The positive transcription elongation factor b (P-TEFb) is a critical coactivator for transcription of most cellular and viral genes, including those of HIV. While P-TEFb is regulated by 7SK snRNA in proliferating cells, P-TEFb is absent due to diminished levels of CycT1 in quiescent and terminally differentiated cells, which has remained unexplored. In these cells, we found that CycT1 not bound to CDK9 is rapidly degraded. Moreover, productive CycT1:CDK9 interactions are increased by PKC-mediated phosphorylation of CycT1 in human cells. Conversely, dephosphorylation of CycT1 by PP1 reverses this process. Thus, PKC inhibitors or removal of PKC by chronic activation results in P-TEFb disassembly and CycT1 degradation. This finding not only recapitulates P-TEFb depletion in resting CD4+ T cells but also in anergic T cells. Importantly, our studies reveal mechanisms of P-TEFb inactivation underlying T cell quiescence, anergy, and exhaustion as well as proviral latency and terminally differentiated cells.

Increasing evidences suggest that the methyltransferase NSUN2 catalyzes 5-methylcytosine (m5C) modifications on viral RNAs, which are essential for the replication of various viruses. Despite the function of m5C deposition is well characterized, other potential roles of NSUN2 in regulating viral replication remain largely unknown. In this study, the m5C modified residues catalyzed by NSUN2 on enterovirus 71 (EV71) RNAs were mapped. NSUN2, along with m5C modifications, played multiple roles during the EV71 life cycle. Functional m5C modified nucleotides increased the translational efficiency and stability of EV71 RNAs. Additionally, NSUN2 was found to target the viral protein VP1 for binding and promote its stability by inhibiting the ubiquitination. Furthermore, both viral replication and pathogenicity in mice were largely attenuated when functional m5C residues were mutated. Taken together, this study characterizes distinct pathways mediated by NSUN2 in regulating EV71 replication, and highlights the importance of its catalyzed m5C modifications on EV71 RNAs for the viral replication and pathogenicity.

Nipah virus (NiV), a highly lethal zoonotic paramyxovirus, displays strain-specific pathogenicity, yet the molecular basis for this divergence remains elusive. Here, we identify N6-methyladenosine (m6A) modification as a pivotal regulator of NiV replication. Higher m6A methylation levels on viral genomic RNA and mRNAs are associated with the increased virulence observed in the NiV-Malaysia (NiV-M) strain compared to NiV-Bangladesh (NiV-B). Underlying this phenomenon, NiV infection orchestrates a reprogramming of the host m6A machinery by downregulating the methyltransferase METTL3 and the demethylase ALKBH5, while concurrently upregulating m6A reader proteins YTHDF1-3. Both METTL3 and ALKBH5 bind directly to NiV RNA, with METTL3 installing m6A to promote viral replication and ALKBH5 removing them to inhibit it. Strikingly, pharmacological inhibition of m6A modification markedly attenuates NiV replication in vitro and in vivo, underscoring the therapeutic potential of targeting the m6A pathway. Our study establishes m6A as a key determinant of NiV pathogenicity and provides a paradigm for host-directed antiviral strategies against high-risk RNA viruses.

Introduction: The beta-coronavirus SARS-CoV-2 has been revealed to infect mammals and other species, which potentially promotes the virus adaptation to broader species and the emergence of new variants. The host range of different SARS-CoV-2 variants are mainly determined by the affinity of the receptor-binding domain (RBD) of the spike protein to the host receptor angiotensin-converting enzyme 2 (ACE2). Thus, this study aims to elucidate the detailed mechanisms of such dynamic adaptation of indicated SARS-CoV-2 variants.Methods: In this study, flow cytometry and surface plasmon resonance (SPR) assays were used to assess the binding affinity between RBDs and avian ACE2. Then, infection assays with MLV-based SARS-CoV-2 spike pseudovirus or authentic viruses were performed to verify the avian ACE2 mediated viral entry. Finally, mutagenesis studies were conducted to identify key amino acids of avian ACE2 orthologs and RBDs.Results: Our previous findings revealed that wild-type SARS-CoV-2 RBD does not bind chicken ACE2. Here, we found that ACE2 orthologs from chicken and mallard were capable to support binding to RBDs of the Alpha, Beta, and Gamma variants, which further enabled the viral entry. On the contrary, the RBD of BA.1 failed to bind avian ACE2. Whereas, a triple-residue reversal mutant (S446G, S496G, H505Y) restored ACE2 binding and enabled efficient viral entry. Additionally, several key residues within RBD were characterized as the determinant of its affinity to avian ACE2.Discussion: Our findings reveal that higher mutation rates in emerging variants might lead to future cross-species receptor usage or even spillover. Understanding such cross-species transmission mechanisms provides new insights to the virological features and potential host range of emerging SARS-CoV-2 variants.