Structure and Function of Hoc-A Novel Environment Sensing Device Encoded by T4 and Other Bacteriophages.

Bacteriophage T4 is decorated with 155 180 Å-long fibers of the highly antigenic outer capsid protein (Hoc). In this study, we describe a near-atomic structural model of Hoc by combining cryo-electron microscopy and AlphaFold structure predictions. It consists of a conserved C-terminal capsid-binding domain attached to a string of three variable immunoglobulin (Ig)-like domains, an architecture well-preserved in hundreds of Hoc molecules found in phage genomes. Each T4-Hoc fiber attaches randomly to the center of gp23* hexameric capsomers in one of the six possible orientations, though at the vertex-proximal hexamers that deviate from 6-fold symmetry, Hoc binds in two preferred orientations related by 180° rotation. Remarkably, each Hoc fiber binds to all six subunits of the capsomer, though the interactions are greatest with three of the subunits, resulting in the off-centered attachment of the C-domain. Biochemical analyses suggest that the acidic Hoc fiber (pI, ~4-5) allows for the clustering of virions in acidic pH and dispersion in neutral/alkaline pH. Hoc appears to have evolved as a sensing device that allows the phage to navigate its movements through reversible clustering-dispersion transitions so that it reaches its destination, the host bacterium, and persists in various ecological niches such as the human/mammalian gut.

Bacteriophage T4 Head: Structure, Assembly, and Genome Packaging.

Bacteriophage (phage) T4 has served as an extraordinary model to elucidate biological structures and mechanisms. Recent discoveries on the T4 head (capsid) structure, portal vertex, and genome packaging add a significant body of new literature to phage biology. Head structures in unexpanded and expanded conformations show dramatic domain movements, structural remodeling, and a ~70% increase in inner volume while creating high-affinity binding sites for the outer decoration proteins Soc and Hoc. Small changes in intercapsomer interactions modulate angles between capsomer planes, leading to profound alterations in head length. The in situ cryo-EM structure of the symmetry-mismatched portal vertex shows the remarkable structural morphing of local regions of the portal protein, allowing similar interactions with the capsid protein in different structural environments. Conformational changes in these interactions trigger the structural remodeling of capsid protein subunits surrounding the portal vertex, which propagate as a wave of expansion throughout the capsid. A second symmetry mismatch is created when a pentameric packaging motor assembles at the outer "clip" domains of the dodecameric portal vertex. The single-molecule dynamics of the packaging machine suggests a continuous burst mechanism in which the motor subunits adjusted to the shape of the DNA fire ATP hydrolysis, generating speeds as high as 2000 bp/s.

Near-atomic, non-icosahedrally averaged structure of giant virus Paramecium bursaria chlorella virus 1.

Giant viruses are a large group of viruses that infect many eukaryotes. Although components that do not obey the overall icosahedral symmetry of their capsids have been observed and found to play critical roles in the viral life cycles, identities and high-resolution structures of these components remain unknown. Here, by determining a near-atomic-resolution, five-fold averaged structure of Paramecium bursaria chlorella virus 1, we unexpectedly found the viral capsid possesses up to five major capsid protein variants and a penton protein variant. These variants create varied capsid microenvironments for the associations of fibers, a vesicle, and previously unresolved minor capsid proteins. Our structure reveals the identities and atomic models of the capsid components that do not obey the overall icosahedral symmetry and leads to a model for how these components are assembled and initiate capsid assembly, and this model might be applicable to many other giant viruses.

Structures of a large prolate virus capsid in unexpanded and expanded states generate insights into the icosahedral virus assembly.

