Significance of hepatitis B virus capsid dephosphorylation via polymerase.

BACKGROUND: It is generally believed that hepatitis B virus (HBV) core protein (HBc) dephosphorylation (de-P) is important for viral DNA synthesis and virion secretion. HBV polymerase contains four domains for terminal protein, spacer, reverse transcriptase, and RNase H activities. METHODS: HBV Polymerase mutants were transfected into HuH-7 cells and assayed for replication and HBc de-P by the Phos-tag gel analysis. Infection assay was performed by using a HepG2-NTCP-AS2 cell line. RESULTS: Here, we show that a novel phosphatase activity responsible for HBc de-P can be mapped to the C-terminal domain of the polymerase overlapping with the RNase H domain. Surprisingly, while HBc de-P is crucial for viral infectivity, it is essential for neither viral DNA synthesis nor virion secretion. The potential origin, significance, and mechanism of this polymerase-associated phosphatase activity are discussed in the context of an electrostatic homeostasis model. The Phos-tag gel analysis revealed an intriguing pattern of "bipolar distribution" of phosphorylated HBc and a de-P HBc doublet. CONCLUSIONS: It remains unknown if such a polymerase-associated phosphatase activity can be found in other related biosystems. This polymerase-associated phosphatase activity could be a druggable target in clinical therapy for hepatitis B.

Locations and structures of influenza A virus packaging-associated signals and other functional elements via an in silico pipeline for predicting constrained features in RNA viruses.

Influenza A virus contains regions of its segmented genome associated with ability to package the segments into virions, but many such regions are poorly characterised. We provide detailed predictions of the key locations within these packaging-associated regions, and their structures, by applying a recently-improved pipeline for delineating constrained regions in RNA viruses and applying structural prediction algorithms. We find and characterise other known constrained regions within influenza A genomes, including the region associated with the PA-X frameshift, regions associated with alternative splicing, and constraint around the initiation motif for a truncated PB1 protein, PB1-N92, associated with avian viruses. We further predict the presence of constrained regions that have not previously been described. The extra characterisation our work provides allows investigation of these key regions for drug target potential, and points towards determinants of packaging compatibility between segments.

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection.

Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d results in accumulation of phage-encoded mRNAs in the phage nucleus, reduces phage protein production, and compromises virion assembly. Taken together, our data show that the conserved ChmC protein plays crucial roles in the viral life cycle, potentially by facilitating phage mRNA translocation through the nuclear shell to promote protein production and virion development.

The structure and physical properties of a packaged bacteriophage particle.

A string of nucleotides confined within a protein capsid contains all the instructions necessary to make a functional virus particle, a virion. Although the structure of the protein capsid is known for many virus species1,2, the three-dimensional organization of viral genomes has mostly eluded experimental probes3,4. Here we report all-atom structural models of an HK97 virion5, including its entire 39,732 base pair genome, obtained through multiresolution simulations. Mimicking the action of a packaging motor6, the genome was gradually loaded into the capsid. The structure of the packaged capsid was then refined through simulations of increasing resolution, which produced a 26 million atom model of the complete virion, including water and ions confined within the capsid. DNA packaging occurs through a loop extrusion mechanism7 that produces globally different configurations of the packaged genome and gives each viral particle individual traits. Multiple microsecond-long all-atom simulations characterized the effect of the packaged genome on capsid structure, internal pressure, electrostatics and diffusion of water, ions and DNA, and revealed the structural imprints of the capsid onto the genome. Our approach can be generalized to obtain complete all-atom structural models of other virus species, thereby potentially revealing new drug targets at the genome-capsid interface.

Bioinformatic analysis of defective viral genomes in SARS-CoV-2 and its impact on population infection characteristics.

