Xinzheng Zhang

The herpesvirus DNA replication machinery comprises a battery of viral enzymes that orchestrate viral genome synthesis. In herpes simplex virus type 1 (HSV-1), the machinery consists of seven essential components, including the origin-binding protein UL9, the single-stranded DNA (ssDNA)-binding protein ICP8, the heterodimeric DNA polymerase complex UL30–UL42, and the heterotrimeric helicase–primase complex UL5–UL8–UL52. UL9, a superfamily 2 (SF2) helicase, functions as a dimer that specifically recognizes replication origins and unwinds duplex DNA to initiate replication. Furthermore, UL9 recruits the replication machinery through interactions with viral components and engages cellular proteins that regulate its function. However, the molecular mechanisms underlying the multifunctionality of UL9 remain incompletely understood due to the lack of structural information. Here, we present cryo-electron microscopy structures of UL9 in both apo and DNA-bound states. Together with biochemical and enzymatic assays, we elucidate the molecular basis of UL9 dimerization, origin recognition and allosteric regulation by ICP8. IMPORTANCE: Herpes simplex virus 1 (HSV-1) is a widespread virus that causes lifelong infections, leading to periodic outbreaks ranging from common cold sores to life-threatening encephalitis, and no current treatment can eradicate the dormant virus. To multiply, HSV-1 relies on a protein-based molecular machine to replicate its genome, where the unwinding of double-stranded DNA at specific replication origins is coordinated by the viral origin-binding protein UL9. Here, we present the high-resolution structures of UL9, both alone and bound to DNA, revealing how it forms a stable homodimer to grab onto the origin. Combined with precise biochemical experiments, we further show how UL9 collaborates with another viral helper protein, ICP8, to unwind DNA efficiently. These discoveries solve a long-standing puzzle in herpesvirus biology and offer a vital structural blueprint for designing new antiviral drugs that can block viral replication at its very earliest stage.

Cryo-electron microscopy (cryo-EM) data acquisition is time-intensive given that a large amount of data is required to obtain a high-resolution reconstruction. Here, we eliminate camera-induced delay time by continuously recording during beam-image shift acquisition using a method called Continuous Recording Beam-Image Shift (CR-BIS). The utilization of CR-BIS with K3 and Falcon 4 direct electron detectors and conventional data acquisition conditions enables the acquisition of ~34,000 micrographs and ~1,000 tilt series per 24 h in single-particle analysis mode and cryo-electron tomography mode, respectively. Three-dimensional reconstructions of single-particle and tomographic datasets show that CR-BIS accelerates data collection and maintains data quality. CR-BIS is broadly applicable for efficient high-resolution cryo-EM since it can be implemented into existing acquisition software through scripting and it does not require hardware modification.

In situ structural analysis allows direct visualization of protein structures in their native cellular environments, but near-atomic resolution in cellular lamellae has been significantly limited to exceptionally large complexes such as ribosomes. A key factor underlying this limitation is the degradation of data quality of thin lamellae caused by substantial subsurface damage from cryo-focused ion beam (cryo-FIB). Here, we developed a cryogenic low-energy polishing in FIB approach, which reliably produces thin lamellae with low damage across different cell types. This advance, combined with in situ single particles analysis, has pushed down the molecular weight lower limit for in situ reconstruction at near-atomic resolution to ∼400 kDa. We demonstrate this by resolving photosynthetic complexes (3.4 Å and 3.3 Å), metabolic enzymes (3.3 Å), chloroplast ribosome (4.0 Å) and respiratory chain complexes (3.7 Å) from Chlamydomonas reinhardtii . Furthermore, the efficient workflow enables rapid structural feedback upon changes in cellular states, offering a practical way to perform multi-condition in situ structural analysis.

Lipid nanoparticles (LNPs) are the most clinically relevant vehicles for mRNA vaccines. Despite the great successes, the toxicity caused by the high dose of lipid components still represents a great challenge. The suboptimal loading capacity of mRNA in LNPs not only compromises the vaccine’s efficacy but also heightens the risk of non-specific immune responses, accelerates clearance caused by anti-PEG IgG/IgM. These problems underscore the urgent need for improving mRNA loading capacity in LNPs to provide dose-sparing effects. Herein, we develop a metal ion mediated mRNA enrichment strategy to efficiently form a high-density mRNA core, and manganese ion (Mn²⁺) exhibits a unique capability to match the need. The prepared Mn-mRNA nanoparticle is subsequently coated with lipids to form the resulting nanosystem, L@Mn-mRNA, which achieved nearly twice the mRNA loading capacity compared to conventional mRNA vaccine formulations (LNP-mRNA). Remarkably, L@Mn-mRNA also demonstrates a 2-fold increase in cellular uptake efficiency compared to LNP-mRNA, attributed to the enhanced stiffness provided by the Mn-mRNA core. By combining improved mRNA loading with superior cellular uptake, L@Mn-mRNA achieves significantly enhanced antigen-specific immune responses and therapeutic efficacy as vaccines. We elucidate the mechanism behind Mn-mRNA construction and optimize the L@Mn-mRNA formulations, and this method is suitable for types of lipids and mRNAs. Moreover, L@Mn-mRNA also reduces the risk of anti-PEG IgG/IgM generation. Thus, this strategy holds significant potential as a platform for the next generation of lipid-based mRNA vaccines.