Many icosahedral viruses assemble proteinaceous precursors called proheads or procapsids. Proheads are metastable structures that undergo a profound structural transition known as expansion that transforms an immature unexpanded head into a mature genome-packaging head. Bacteriophage T4 is a model virus, well studied genetically and biochemically, but its structure determination has been challenging because of its large size and unusually prolate-shaped, ∼1,200-Å-long and ∼860-Å-wide capsid. Here, we report the cryogenic electron microscopy (cryo-EM) structures of T4 capsid in both of its major conformational states: unexpanded at a resolution of 5.1 Å and expanded at a resolution of 3.4 Å. These are among the largest structures deposited in Protein Data Bank to date and provide insights into virus assembly, head length determination, and shell expansion. First, the structures illustrate major domain movements and ∼70% additional gain in inner capsid volume, an essential transformation to contain the entire viral genome. Second, intricate intracapsomer interactions involving a unique insertion domain dramatically change, allowing the capsid subunits to rotate and twist while the capsomers remain fastened at quasi-threefold axes. Third, high-affinity binding sites emerge for a capsid decoration protein that clamps adjacent capsomers, imparting extraordinary structural stability. Fourth, subtle conformational changes at capsomers' periphery modulate intercapsomer angles between capsomer planes that control capsid length. Finally, conformational changes were observed at the symmetry-mismatched portal vertex, which might be involved in triggering head expansion. These analyses illustrate how small changes in local capsid subunit interactions lead to profound shifts in viral capsid morphology, stability, and volume.

Structural and functional insight into regulation of kinesin-1 by microtubule-associated protein MAP7.

Microtubule (MT)-associated protein 7 (MAP7) is a required cofactor for kinesin-1-driven transport of intracellular cargoes. Using cryo-electron microscopy and single-molecule imaging, we investigated how MAP7 binds MTs and facilitates kinesin-1 motility. The MT-binding domain (MTBD) of MAP7 bound MTs as an extended α helix between the protofilament ridge and the site of lateral contact. Unexpectedly, the MTBD partially overlapped with the binding site of kinesin-1 and inhibited its motility. However, by tethering kinesin-1 to the MT, the projection domain of MAP7 prevented dissociation of the motor and facilitated its binding to available neighboring sites. The inhibitory effect of the MTBD dominated as MTs became saturated with MAP7. Our results reveal biphasic regulation of kinesin-1 by MAP7 in the context of their competitive binding to MTs.

COVID-19 vaccine uptake and hesitancy among HIV-infected men who have sex with men in mainland China: a cross-sectional survey.

BACKGROUND: Men who have sex with men (MSM), a population bearing the greatest HIV burden in many countries, may also be vulnerable to COVID-19. COVID-19 vaccines are essential to containing the pandemic. However, vaccine hesitancy may compromise vaccine coverage. We aimed to understand the uptake of COVID-19 vaccine and factors associated with COVID-19 vaccine hesitancy among HIV-infected MSM in mainland China. METHODS: A cross-sectional online survey among HIV-infected MSM was conducted between 13 and 21 February 2021 in mainland China. Variables including demographics, mental health status, HIV characteristics, and knowledge of and attitudes toward COVID-19 pandemic and COVID-19 vaccine were collected. Chi-square tests and multivariable logistic regression were used to analyze factors associated with COVID-19 vaccine hesitancy. RESULTS: A total of 1295 participants were included. The median age was 29.3 years (interquartile range [IQR] 25.2-34.0 years). The uptake of COVID-19 vaccine was 8.7%. Two main reasons for receiving vaccines were "regarded vaccination as self-health protection" (67.3%) and "trust in domestic medical technology" (67.3%). Among participants who did not initiate vaccination, concern about side effects (46.4%) and disclosure of HIV infection (38.6%) were top two reasons, and 47.2% had higher vaccine hesitancy. Men who had with high antiretroviral therapy (ART) adherence (adjusted odds ratio [aOR] 0.53, 95% confidence interval [CI] 0.35-0.80), often (0.26, 0.17-0.40) or sometimes (0.46, 0.31-0.67) paid attention to information about the COVID-19 vaccine, preferred domestic vaccines (0.37, 0.24-0.59), thought the pandemic had moderate (0.58, 0.38-0.90) and moderately severe or severe impact (0.54, 0.38-0.78) on immunity, who were waiting for vaccination programs organized at workplace (0.60, 0.44-0.81) and who were unaware of where to get COVID-19 vaccine (0.61, 0.45-0.82) had lower degree of COVID-19 vaccine hesitancy. Men who were concerned about the efficacy (1.72, 1.16-2.54) and side effects (2.44, 1.78-3.35) had higher degree of COVID-19 vaccine hesitancy. CONCLUSION: COVID-19 vaccine uptake among HIV-infected MSM is still suboptimal. Understanding influencing factors of vaccine hesitancy among this group and making tailored measures to alleviate hesitancy would help improve the coverage of COVID-19 vaccination in this population.

