Coronavirus spike proteins mediate receptor binding and membrane fusion, making them prime targets for neutralizing antibodies. In the cases of severe acute respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus 2 and Middle East respiratory syndrome coronavirus, spike proteins transition freely between open and closed conformations to balance host cell attachment and immune evasion^1–5. Spike opening exposes domain S1^B, allowing it to bind to proteinaceous receptors^6,7, and is also thought to enable protein refolding during membrane fusion^4,5. However, with a single exception, the pre-fusion spike proteins of all other coronaviruses studied so far have been observed exclusively in the closed state. This raises the possibility of regulation, with spike proteins more commonly transitioning to open states in response to specific cues, rather than spontaneously. Here, using cryogenic electron microscopy and molecular dynamics simulations, we show that the spike protein of the common cold human coronavirus HKU1 undergoes local and long-range conformational changes after binding a sialoglycan-based primary receptor to domain S1^A. This binding triggers the transition of S1^B domains to the open state through allosteric interdomain crosstalk. Our findings provide detailed insight into coronavirus attachment, with possibilities of dual receptor usage and priming of entry as a means of immune escape.

Influenza A virus (IAV) neuraminidase (NA) receptor-destroying activity and hemagglutinin (HA) receptor-binding affinity need to be balanced with the host receptor repertoire for optimal viral fitness. NAs of avian, but not human viruses, contain a functional 2^nd sialic acid (SIA)-binding site (2SBS) adjacent to the catalytic site, which contributes to sialidase activity against multivalent substrates. The receptor-binding specificity and potentially crucial contribution of the 2SBS to the HA-NA balance of virus particles is, however, poorly characterized. Here, we elucidated the receptor-binding specificity of the 2SBS of N2 NA and established an important role for this site in the virion HA-NA-receptor balance. NAs of H2N2/1957 pandemic virus with or without a functional 2SBS and viruses containing this NA were analysed. Avian-like N2, with a restored 2SBS due to an amino acid substitution at position 367, was more active than human N2 on multivalent substrates containing α2,3-linked SIAs, corresponding with the pronounced binding-specificity of avian-like N2 for these receptors. When introduced into human viruses, avian-like N2 gave rise to altered plaque morphology and decreased replication compared to human N2. An opposite replication phenotype was observed when N2 was combined with avian-like HA. Specific bio-layer interferometry assays revealed a clear effect of the 2SBS on the dynamic interaction of virus particles with receptors. The absence or presence of a functional 2SBS affected virion-receptor binding and receptor cleavage required for particle movement on a receptor-coated surface and subsequent NA-dependent self-elution. The contribution of the 2SBS to virus-receptor interactions depended on the receptor-binding properties of HA and the identity of the receptors used. We conclude that the 2SBS is an important and underappreciated determinant of the HA-NA-receptor balance. The rapid loss of a functional 2SBS in pandemic viruses may have served to balance the novel host receptor-repertoire and altered receptor-binding properties of the corresponding HA protein. Influenza A viruses infect birds and mammals. They contain receptor-binding (HA) and receptor-destroying (NA) proteins, which are crucial determinants of host tropism and pathogenesis. It is generally accepted that the functional properties of HA and NA need to be well balanced to enable virion penetration of the receptor-rich mucus layer, binding to host cells, and release of newly assembled particles. This HA-NA-receptor balance is, however, poorly characterized resulting in part from a lack of suitable assays to measure this balance. In addition, NA is much less studied than HA. NA contains, besides its receptor-cleavage site, a 2^nd receptor-binding site, which is functional in avian, but not in human viruses. We now show that this 2^nd receptor-binding site prefers binding to avian-type receptors and promotes cleavage of substrates carrying this receptor. Furthermore, by using novel assays, we established an important role for this site in the HA-NA-receptor balance of virus particles as it contributes to receptor binding and cleavage by virions, the latter of which is required for virion movement and self-elution from receptors. The results may provide an explanation for the rapid loss of a functional 2^nd receptor-binding site in human pandemic viruses.

O-acetylation is a common modification of sialic acids that has been implicated in a multitude of biological and disease processes. A lack of analytical methods that can determine exact structures of sialic acid variants is a hurdle to determine roles of distinct O-acetylated sialosides. Here, we describe a drift tube ion mobility-mass spectrometry approach that can elucidate exact O-acetylation patterns as well as glycosidic linkage types of sialosides isolated from complex biological samples. It is based on the use of a library of synthetic O-acetylated sialosides to establish intrinsic collision cross section (CCS) values of diagnostic fragment ions. The CCS values were used to characterize O-acetylated sialosides from mucins and N-linked glycans from biologicals as well as equine tracheal and nasal tissues. It uncovered contrasting sialic acid linkage types of acetylated and non-acetylated sialic acids and provided a rationale for sialic acid binding preferences of equine H7 influenza A viruses.

A recent ground-breaking study suggested that small RNA from mammalian cells can undergo N-glycan modifications (termed glycoRNA) ¹ . The discovery relied upon a metabolic glycan labeling strategy in combination with commonly used phase-separation-based RNA isolation. Following the reported procedure, here we likewise identify an N-glycosylated species in the RNA fraction. However, our results suggest that the reported RNase sensitivity of the glycosylated species depends on the specific RNA purification method. This suggests the possibility of copurifying unexpected RNase-insensitive N-glycoconjugates during glycoRNA isolation. The co-existence of two independent, yet highly similar molecular entities, complicates biochemical assays on glycoRNA and calls for more specific approaches for glycoRNA analysis. To address this, we propose a control experiment that can help distinguish genuine glycoRNA species from copurified glycoconjugates.