Ribonucleic acid (RNA) viruses pose heavy burdens on public-health systems. Synthetic biology holds great potential for artificially controlling their replication, a strategy that could be used to attenuate infectious viruses but is still in the exploratory stage. Herein, we used the genetic-code expansion technique to convert Enterovirus 71 (EV71), a prototypical RNA virus, into a controllable EV71 strain carrying the unnatural amino acid (UAA) Nε-2-azidoethyloxycarbonyl-L-lysine (NAEK), which we termed an EV71-NAEK virus. After NAEK supplementation, EV71-NAEK could recapitulate an authentic NAEK time- and dose-dependent infection in vitro, which could serve as a novel method to manipulate virulent viruses in conventional laboratories. We further validated the prophylactic effect of EV71-NAEK in two mouse models. In susceptible parent mice, vaccination with EV71-NAEK elicited a strong immune response and protected their neonatal offspring from lethal challenges similar to that of commercial vaccines. Meanwhile, in transgenic mice harboring a PylRS-${\mathrm{tRNA}}_{\mathrm{CUA}}^{\mathrm{Pyl}}$ pair, substantial elements of genetic-code expansion technology, EV71-NAEK evoked an adjustable neutralizing-antibody response in a strictly external NAEK dose-dependent manner. These findings suggested that EV71-NAEK could be the basis of a feasible immunization program for populations with different levels of immunity. Moreover, we expanded the strategy to generate controllable coxsackieviruses for conceptual verification. In combination, these results could underlie a competent strategy for attenuating viruses and priming the immune system via artificial control, which might be a promising direction for the development of amenable vaccine candidates and be broadly applied to other RNA viruses.

Human respirovirus 3 (also known as human parainfluenza virus 3; hPIV3) is a major cause of severe acute respiratory infections in vulnerable populations. Here we conducted a genome-wide CRISPR/Cas9 library screen to identify key host factors for hPIV3 infection. In addition to identifying several host proteins involved in glycosylation as proviral factors, we identified β-1,4-N-Acetyl-Galactosaminyltransferase 2 (β4GALNT2) as a potent restriction factor. Further investigation demonstrated that the addition of a GalNAc residue to α2-3-sialylated glycans by β4GALNT2, resulting in the Sd^a glycotope, disrupted the interaction between the viral hemagglutinin-neuraminidase (HN) attachment protein and sialoglycan receptors. Specifically, the additional GalNAc residue interfered with the interaction of residue W371 in HN with sub-terminal glycan moieties. β4GALNT2-mediated Sd^a epitope expression also negatively affected infection by other respiroviruses, with the strongest effect being observed for hPIV3.

Sialic acids (Sia) on glycoproteins and glycosphingolipids act as receptors for many respiratory viruses, including human influenza A viruses and respiroviruses, which typically bind to α2-6- and α2-3-linked Sia (2-6Sia and 2-3Sia), respectively. In view of this binding discrepancy and as the exact sialoglycoconjugates supporting respirovirus infection remain unclear, we assessed the receptor requirements for infection and binding of several respiroviruses and avian Newcastle disease virus (NDV). To this end, we employed a library of isogenic HEK293 cells displaying defined sialoglycoconjugates. Respirovirus infection was shown to strictly depend on 2-3Sia, with a preference for the terminal Siaα2-3Galβ1-4GlcNAc sialoglycotope regardless of the underlying glycoconjugate structure, whereas NDV showed a broader sialoglycotope usage. Notably, 2-6Sia enhanced respirovirus infection in low 2-3Sia contexts via heteromultivalent surface binding, which resulted in prolonged virus–receptor interactions. This heteromultivalent binding is expected to aid respirovirus infection of the 2-6Sia-rich human upper respiratory tract.

