A narrow ratio of nucleic acid to SARS-CoV-2 N-protein enables phase separation.

SARS-CoV-2 Nucleocapsid protein (N) is a viral structural protein that packages the 30kb genomic RNA inside virions and forms condensates within infected cells through liquid-liquid phase separation (LLPS). N, in both soluble and condensed forms, has accessory roles in the viral life cycle including genome replication and immunosuppression. The ability to perform these tasks depends on phase separation and its reversibility. The conditions that stabilize and destabilize N condensates and the role of N-N interactions are poorly understood. We have investigated LLPS formation and dissolution in a minimalist system comprised of N protein and an ssDNA oligomer just long enough to support assembly. The short oligo allows us to focus on the role of N-N interaction. We have developed a sensitive FRET assay to interrogate LLPS assembly reactions from the perspective of the oligonucleotide. We find that N alone can form oligomers but that oligonucleotide enables their assembly into a three-dimensional phase. At a ~1:1 ratio of N to oligonucleotide LLPS formation is maximal. We find that a modest excess of N or of nucleic acid causes the LLPS to break down catastrophically. Under the conditions examined here assembly has a critical concentration of about 1 µM. The responsiveness of N condensates to their environment may have biological consequences. A better understanding of how nucleic acid modulates N-N association will shed light on condensate activity and could inform antiviral strategies targeting LLPS.

Mutations at the Alphavirus E1'-E2 Interdimer Interface Have Host-Specific Phenotypes.

Alphaviruses are enveloped viruses transmitted by arthropod vectors to vertebrate hosts. The surface of the virion contains 80 glycoprotein spikes embedded in the membrane, and these spikes mediate attachment to the host cell and initiate viral fusion. Each spike consists of a trimer of E2-E1 heterodimers. These heterodimers interact at the following two interfaces: (i) the intradimer interactions between E2 and E1 of the same heterodimer and (ii) the interdimer interactions between E2 of one heterodimer and E1 of the adjacent heterodimer (E1'). We hypothesized that the interdimer interactions are essential for trimerization of the E2-E1 heterodimers into a functional spike. In this work, we made a mutant virus (chikungunya piggyback [CPB]) where we replaced six interdimeric residues in the E2 protein of Sindbis virus (wild-type [WT] SINV) with those from the E2 protein from chikungunya virus and studied its effect in both mammalian and mosquito cell lines. CPB produced fewer infectious particles in mammalian cells than in mosquito cells, relative to WT SINV. When CPB virus was purified from mammalian cells, particles showed reduced amounts of glycoproteins relative to the capsid protein and contained defects in particle morphology compared with virus derived from mosquito cells. Using cryo-electron microscopy (cryo-EM), we determined that the spikes of CPB had a different conformation than WT SINV. Last, we identified two revertants, E2-H333N and E1-S247L, that restored particle growth and assembly to different degrees. We conclude the interdimer interface is critical for spike trimerization and is a novel target for potential antiviral drug design. IMPORTANCE Alphaviruses, which can cause disease when spread to humans by mosquitoes, have been classified as emerging pathogens, with infections occurring worldwide. The spikes on the surface of the alphavirus particle are absolutely required for the virus to enter a new host cell and initiate an infection. Using a structure-guided approach, we made a mutant virus that alters spike assembly in mammalian cells but not mosquito cells. This finding is important because it identifies a region in the spike that could be a target for antiviral drug design.

Dissecting the Components of Sindbis Virus from Arthropod and Vertebrate Hosts: Implications for Infectivity Differences.

Sindbis virus (SINV) is an enveloped, single-stranded RNA virus, which is transmitted via mosquitos to a wide range of vertebrate hosts. SINV produced by vertebrate, baby hamster kidney (BHK) cells is more than an order of magnitude less infectious than SINV produced from mosquito (C6/36) cells. The cause of this difference is poorly understood. In this study, charge detection mass spectrometry was used to determine the masses of intact SINV particles isolated from BHK and C6/36 cells. The measured masses are substantially different: 52.88 MDa for BHK derived SINV and 50.69 MDa for C6/36 derived. Further analysis using several mass spectrometry-based methods and biophysical approaches indicates that BHK derived SINV has a substantially higher mass than C6/36 derived because in the lipid bilayer, there is a higher portion of lipids containing long chain fatty acids. The difference in lipid composition could influence the organization of the lipid bilayer. As a result, multiple stages of the viral lifecycle may be affected including assembly and budding, particle stability during transmission, and fusion events, all of which could contribute to the differences in infectivity.

Charge Detection Mass Spectrometry Identifies Preferred Non-Icosahedral Polymorphs in the Self-Assembly of Woodchuck Hepatitis Virus Capsids.