Oxidative stress transforms 3CLpro into an insoluble and more active form to promote SARS-CoV-2 replication.

3CLpro is a key proteinase for SARS-CoV-2 replication and serves as an important target for antiviral drug development. However, how its activity is regulated intracellularly is still obscure. In this study, we developed a 3CLpro protease activity reporter system to examine the impact of various factors, including nutrient supplements, ions, pHs, or oxidative stress inducers, on 3CLpro protease activity. We found that oxidative stress could increase the overall activity of 3CLpro. Not altering the expression, oxidative stress decreased the solubility of 3CLpro in the lysis buffer containing 1% Triton-X-100. The Triton-X-100-insoluble 3CLpro was correlated with aggregates' formation and responsible for the increased enzymatic activity. The disulfide bonds formed between Cys85 sites of 3CLpro protomers account for the insolubility and the aggregation of 3CLpro. Besides being regulated by oxidative stress, 3CLpro impaired the cellular antioxidant capacity by regulating the cleavage of GPx1 at its N-terminus. This cleavage could further elevate the 3CLpro-proximate oxidative activity, favor aggregation and activation of 3CLpro, and thus lead to a positive feedback loop. In summary, we reported that oxidative stress transforms 3CLpro into a detergent-insoluble form that is more enzymatically active, leading to increased viral replication/transcription. Our study provided mechanistic evidence that suggests the therapeutic potential of antioxidants in the clinical treatment of COVID-19 patients.

Structure of the human SAGA coactivator complex.

The SAGA complex is a regulatory hub involved in gene regulation, chromatin modification, DNA damage repair and signaling. While structures of yeast SAGA (ySAGA) have been reported, there are noteworthy functional and compositional differences for this complex in metazoans. Here we present the cryogenic-electron microscopy (cryo-EM) structure of human SAGA (hSAGA) and show how the arrangement of distinct structural elements results in a globally divergent organization from that of yeast, with a different interface tethering the core module to the TRRAP subunit, resulting in a dramatically altered geometry of functional elements and with the integration of a metazoan-specific splicing module. Our hSAGA structure reveals the presence of an inositol hexakisphosphate (InsP6) binding site in TRRAP and an unusual property of its pseudo-(Ψ)PIKK. Finally, we map human disease mutations, thus providing the needed framework for structure-guided drug design of this important therapeutic target for human developmental diseases and cancer.

The remarkable viral portal vertex: structure and a plausible model for mechanism.

Many icosahedral viruses including tailed bacteriophages and herpes viruses have a unique portal vertex where a dodecameric protein ring is associated with a fivefold capsid shell. While the peripheral regions of the portal ring are involved in capsid assembly, its central channel is used to transport DNA into and out of capsid during genome packaging and infection. Though the atomic structure of this highly conserved, turbine-shaped, portal is known for nearly two decades, its molecular mechanism remains a mystery. Recent high-resolution in situ structures reveal various conformational states of the portal and the asymmetric interactions between the 12-fold portal and the fivefold capsid. These lead to a valve-like mechanism for this symmetry-mismatched portal vertex that regulates DNA flow through the channel, a critical function for high fidelity assembly of an infectious virion.

Structure of Usutu virus SAAR-1776 displays fusion loop asymmetry.

Usutu virus (USUV) is an emerging arbovirus in Europe that has been increasingly identified in asymptomatic humans and donated blood samples and is a cause of increased incidents of neuroinvasive human disease. Treatment or prevention options for USUV disease are currently nonexistent, the result of a lack of understanding of the fundamental elements of USUV pathogenesis. Here, we report two structures of the mature USUV virus, determined at a resolution of 2.4 Å, using single-particle cryogenic electron microscopy. Mature USUV is an icosahedral shell of 180 copies of envelope (E) and membrane (M) proteins arranged in the classic herringbone pattern. However, unlike previous reports of flavivirus structures, we observe virus subpopulations and differences in the fusion loop disulfide bond. Presence of a second, unique E glycosylation site could elucidate host interactions, contributing to the broad USUV tissue tropism. The structures provide a basis for exploring USUV interactions with glycosaminoglycans and lectins, the role of the RGD motif as a receptor, and the inability of West Nile virus therapeutic antibody E16 to neutralize the mature USUV strain SAAR-1776. Finally, we identify three lipid binding sites and predict key residues that likely participate in virus stability and flexibility during membrane fusion. Our findings provide a framework for the development of USUV therapeutics and expand the current knowledge base of flavivirus biology.