DVGs (Defective Viral Genomes) are prevalent in RNA virus infections. In this investigation, we conducted an analysis of high-throughput sequencing data and observed widespread presence of DVGs in SARS-CoV-2. Comparative analysis between SARS-CoV-2 and diverse DNA viruses revealed heightened susceptibility to damage and increased sequencing sample heterogeneity within the SARS-CoV-2 genome. Whole-genome sequencing depth variability analysis exhibited a higher coefficient of variation for SARS-CoV-2, while DVG analysis indicated a significant proportion of recombination sites, signifying notable genome heterogeneity and suggesting that a large proportion of assembled virus particles contain incomplete RNA sequences. Moreover, our investigation explored the sequencing depth and DVG content differences among various strains. Our findings revealed that as the virus evolves, there is a notable increase in the proportion of intact genomes within virus particles, as evidenced by third-generation sequencing data. Specifically, the proportion of intact genome in the Omicron strain surpassed that of the Delta and Alpha strains. This observation effectively elucidates the heightened infectiousness of the Omicron strain compared to the Delta and Alpha strains. We also postulate that this improvement in completeness stems from enhanced virus assembly capacity, as the Omicron strain can promptly facilitate the binding of RNA and capsid protein, thereby reducing the exposure time of vulnerable virus RNA in the host environment and significantly mitigating its degradation. Finally, employing mathematical modeling, we simulated the impact of DVG effects under varying environmental factors on infection characteristics and population evolution. Our findings provide an explanation for the close association between symptom severity and the extent of virus invasion, as well as the substantial disparity in population infection characteristics caused by the same strain under distinct environmental conditions. This study presents a novel approach for future virus research and vaccine development.

Identification of a critical role for ZIKV capsid α3 in virus assembly and its genetic interaction with M protein.

Flaviviruses such as Zika and dengue viruses are persistent health concerns in endemic regions worldwide. Efforts to combat the spread of flaviviruses have been challenging, as no antivirals or optimal vaccines are available. Prevention and treatment of flavivirus-induced diseases require a comprehensive understanding of their life cycle. However, several aspects of flavivirus biogenesis, including genome packaging and virion assembly, are not well characterized. In this study, we focused on flavivirus capsid protein (C) using Zika virus (ZIKV) as a model to investigate the role of the externally oriented α3 helix (C α3) without a known or predicted function. Alanine scanning mutagenesis of surface-exposed amino acids on C α3 revealed a critical CN67 residue essential for ZIKV virion production. The CN67A mutation did not affect dimerization or RNA binding of purified C protein in vitro. The virus assembly is severely affected in cells transfected with an infectious cDNA clone of ZIKV with CN67A mutation, resulting in a highly attenuated phenotype. We isolated a revertant virus with a partially restored phenotype by continuous passage of the CN67A mutant virus in Vero E6 cells. Sequence analysis of the revertant revealed a second site mutation in the viral membrane (M) protein MF37L, indicating a genetic interaction between the C and M proteins of ZIKV. Introducing the MF37L mutation on the mutant ZIKV CN67A generated a double-mutant virus phenotypically consistent with the isolated genetic revertant. Similar results were obtained with analogous mutations on C and M proteins of dengue virus, suggesting the critical nature of C α3 and possible C and M residues contributing to virus assembly in other Aedes-transmitted flaviviruses. This study provides the first experimental evidence of a genetic interaction between the C protein and the viral envelope protein M, providing a mechanistic understanding of the molecular interactions involved in the assembly and budding of Aedes-transmitted flaviviruses.

Are kuravirus capsid diameters quantized? The first all-atom genome tracing method for double-stranded DNA viruses.

The revolution in cryo-electron microscopy has resulted in unprecedented power to resolve large macromolecular complexes including viruses. Many methods exist to explain density corresponding to proteins and thus entire protein capsids have been solved at the all-atom level. However methods for nucleic acids lag behind, and no all-atom viral double-stranded DNA genomes have been published at all. We here present a method which exploits the spiral winding patterns of DNA in icosahedral capsids. The method quickly generates shells of DNA wound in user-specified, idealized spherical or cylindrical spirals. For transition regions, the method allows guided semiflexible fitting. For the kuravirus SU10, our method explains most of the density in a semiautomated fashion. The results suggest rules for DNA turns in the end caps under which two discrete parameters determine the capsid inner diameter. We suggest that other kuraviruses viruses may follow the same winding scheme, producing a discrete rather than continuous spectrum of capsid inner diameters. Our software may be used to explain the published density maps of other double-stranded DNA viruses and uncover their genome packaging principles.

