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SignificanceProtein and mRNA expression are in most cases poorly correlated, which suggests that the posttranscriptional regulatory program of a cell is an important component of gene expression. This regulatory network is still poorly understood, including how RNA structure quantitatively contributes to translational control. We present here a series of structural and functional experiments that together allow us to derive a quantitative, structure-dependent model of translation that accurately predicts translation efficiency in reporter assays and primary human tissue for a complex and medically important protein, α-1-antitrypsin. Our model demonstrates the importance of accurate, experimentally derived RNA structural models partnered with Kozak sequence information to explain protein expression and suggests a strategy by which α-1-antitrypsin expression may be increased in diseased individuals.

27Coronaviruses, including SARS-CoV-2 the etiological agent of COVID-19 disease, have 28 caused multiple epidemic and pandemic outbreaks in the past 20 years 1-3 . With no vaccines, 29 and only recently developed antiviral therapeutics, we are ill equipped to handle coronavirus 30 outbreaks 4 . A better understanding of the molecular mechanisms that regulate coronavirus 31 replication and pathogenesis is needed to guide the development of new antiviral therapeutics 32 and vaccines. RNA secondary structures play critical roles in multiple aspects of coronavirus 33 replication, but the extent and conservation of RNA secondary structure across coronavirus 34 genomes is unknown 5 . Here, we define highly structured RNA regions throughout the MERS- 35CoV, SARS-CoV, and SARS-CoV-2 genomes. We find that highly stable RNA structures are 36 pervasive throughout coronavirus genomes, and are conserved between the SARS-like CoV. 37Our data suggests that selective pressure helps preserve RNA secondary structure in 38 coronavirus genomes, suggesting that these structures may play important roles in virus 39 replication and pathogenesis. Thus, disruption of conserved RNA secondary structures could be 40 a novel strategy for the generation of attenuated SARS-CoV-2 vaccines for use against the 41 current COVID-19 pandemic. 42 43 Main 44 Severe acute respiratory syndrome coronavirus (SARS-CoV), Middle Eastern respiratory 45 syndrome coronavirus (MERS-CoV), and SARS-CoV-2, the etiological agent of the current 46 COVID-19 pandemic, have caused widespread disease, death, and economic hardship in the 47 past 20 years 1 , highlighting the pandemic potential of the CoV genus. While recently developed 48 antivirals show promise against MERS and SARS-CoV-2, further understanding of coronavirus 49 molecular virology is necessary to inform the design of more effective antiviral therapeutics and 50 vaccines 6, 7 . 51RNA structures in the ~30kb of single-stranded RNA 8 genomes of Coronavirus play 52 important roles in coronavirus replication 9, 5, 10, 11, 12, 13 . Given the length of coronavirus RNA 53 genomes, additional RNA structures likely exist that regulate CoV replication and disease 14 . In 54 this study, we used selective 2'-hydroxyl acylation by primer extension and mutational profiling 55 (SHAPE-MaP) 15 to identify highly stable, structured regions of the SARS-CoV, MERS-CoV, and 56 SARS-CoV-2 genomes. Our results revealed novel areas of RNA structure across the genomes 57 of all three viruses. SHAPE-MaP analysis confirmed previously described structures, and also 58 revealed that SARS-like coronaviruses contain a greater number of highly structured RNA 59 regions than MERS-CoV. Comparing nucleotide variation across multiple strains of each virus, 60 we find that highly variable nucleotides rarely impact RNA secondary structure, suggesting the 61 existence of selective pressure against RNA secondary structure disruption. We also identified 62 dozens of conserved highly stable structured regions in SARS-CoV and SARS-CoV-2 that share...

Expression of the human cytomegalovirus (HCMV) IE1 and IE2 proteins is critical for the establishment of lytic infection and reactivation from viral latency. Defining the mechanisms controlling IE1 and IE2 expression is therefore important for understanding how HCMV regulates its replicative cycle. Here we identify several novel transcripts encoding full-length IE1 and IE2 proteins during HCMV lytic replication. Two of the alternative major immediate early (MIE) transcripts initiate in the first intron, intron A, of the previously defined MIE transcript, while others extend the 5= untranslated region. Each of the MIE transcripts associates with polyribosomes in infected cells and therefore contributes to IE1 and IE2 protein levels. Surprisingly, deletion of the core promoter region of the major immediate early promoter (MIEP) from a plasmid containing the MIE genomic locus did not completely abrogate IE1 and IE2 expression. Instead, deletion of the MIEP core promoter resulted in increased expression of alternative MIE transcripts, suggesting that the MIEP suppresses the activity of the alternative MIE promoters. While the canonical MIE mRNA was the most abundant transcript at immediate early times, the novel MIE transcripts accumulated to levels equivalent to that of the known MIE transcript later in infection. Using two HCMV recombinants, we found that sequences in intron A of the previously defined MIE transcript are required for efficient IE1 and IE2 expression and viral replication. Together, our results identify new regulatory sequences controlling IE1 and IE2 expression and suggest that multiple transcription units act in concert to regulate IE1 and IE2 expression during lytic infection. IMPORTANCEThe HCMV IE1 and IE2 proteins are critical regulators of HCMV replication, both during primary infection and reactivation from viral latency. This study expands our understanding of the sequences controlling IE1 and IE2 expression by defining novel transcriptional units controlling the expression of full-length IE1 and IE2 proteins. Our results suggest that alternative promoters may allow for IE1 and IE2 expression when MIEP activity is limiting, as occurs in latently infected cells. The human cytomegalovirus (HCMV) IE1 and IE2 proteins are critical regulators of the viral replicative cycle. Both proteins are immediately expressed upon infection and together stimulate the expression of host and viral genes necessary for virus replication (1). IE2 acts as a general transcription factor that broadly transactivates host genes and viral early and late genes to facilitate virus replication (2-12). IE1 promotes transcription from the HCMV genome by inhibiting histone deactylases (HDACs) (13-15), which otherwise limit virus transcription by forming inhibitory chromatin structures on the viral genome. Reexpression of IE1 and IE2 is also thought to be critical for the reactivation of quiescent HCMV genomes from latent infection. Thus, understanding the regulatory mechanisms controlling IE1 and IE2 expression is im...

Alphaviruses are mosquito-borne pathogens that cause human diseases ranging from debilitating arthritis to lethal encephalitis. Studies with Sindbis virus (SINV), which causes fever, rash, and arthralgia in humans, and Venezuelan equine encephalitis virus (VEEV), which causes encephalitis, have identified RNA structural elements that play key roles in replication and pathogenesis. However, a complete genomic structural profile has not been established for these viruses. We used the structural probing technique SHAPE-MaP to identify structured elements within the SINV and VEEV genomes. Our SHAPE-directed structural models recapitulate known RNA structures, while also identifying novel structural elements, including a new functional element in the nsP1 region of SINV whose disruption causes a defect in infectivity. Although RNA structural elements are important for multiple aspects of alphavirus biology, we found the majority of RNA structures were not conserved between SINV and VEEV. Our data suggest that alphavirus RNA genomes are highly divergent structurally despite similar genomic architecture and sequence conservation; still, RNA structural elements are critical to the viral life cycle. These findings reframe traditional assumptions about RNA structure and evolution: rather than structures being conserved, alphaviruses frequently evolve new structures that may shape interactions with host immune systems or co-evolve with viral proteins.