Structural morphing in a symmetry-mismatched viral vertex.

Large biological structures are assembled from smaller, often symmetric, sub-structures. However, asymmetry among sub-structures is fundamentally important for biological function. An extreme form of asymmetry, a 12-fold-symmetric dodecameric portal complex inserted into a 5-fold-symmetric capsid vertex, is found in numerous icosahedral viruses, including tailed bacteriophages, herpesviruses, and archaeal viruses. This vertex is critical for driving capsid assembly, DNA packaging, tail attachment, and genome ejection. Here, we report the near-atomic in situ structure of the symmetry-mismatched portal vertex from bacteriophage T4. Remarkably, the local structure of portal morphs to compensate for symmetry-mismatch, forming similar interactions in different capsid environments while maintaining strict symmetry in the rest of the structure. This creates a unique and unusually dynamic symmetry-mismatched vertex that is central to building an infectious virion.

Cryo-EM structure of rhinovirus C15a bound to its cadherin-related protein 3 receptor.

Infection by Rhinovirus-C (RV-C), a species of Picornaviridae Enterovirus, is strongly associated with childhood asthma exacerbations. Cellular binding and entry by all RV-C, which trigger these episodes, is mediated by the first extracellular domain (EC1) of cadherin-related protein 3 (CDHR3), a surface cadherin-like protein expressed primarily on the apical surfaces of ciliated airway epithelial cells. Although recombinant EC1 is a potent inhibitor of viral infection, there is no molecular description of this protein or its binding site on RV-C. Here we present cryo-electron microscopy (EM) data resolving the EC1 and EC1+2 domains of human CDHR3 complexed with viral isolate C15a. Structure-suggested residues contributing to required interfaces on both EC1 and C15a were probed and identified by mutagenesis studies with four different RV-C genotypes. In contrast to most other rhinoviruses, which bind intercellular adhesion molecule 1 receptors via a capsid protein VP1-specific fivefold canyon feature, the CDHR3 EC1 contacts C15a, and presumably all RV-Cs, in a unique cohesive footprint near the threefold vertex, encompassing residues primarily from viral protein VP3, but also from VP1 and VP2. The EC1+2 footprint on C15a is similar to that of EC1 alone but shows that steric hindrance imposed by EC2 would likely prevent multiprotein binding by the native receptor at any singular threefold vertex. Definition of the molecular interface between the RV-Cs and their receptors provides new avenues that can be explored for potential antiviral therapies.

A sequestered fusion peptide in the structure of an HIV-1 transmitted founder envelope trimer.

The envelope protein of human immunodeficiency virus-1 (HIV-1) and its fusion peptide are essential for cell entry and vaccine design. Here, we describe the 3.9-Å resolution structure of an envelope protein trimer from a very early transmitted founder virus (CRF01_AE T/F100) complexed with Fab from the broadly neutralizing antibody (bNAb) 8ANC195. The overall T/F100 trimer structure is similar to other reported "closed" state prefusion trimer structures. In contrast, the fusion peptide, which is exposed to solvent in reported closed structures, is sequestered (buried) in the hydrophobic core of the T/F100 trimer. A buried conformation has previously been observed in "open" state structures formed after CD4 receptor binding. The T/F100 trimer binds poorly to bNAbs including the fusion peptide-specific bNAbs PGT151 and VRC34.01. The T/F100 structure might represent a prefusion state, intermediate between the closed and open states. These observations are relevant to mechanisms of HIV-1 transmission and vaccine design.

Near-atomic structure of a giant virus.