Woodchuck hepatitis virus (WHV) is prone to aberrant assembly in vitro and can form a broad distribution of oversized particles. Characterizing aberrant assembly products is challenging because they are both large and heterogeneous. In this work, charge detection mass spectrometry (CDMS) is used to measure the distribution of WHV assembly products. CDMS is a single-particle technique where the masses of individual ions are determined from simultaneous measurement of each ion's charge and m/z (mass-to-charge) ratio. Under relatively aggressive, assembly promoting conditions, roughly half of the WHV assembly products are T=4 capsids composed of exactly 120 dimers while the other half are a broad distribution of larger species that extends to beyond 210 dimers. There are prominent peaks at around 132 dimers and at 150 dimers. In part, the 150 dimer complex can be attributed to elongating a T=4 capsid along its 5-fold axis by adding a ring of hexamers. However, most of the other features cannot be explained by existing models for hexameric defects. Cryo-electron microscopy provides evidence of elongated capsids. However, image analysis reveals that many of them are not closed but have "spiral-like" morphologies. The CDMS data indicate that oversized capsids have a preference for growth by addition of 3 or 4 dimers, probably by completion of hexameric vertices.

Hepatitis B Virus Core Protein Phosphorylation Sites Affect Capsid Stability and Transient Exposure of the C-terminal Domain.

Hepatitis B virus core protein has 183 amino acids divided into an assembly domain and an arginine-rich C-terminal domain (CTD) that regulates essential functions including genome packaging, reverse transcription, and intracellular trafficking. Here, we investigated the CTD in empty hepatitis B virus (HBV) T=4 capsids. We examined wild-type core protein (Cp183-WT) and a mutant core protein (Cp183-EEE), in which three CTD serines are replaced with glutamate to mimic phosphorylated protein. We found that Cp183-WT capsids were less stable than Cp183-EEE capsids. When we tested CTD sensitivity to trypsin, we detected two different populations of CTDs differentiated by their rate of trypsin cleavage. Interestingly, CTDs from Cp183-EEE capsids exhibited a much slower rate of proteolytic cleavage when compared with CTDs of Cp183-WT capsids. Cryo-electron microscopy studies of trypsin-digested capsids show that CTDs at five-fold symmetry vertices are most protected. We hypothesize that electrostatic interactions between glutamates and arginines in Cp183-EEE, particularly at five-fold, increase capsid stability and reduce CTD exposure. Our studies show that quasi-equivalent CTDs exhibit different rates of exposure and thus might perform distinct functions during the hepatitis B virus lifecycle. Our results demonstrate a structural role for CTD phosphorylation and indicate crosstalk between CTDs within a capsid particle.

Structurally similar woodchuck and human hepadnavirus core proteins have distinctly different temperature dependences of assembly.

UNLABELLED: Woodchuck hepatitis virus (WHV), a close relative of human hepatitis B virus (HBV), has been a key model for disease progression and clinical studies. Sequences of the assembly domain of WHV and HBV core proteins (wCp149 and hCp149, respectively) have 65% identity, suggesting similar assembly behaviors. We report a cryo-electron microscopy (cryo-EM) structure of the WHV capsid at nanometer resolution and characterization of wCp149 assembly. At this resolution, the T=4 capsid structures of WHV and HBV are practically identical. In contrast to their structural similarity, wCp149 demonstrates enhanced assembly kinetics and stronger dimer-dimer interactions than hCp149: at 23 °C and at 100 mM ionic strength, the pseudocritical concentrations of assembly of wCp149 and hCp149 are 1.8 µM and 43.3 µM, respectively. Transmission electron microscopy reveals that wCp149 assembles into predominantly T=4 capsids with a sizeable population of larger, nonicosahedral structures. Charge detection mass spectrometry indicates that T=3 particles are extremely rare compared to the ∼ 5% observed in hCp149 reactions. Unlike hCp149, wCp149 capsid assembly is favorable over a temperature range of 4 °C to 37 °C; van't Hoff analyses relate the differences in temperature dependence to the high positive values for heat capacity, enthalpy, and entropy of wCp149 assembly. Because the final capsids are so similar, these findings suggest that free wCp149 and hCp149 undergo different structural transitions leading to assembly. The difference in the temperature dependence of wCp149 assembly may be related to the temperature range of its hibernating host. IMPORTANCE: In this paper, we present a cryo-EM structure of a WHV capsid showing its similarity to HBV. We then observe that the assembly properties of the two homologous proteins are very different. Unlike human HBV, the capsid protein of WHV has evolved to function in a nonhomeostatic environment. These studies yield insight into the interplay between core protein self-assembly and the host environment, which may be particularly relevant to plant viruses and viruses with zoonotic cycles involving insect vectors.

Detection of late intermediates in virus capsid assembly by charge detection mass spectrometry.

The assembly of hundreds of identical proteins into an icosahedral virus capsid is a remarkable feat of molecular engineering. How this occurs is poorly understood. Key intermediates have been anticipated at the end of the assembly reaction, but it has not been possible to detect them. In this work we have used charge detection mass spectrometry to identify trapped intermediates from late in the assembly of the hepatitis B virus T = 4 capsid, a complex of 120 protein dimers. Prominent intermediates are found with 104/105, 110/111, and 117/118 dimers. Cryo-EM observations indicate the intermediates are incomplete capsids and, hence, on the assembly pathway. On the basis of their stability and kinetic accessibility we have proposed plausible structures. The prominent trapped intermediate with 104 dimers is attributed to an icosahedron missing two neighboring facets, the 111-dimer species is assigned to an icosahedron missing a single facet, and the intermediate with 117 dimers is assigned to a capsid missing a ring of three dimers in the center of a facet.