Neutralizing antibodies have been designed to specifically target and bind to the receptor binding domain (RBD) of spike (S) protein to block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus from attaching to angiotensin converting enzyme 2 (ACE2). This study reports a distinctive nanobody, designated as VHH21, that directly catalyzes the S-trimer into an irreversible transition state through postfusion conformational changes. Derived from camels immunized with multiple antigens, a set of nanobodies with high affinity for the S1 protein displays abilities to neutralize pseudovirion infections with a broad resistance to variants of concern of SARS-CoV-2, including SARS-CoV and BatRaTG13. Importantly, a super-resolution screening and analysis platform based on visual fluorescence probes was designed and applied to monitor single proteins and protein subunits. A spontaneously occurring dimeric form of VHH21 was obtained to rapidly destroy the S-trimer. Structural analysis via cryogenic electron microscopy revealed that VHH21 targets specific conserved epitopes on the S protein, distinct from the ACE2 binding site on the RBD, which destabilizes the fusion process. This research highlights the potential of VHH21 as an abzyme-like nanobody (nanoabzyme) possessing broad-spectrum binding capabilities and highly effective anti-viral properties and offers a promising strategy for combating coronavirus outbreaks.

Many protein complexes are highly dynamic in cells; thus, characterizing their conformational changes in cells is crucial for unraveling their functions. Here, using cryo-electron microscopy, 451,700 ribosome particles from Saccharomyces cerevisiae cell lamellae were obtained to solve the 60S region to 2.9-Å resolution by in situ single-particle analysis. Over 20 distinct conformations were identified by three-dimensional classification with resolutions typically higher than 4 Å. These conformations were used to reconstruct a complete elongation cycle of eukaryotic translation with elongation factors (eEFs). We found that compact eEF2 anchors to the partially rotated ribosome after subunit rolling and hypothesize that it stabilizes the local conformation for peptidyl transfer. Moreover, open-eEF3 binding to a fully rotated ribosome was observed, whose conformational change was coupled with head swiveling and body back-rotation of the 40S subunit.

Eastern Equine Encephalitis virus (EEEV) is an alphavirus that can cause severe diseases in infected humans. The very low-density lipoprotein receptor (VLDLR) was recently identified as a receptor of EEEV. Herein, we performed cryo-electron microscopy structural and biochemistry studies on the specific interactions between EEEV and VLDLR. Our results show that VLDLR binds EEEV at three different sites A, B and C through its membrane-distal LDLR class A (LA) repeats. Site A is located in the cleft in between the E1-E2 heterodimers. Site B is located near the connecting β ribbon of E2 and is in proximity to site A, while site C is on the domain B of E2. The binding of VLDLR LAs to EEEV is in complex modes, including the LA1-2 and LA3-5 mediated two major modes. Disruption of the LA1-2 mediated binding significantly affect the cell attachment of EEEV. However, the mutation W132G of VLDLR impairs the binding of LA3, drives the switch of the binding modes, and significantly enhances the attachment of EEEV to the cell. The W132G variant of VLDLR could be identified in human genome and SNP sequences, implying that people with similar mutations in VLDLR may be highly susceptible to EEEV infection.

Lipid nanoparticles (LNPs) are the most clinically relevant vehicles for mRNA vaccines. Despite the great successes, the toxicity caused by the high dose of lipid components still represents a great challenge. The suboptimal loading efficiency of mRNA in LNPs not only compromises the vaccine’s efficacy but also heightens the risk of non-specific immune responses, accelerates clearance from the bloodstream, and exacerbates side effects associated with the lipid carriers. These problems underscore the urgent need for improving mRNA loading in LNPs to provide dose-sparing effects. Herein, we developed a manganese ion (Mn²⁺) mediated mRNA enrichment strategy to efficiently form a high-density mRNA core, termed Mn-mRNA nanoparticle, which is subsequently coated with lipids. The resulting nanosystem, L@Mn-mRNA, achieved over twice the mRNA loading compared to conventional mRNA vaccine formulations (LNP-mRNA). Remarkably, L@Mn-mRNA also demonstrated a 2-fold increase in cellular uptake efficiency compared to LNP-mRNA, attributed to the enhanced stiffness provided by the Mn-mRNA core. By combining improved mRNA loading with superior cellular uptake, L@Mn-mRNA achieved significantly enhanced antigen-specific immune responses and therapeutic efficacy as vaccines. We elucidated the mechanism behind Mn-mRNA construction and optimized the L@Mn-mRNA formulations, and this method is suitable for types of lipids and mRNAs. Thus, this strategy holds significant potential as a platform for the next generation of lipid-based mRNA vaccines.