Biophysical and structural characterization of a multifunctional viral genome packaging motor.

The large dsDNA viruses replicate their DNA as concatemers consisting of multiple covalently linked genomes. Genome packaging is catalyzed by a terminase enzyme that excises individual genomes from concatemers and packages them into preassembled procapsids. These disparate tasks are catalyzed by terminase alternating between two distinct states-a stable nuclease that excises individual genomes and a dynamic motor that translocates DNA into the procapsid. It was proposed that bacteriophage λ terminase assembles as an anti-parallel dimer-of-dimers nuclease complex at the packaging initiation site. In contrast, all characterized packaging motors are composed of five terminase subunits bound to the procapsid in a parallel orientation. Here, we describe biophysical and structural characterization of the λ holoenzyme complex assembled in solution. Analytical ultracentrifugation, small angle X-ray scattering, and native mass spectrometry indicate that 5 subunits assemble a cone-shaped terminase complex. Classification of cryoEM images reveals starfish-like rings with skewed pentameric symmetry and one special subunit. We propose a model wherein nuclease domains of two subunits alternate between a dimeric head-to-head arrangement for genome maturation and a fully parallel arrangement during genome packaging. Given that genome packaging is strongly conserved in both prokaryotic and eukaryotic viruses, the results have broad biological implications.

Molecular frustration: a hypothesis for regulation of viral infections.

The recent revolution in imaging techniques and results from RNA footprinting in situ reveal how the bacteriophage MS2 genome regulates both particle assembly and genome release. We have proposed a model in which multiple packaging signal (PS) RNA-coat protein (CP) contacts orchestrate different stages of a viral life cycle. Programmed formation and release of specific PS contacts with CP regulates viral particle assembly and genome uncoating during cell entry. We hypothesize that molecular frustration, a concept introduced to understand protein folding, can be used to better rationalize how PSs function in both particle assembly and genome release. More broadly this concept may explain the directionality of viral life cycles, for example, the roles of host cofactors in HIV infection. We propose that this is a universal principle in virology that explains mechanisms of host-virus interaction and suggests diverse therapeutic interventions.

Packing up the genome.

Nucleotide and force-dependent mechanisms control how the viral genome of lambda bacteriophage is inserted into capsids.

The KxGxYR and DxE motifs in the C-tail of the Middle East respiratory syndrome coronavirus membrane protein are crucial for infectious virus assembly.

The coronavirus' (CoV) membrane (M) protein is the driving force during assembly, but this process remains poorly characterized. Previously, we described two motifs in the C-tail of the Middle East respiratory syndrome CoV (MERS-CoV) M protein involved in its endoplasmic reticulum (ER) exit (211DxE213) and trans-Golgi network (TGN) retention (199KxGxYR204). Here, their function in virus assembly was investigated by two different virus-like particle (VLP) assays and by mutating both motifs in an infectious MERS-CoV cDNA clone. It was shown that the 199KxGxYR204 motif was essential for VLP and infectious virus assembly. Moreover, the mislocalization of the M protein induced by mutation of this motif prevented M-E interaction. Hampering the ER export of M by mutating its 211DxE213 motif still allowed the formation of nucleocapsid-empty VLPs, but prevented the formation of fully assembled VLPs and infectious particles. Taken together, these data show that the MERS-CoV assembly process highly depends on the correct intracellular trafficking of its M protein, and hence that not only specific protein-protein interacting motifs but also correct subcellular localization of the M protein in infected cells is essential for virus formation and should be taken into consideration when studying the assembly process.

Identification of a putative Gag binding site critical for feline immunodeficiency virus genomic RNA packaging.