Influenza A virus neuraminidase (NA) is central to the viral life cycle and a key target for studying sialoside recognition and hydrolysis and its impact on viral uptake, tropism, and pathogenesis. Here, we report the design, synthesis, and evaluation of 2,3‐difluorosialic acid‐based activity‐based probes for NA profiling. The probes carry a C4‐amino substituent to promote active‐site engagement and stabilize covalent trapping through a tyrosine‐sialosyl intermediate. A C5 azidoacetamide handle enables bioorthogonal tagging by CuAAC for detection, while a preclicked biotin variant supports one‐step labeling. We synthesized both the azide and biotin formats and assessed their inhibitory activity against recombinant influenza A NAs. A reactivation assay showed prolonged, hour‐scale recovery relative to related 2,3‐difluoro analogs, although the C5 modification reduced NA affinity and covalent half‐life compared with 4‐amino‐2,3‐difluoro‐Neu5Ac. In labeling experiments, the probes tagged multiple recombinant viral neuraminidases and NA present in virus samples. In addition, 9‐azido‐2,3‐difluoro‐Neu5Ac and its biotin preclicked counterpart proved potent activity‐based probes for NAs. Together, these four probes provide lead structures for further development of molecular tools for cellular profiling, viral NA activity detection, and diagnostics.

Swine are a crucial species in influenza A virus (IAV) ecology, capable of supporting replication and consequently evolution of human, avian, and swine strains. They are also the source of the 2009 pandemic H1N1 virus (H1N1pdm09). Understanding how swine IAV breach the species barrier is essential for detection of potentially pandemic viruses, but is often overlooked compared to avian IAV. We hypothesize that the HA–NA balance of (pre)pandemic swine viruses is a key feature that needs adjustment as swine and humans differ in their sialoglycome. We focused on a set of (pre)pandemic swine viruses collected in Mexico following the H1N1pdm09 emergence. Phylogenetic analysis showed that the emergence was preceded by substitutions in HA and NA predicted to affect their function. Thus, HA acquired substitution A227E, previously shown to reduce receptor binding in H1N1pdm09, while NA obtained substitution S369N in the 2nd sialic acid binding site (2SBS) predicted to reduce NA cleavage. The substitutions’ effect on the HA–NA balance, was confirmed with biolayer interferometry using recombinant proteins attached to nanoparticles. Engineering recombinant attenuated influenza viruses with swine HA and NA with/without the mentioned substitutions, showed that the 2SBS substitution negatively affected replication in primary human and swine cultures. Interestingly, replication was restored by the HA substitution. Overall, we show that the emergence of H1N1pdm09 in humans was preceded by substitutions in HA and NA that were acquired in swine and adjusted the HA–NA balance. We hypothesize that adjustment of the HA–NA balance was a critical step for crossing the host–species barrier.

Respiratory viruses, including coronaviruses, generally initiate infection in the upper respiratory tract, where mechanistic studies of host-virus interactions require models amenable to genetic manipulation. Human nasal air-liquid interface (ALI) cultures are realistic models of the nasal epithelium, but robust CRISPR/Cas9 engineering workflows for this primary cell system have been lacking. Here, we established an integration-free CRISPR/Cas9 workflow for genetic engineering of human nasal basal cells, which are subsequently differentiated into gene-edited ALI cultures. GFP-based sorting of RNP-transfected nasal basal cells substantially increases editing, achieving knockout efficiencies of 70–90% across gene targets (B2M, SLC35A1, ACE2, and TMPRSS2). Genetic disruption of ACE2 markedly reduces infection by SARS-CoV-2 and hCoV-NL63, confirming its important role for both viruses in the human nasal epithelium. TMPRSS2 knockout substantially reduces replication of both viruses, demonstrating SARS-CoV-2 depending on TMPRSS2 in the human nasal epithelium and providing genetic evidence in primary human cell cultures that hCoV-NL63 relies on TMPRSS2. This platform enables systematic genetic perturbation in a physiologically relevant model of the primary site of respiratory virus infection, validates ACE2 and TMPRSS2 as targets for medical countermeasures against SARS-CoV-2 and hCoV-NL63, and provides a versatile approach to identify host dependency factors for both circulating and emerging respiratory viruses.