Although the nucleocytoplasmic large DNA viruses (NCLDVs) are one of the largest group of viruses that infect many eukaryotic hosts, the near-atomic resolution structures of these viruses have remained unknown. Here we describe a 3.5 Å resolution icosahedrally averaged capsid structure of Paramecium bursaria chlorella virus 1 (PBCV-1). This structure consists of 5040 copies of the major capsid protein, 60 copies of the penton protein and 1800 minor capsid proteins of which there are 13 different types. The minor capsid proteins form a hexagonal network below the outer capsid shell, stabilizing the capsid by binding neighboring capsomers together. The size of the viral capsid is determined by a tape-measure, minor capsid protein of which there are 60 copies in the virion. Homologs of the tape-measure protein and some of the other minor capsid proteins exist in other NCLDVs. Thus, a similar capsid assembly pathway might be used by other NCLDVs.

Pushing the resolution limit by correcting the Ewald sphere effect in single-particle Cryo-EM reconstructions.

The Ewald sphere effect is generally neglected when using the Central Projection Theorem for cryo electron microscopy single-particle reconstructions. This can reduce the resolution of a reconstruction. Here we estimate the attainable resolution and report a "block-based" reconstruction method for extending the resolution limit. We find the Ewald sphere effect limits the resolution of large objects, especially large viruses. After processing two real datasets of large viruses, we show that our procedure can extend the resolution for both datasets and can accommodate the flexibility associated with large protein complexes.

Human cytomegalovirus IE1 protein alters the higher-order chromatin structure by targeting the acidic patch of the nucleosome.

Human cytomegalovirus (hCMV) immediate early 1 (IE1) protein associates with condensed chromatin of the host cell during mitosis. We have determined the structure of the chromatin-tethering domain (CTD) of IE1 bound to the nucleosome core particle, and discovered that IE1-CTD specifically interacts with the H2A-H2B acidic patch and impairs the compaction of higher-order chromatin structure. Our results suggest that IE1 loosens up the folding of host chromatin during hCMV infections.

Hat2p recognizes the histone H3 tail to specify the acetylation of the newly synthesized H3/H4 heterodimer by the Hat1p/Hat2p complex.

Post-translational modifications of histones are significant regulators of replication, transcription, and DNA repair. Particularly, newly synthesized histone H4 in H3/H4 heterodimers becomes acetylated on N-terminal lysine residues prior to its incorporation into chromatin. Previous studies have established that the histone acetyltransferase (HAT) complex Hat1p/Hat2p medicates this modification. However, the mechanism of how Hat1p/Hat2p recognizes and facilitates the enzymatic activities on the newly assembled H3/H4 heterodimer remains unknown. Furthermore, Hat2p is a WD40 repeat protein, which is found in many histone modifier complexes. However, how the WD40 repeat proteins facilitate enzymatic activities of histone modification enzymes is unclear. In this study, we first solved the high-resolution crystal structure of a Hat1p/Hat2p/CoA/H4 peptide complex and found that the H4 tail interacts with both Hat1p and Hat2p, by which substrate recruitment is facilitated. We further discovered that H3 N-terminal peptides can bind to the Hat2p WD40 domain and solved the structure of the Hat1p/Hat2p/CoA/H4/H3 peptide complex. Moreover, the interaction with Hat2p requires unmodified Arg2/Lys4 and Lys9 on the H3 tail, suggesting a novel model to specify the activity of Hat1p/Hat2p toward newly synthesized H3/H4 heterodimers. Together, our study demonstrated the substrate recognition mechanism by the Hat1p/Hat2p complex, which is critical for DNA replication and other chromatin remodeling processes.

Nα-acetylated Sir3 stabilizes the conformation of a nucleosome-binding loop in the BAH domain.

In Saccharomyces cerevisiae, acetylation of the Sir3 N terminus is important for transcriptional silencing. This covalent modification promotes the binding of the Sir3 BAH domain to the nucleosome, but a mechanistic understanding of this phenomenon is lacking. By X-ray crystallography, we show here that the acetylated N terminus of Sir3 does not interact with the nucleosome directly. Instead, it stabilizes a nucleosome-binding loop in the BAH domain.