Designing two self-assembly mechanisms into one viral capsid protein.

ELP-CP, a structural fusion protein of the thermally responsive elastin-like polypeptide (ELP) and a viral capsid protein (CP), was designed, and its assembly properties were investigated. Interestingly, this protein-based block copolymer could be self-assembled via two mechanisms into two different, well-defined nanocapsules: (1) pH-induced assembly yielded 28 nm virus-like particles, and (2) ELP-induced assembly yielded 18 nm virus-like particles. The latter were a result of the emergent properties of the fusion protein. This work shows the feasibility of creating a self-assembly system with new properties by combining two structural protein elements.

Structural organization of pregenomic RNA and the carboxy-terminal domain of the capsid protein of hepatitis B virus.

The Hepatitis B Virus (HBV) double-stranded DNA genome is reverse transcribed from its RNA pregenome (pgRNA) within the virus core (or capsid). Phosphorylation of the arginine-rich carboxy-terminal domain (CTD) of the HBV capsid protein (Cp183) is essential for pgRNA encapsidation and reverse transcription. However, the structure of the CTD remains poorly defined. Here we report sub-nanometer resolution cryo-EM structures of in vitro assembled empty and pgRNA-filled Cp183 capsids in unphosphorylated and phosphorylation-mimic states. In empty capsids, we found unexpected evidence of surface accessible CTD density partially occluding pores in the capsid surface. We also observed that CTD organization changed substantively as a function of phosphorylation. In RNA-filled capsids, unphosphorylated CTDs favored thick ropes of RNA, while the phosphorylation-mimic favored a mesh of thin, high-density strands suggestive of single stranded RNA. These results demonstrate that the CTD can regulate nucleic acid structure, supporting the hypothesis that the HBV capsid has a functional role as a nucleic acid chaperone.

Differential assembly of Hepatitis B Virus core protein on single- and double-stranded nucleic acid suggest the dsDNA-filled core is spring-loaded.

Hepatitis B Virus (HBV) cores assemble on viral RNA, which is reverse transcribed within the core to the partially dsDNA genome of mature HBV. However, constraining dsDNA, a stiff polymer, within a core necessarily requires far greater capsid stability than constraining ssRNA. We hypothesized that, unlike ssRNA, dsDNA would be a poor substrate for assembly. We examined titrations of ssDNA and dsDNA with purified HBV core protein, Cp183, by EMSA, EM, DLS, and etheno-DNA fluorescence. Cp183 bound ssDNA with high affinity to form virus-like capsids. However, Cp183 bound dsDNA poorly, forming a mixture of irregular complexes. Nonetheless, we observed some normal cores in dsDNA assembly reactions, indicating that the energy required to bend DNA could be similar to the protein-protein association energy. This similarity of energies suggests that dsDNA stresses mature HBV cores, in agreement with calculation, which may be the basis for the virus maturation signal and DNA release.

Essential elements of the capsid protein for self-assembly into empty virus-like particles of hepatitis E virus.

Hepatitis E virus (HEV) is a noncultivable virus that causes acute liver failure in humans. The virus's major capsid protein is encoded by an open reading frame 2 (ORF2) gene. When the recombinant protein consisting of amino acid (aa) residues 112 to 660 of ORF2 is expressed with a recombinant baculovirus, the protein self-assembles into virus-like particles (VLPs) (T.-C. Li, Y. Yamakawa, K. Suzuki, M. Tatsumi, M. A. Razak, T. Uchida, N. Takeda, and T. Miyamura, J. Virol. 71:7207-7213, 1997). VLPs can be found in the culture medium of infected Tn5 cells but not in that of Sf9 cells, and the major VLPs have lost the C-terminal 52 aa. To investigate the protein requirement for HEV VLP formation, we prepared 14 baculovirus recombinants to express the capsid proteins truncated at the N terminus, the C terminus, or both. The capsid protein consisting of aa residues 112 to 608 formed VLPs in Sf9 cells, suggesting that particle formation is dependent on the modification process of the ORF2 protein. In the present study, electron cryomicroscopy and image processing of VLPs produced in Sf9 and Tn5 cells indicated that they possess the same configurations and structures. Empty VLPs were found in both Tn5 and Sf9 cells infected with the recombinant containing an N-terminal truncation up to aa residue 125 and C-terminal to aa residue 601, demonstrating that the aa residues 126 to 601 are the essential elements required for the initiation of VLP assembly. The recombinant HEV VLPs are potential mucosal vaccine carrier vehicles for the presentation of foreign antigenic epitopes and may also serve as vectors for the delivery of genes to mucosal tissue for DNA vaccination and gene therapy. The results of the present study provide useful information for constructing recombinant HEV VLPs having novel functions.