Few human pathogens have been the focus of as much concentrated worldwide attention as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID-19. Its emergence into the human population and ensuing pandemic came on the heels of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), two other highly pathogenic coronavirus spillovers, which collectively have reshaped our view of a virus family previously associated primarily with the common cold. It has placed intense pressure on the collective scientific community to develop therapeutics and vaccines, whose engineering relies on a detailed understanding of coronavirus biology. Here, we present the molecular virology of coronavirus infection, including its entry into cells, its remarkably sophisticated gene expression and replication mechanisms, its extensive remodeling of the intracellular environment, and its multifaceted immune evasion strategies. We highlight aspects of the viral life cycle that may be amenable to antiviral targeting as well as key features of its biology that await discovery.

Methylation at the N⁶ position of adenosine (m⁶A) is a highly prevalent and reversible modification within eukaryotic mRNAs that has been linked to many stages of RNA processing and fate. Recent studies suggest that m⁶A deposition and proteins involved in the m⁶A pathway play a diverse set of roles in either restricting or modulating the lifecycles of select viruses. Here, we report that m⁶A levels are significantly increased in cells infected with the oncogenic human DNA virus Kaposi’s sarcoma-associated herpesvirus (KSHV). Transcriptome-wide m⁶A-sequencing of the KSHV-positive renal carcinoma cell line iSLK.219 during lytic reactivation revealed the presence of m⁶A across multiple kinetic classes of viral transcripts, and a concomitant decrease in m⁶A levels across much of the host transcriptome. However, we found that depletion of the m⁶A machinery had differential pro- and anti-viral impacts on viral gene expression depending on the cell-type analyzed. In iSLK.219 and iSLK.BAC16 cells the pathway functioned in a pro-viral manner, as depletion of the m⁶A writer METTL3 and the reader YTHDF2 significantly impaired virion production. In iSLK.219 cells the defect was linked to their roles in the post-transcriptional accumulation of the major viral lytic transactivator ORF50, which is m⁶A modified. In contrast, although the ORF50 mRNA was also m⁶A modified in KSHV infected B cells, ORF50 protein expression was instead increased upon depletion of METTL3, or, to a lesser extent, YTHDF2. These results highlight that the m⁶A pathway is centrally involved in regulating KSHV gene expression, and underscore how the outcome of this dynamically regulated modification can vary significantly between cell types. In addition to its roles in regulating cellular RNA fate, methylation at the N⁶ position of adenosine (m⁶A) of mRNA has recently emerged as a mechanism for regulating viral infection. While it has been known for over 40 years that the mRNA of nuclear replicating DNA viruses contain m⁶A, only recently have studies began to examine the distribution of this modification across viral transcripts, as well as characterize its functional impact upon viral lifecycles. Here, we apply m⁶A-sequencing to map the location of m⁶A modifications throughout the transcriptome of the oncogenic human DNA virus Kaposi’s sarcoma-associated herpesvirus (KSHV). We show that the m⁶A machinery functions in a cell type specific manner to either promote or inhibit KSHV gene expression. Thus, the KSHV lifecycle is impacted by the m⁶A pathway, but the functional outcome may depend on cell lineage specific differences in m⁶A-based regulation.

Infection with gammaherpesviruses, alphaherpesviruses, and betacoronaviruses can result in widespread mRNA degradation, in each case initiated predominantly by a single viral factor. Although not homologous, these factors exhibit significant mechanistic similarities. In cells, each targets translatable RNAs for cleavage and requires host Xrn1 to complete RNA degradation, although the mechanism of targeting and the position of the primary cleavage differ. Thus, multiple host shutoff factors have converged upon a common mRNA degradation pathway.

Alterations in global mRNA decay broadly impact multiple stages of gene expression, although signals that connect these processes are incompletely defined. Here, we used tandem mass tag labeling coupled with mass spectrometry to reveal that changing the mRNA decay landscape, as frequently occurs during viral infection, results in subcellular redistribution of RNA binding proteins (RBPs) in human cells. Accelerating Xrn1-dependent mRNA decay through expression of a gammaherpesviral endonuclease drove nuclear translocation of many RBPs, including poly(A) tail-associated proteins. Conversely, cells lacking Xrn1 exhibited changes in the localization or abundance of numerous factors linked to mRNA turnover. Using these data, we uncovered a new role for relocalized cytoplasmic poly(A) binding protein in repressing recruitment of TATA binding protein and RNA polymerase II to promoters. Collectively, our results show that changes in cytoplasmic mRNA decay can directly impact protein localization, providing a mechanism to connect seemingly distal stages of gene expression.

Nonstructural protein 1 (nsp1) is a coronavirus (CoV) virulence factor that restricts cellular gene expression by inhibiting translation through blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting mRNA degradation. We perform a detailed structure-guided mutational analysis of severe acute respiratory syndrome (SARS)-CoV-2 nsp1, revealing insights into how it coordinates these activities against host but not viral mRNA. We find that residues in the N-terminal and central regions of nsp1 not involved in docking into the 40S mRNA entry channel nonetheless stabilize its association with the ribosome and mRNA, both enhancing its restriction of host gene expression and enabling mRNA containing the SARS-CoV-2 leader sequence to escape translational repression. These data support a model in which viral mRNA binding functionally alters the association of nsp1 with the ribosome, which has implications for drug targeting and understanding how engineered or emerging mutations in SARS-CoV-2 nsp1 could attenuate the virus.