of the NES impaired the nuclear export of M1 and significantly reduced the virus titer compared to titers of wild-type viruses. The NES-defective M1 protein was retained in the nucleus during infection, accompanied by a lowered efficiency of the nuclear export of viral RNPs (vRNPs). In conclusion, M1 nuclear export was specifically dependent on the Flu-A-M1 NES and critical for influenza A virus replication. Influenza A virus is a negative-strand RNA virus composed of eight segmented strands of an RNA genome (21). The viral structural components include the viral envelope, the transmembrane proteins (hemagglutinin [HA], neuraminidase [NA], and M2), M1, NS2, and the viral ribonucleoprotein (vRNP), which contains viral RNA (vRNA), nucleoprotein (NP), and viral polymerase proteins (PB1, PB2, and PA) (44).M1 is the most abundant protein in the virus particle, sustaining the virion structure by forming a shell connecting the viral envelope and the nucleocapsid (40). M1 is synthesized in the late stages of infection, shuttles between the nucleus and the cytoplasm (45), and plays multiple roles in various steps of the influenza virus life cycle.Newly synthesized M1 is transported from the cytoplasm into the nucleus via a nuclear localization signal (NLS) located in its N-terminal domain (residues 101 to 105) (38, 48). In the nucleus, M1 is involved in the blocking of the transcription of viral mRNA by binding to vRNPs (4, 49). Aside from terminating viral transcription, M1 also plays an important role in the nuclear export of vRNPs. Indeed, the nuclear presence of M1 is required for vRNPs to be transported to the cytoplasm (8, 27), where the vRNPs are subsequently transported to the budding site. First, M1 binds to histones in the nucleus (51) and may participate in releasing vRNPs from the nuclear matrix. Second, M1 likely bridges the NEP and vRNPs, forming a complex that is in turn exported from the nucleus by the nuclear export signal (NES) ...

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

Metabolism is a crucial frontier of host-virus interaction as viruses rely on their host cells to provide nutrients and energy for propagation. Vaccinia virus (VACV) is the prototype poxvirus. It makes intensive demands for energy and macromolecules in order to build hundreds and thousands of viral particles in a single cell within hours of infection. Our comprehensive metabolic profiling reveals profound reprogramming of cellular metabolism by VACV infection, including increased levels of the intermediates of the tri-carboxylic acid (TCA) cycle independent of glutaminolysis. By investigating the level of citrate, the first metabolite of the TCA cycle, we demonstrate that the elevation of citrate depends on VACV-encoded viral growth factor (VGF), a viral homolog of cellular epidermal growth factor. Further, the upregulation of citrate is dependent on STAT3 signaling, which is activated non-canonically at the serine727 upon VACV infection. The STAT3 activation is dependent on VGF, and VGF-dependent EGFR and MAPK signaling. Together our study reveals a novel mechanism by which VACV manipulates cellular metabolism through a specific viral factor and by selectively activating a series of cellular signaling pathways.

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.

Polyploidy is a prominent feature for genome evolution in many animals and all flowering plants. Plant polyploids often show enhanced fitness in diverse and extreme environments, but the molecular basis for this remains elusive. Soil salinity presents challenges for many plants including agricultural crops. Here we report that salt tolerance is enhanced in tetraploid rice through lower sodium uptake and correlates with epigenetic regulation of jasmonic acid (JA)–related genes. Polyploidy induces DNA hypomethylation and potentiates genomic loci coexistent with many stress-responsive genes, which are generally associated with proximal transposable elements (TEs). Under salt stress, the stress-responsive genes including those in the JA pathway are more rapidly induced and expressed at higher levels in tetraploid than in diploid rice, which is concurrent with increased jasmonoyl isoleucine (JA-Ile) content and JA signaling to confer stress tolerance. After stress, elevated expression of stress-responsive genes in tetraploid rice can induce hypermethylation and suppression of the TEs adjacent to stress-responsive genes. These induced responses are reproducible in a recurring round of salt stress and shared between two japonica tetraploid rice lines. The data collectively suggest a feedback relationship between polyploidy-induced hypomethylation in rapid and strong stress response and stress-induced hypermethylation to repress proximal TEs and/or TE-associated stress-responsive genes. This feedback regulation may provide a molecular basis for selection to enhance adaptation of polyploid plants and crops during evolution and domestication.