The retroviral Gag precursor plays a central role in the selection and packaging of viral genomic RNA (gRNA) by binding to virus-specific packaging signal(s) (psi or ψ). Previously, we mapped the feline immunodeficiency virus (FIV) ψ to two discontinuous regions within the 5' end of the gRNA that assumes a higher order structure harboring several structural motifs. To better define the region and structural elements important for gRNA packaging, we methodically investigated these FIV ψ sequences using genetic, biochemical, and structure-function relationship approaches. Our mutational analysis revealed that the unpaired U85CUG88 stretch within FIV ψ is crucial for gRNA encapsidation into nascent virions. High-throughput selective 2' hydroxyl acylation analyzed by primer extension (hSHAPE) performed on wild type (WT) and mutant FIV ψ sequences, with substitutions in the U85CUG88 stretch, revealed that these mutations had limited structural impact and maintained nucleotides 80-92 unpaired, as in the WT structure. Since these mutations dramatically affected packaging, our data suggest that the single-stranded U85CUG88 sequence is important during FIV RNA packaging. Filter-binding assays performed using purified FIV Pr50Gag on WT and mutant U85CUG88 ψ RNAs led to reduced levels of Pr50Gag binding to mutant U85CUG88 ψ RNAs, indicating that the U85CUG88 stretch is crucial for ψ RNA-Pr50Gag interactions. Delineating sequences important for FIV gRNA encapsidation should enhance our understanding of both gRNA packaging and virion assembly, making them potential targets for novel retroviral therapeutic interventions, as well as the development of FIV-based vectors for human gene therapy.

Assembly of SARS-CoV-2 ribonucleosomes by truncated N∗ variant of the nucleocapsid protein.

The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) compacts the RNA genome into viral ribonucleoprotein (vRNP) complexes within virions. Assembly of vRNPs is inhibited by phosphorylation of the N protein serine/arginine (SR) region. Several SARS-CoV-2 variants of concern carry N protein mutations that reduce phosphorylation and enhance the efficiency of viral packaging. Variants of the dominant B.1.1 viral lineage also encode a truncated N protein, termed N∗ or Δ(1-209), that mediates genome packaging despite lacking the N-terminal RNA-binding domain and SR region. Here, we use mass photometry and negative stain electron microscopy to show that purified Δ(1-209) and viral RNA assemble into vRNPs that are remarkably similar in size and shape to those formed with full-length N protein. We show that assembly of Δ(1-209) vRNPs requires the leucine-rich helix of the central disordered region and that this helix promotes N protein oligomerization. We also find that fusion of a phosphomimetic SR region to Δ(1-209) inhibits RNA binding and vRNP assembly. Our results provide new insights into the mechanisms by which RNA binding promotes N protein self-association and vRNP assembly, and how this process is modulated by phosphorylation.

Giant variations in giant virus genome packaging.

Giant viruses (Nucleocytoviricota) have a largely conserved lifecycle, yet how they cram their large genomes into viral capsids is mostly unknown. The major capsid protein and the packaging ATPase (pATPase) comprise a highly conserved morphogenesis module in giant viruses, yet some giant viruses dispense with an icosahedral capsid, and others encode multiple versions of pATPases, including conjoined ATPase doublets, or encode none. Some giant viruses have acquired DNA-condensing proteins to compact their genomes, including sheath-like structures encasing folded DNA or densely packed viral nucleosomes that show a resemblance to eukaryotic nucleosomes at the telomeres. Here, we review what is known and unknown about these ATPases and condensing proteins, and place these variations in the context of viral lifecycles.

Cryo-electron microscopy structures of capsids and in situ portals of DNA-devoid capsids of human cytomegalovirus.

The portal-scaffold complex is believed to nucleate the assembly of herpesvirus procapsids. During capsid maturation, two events occur: scaffold expulsion and DNA incorporation. The portal-scaffold interaction and the conformational changes that occur to the portal during the different stages of capsid formation have yet to be elucidated structurally. Here we present high-resolution structures of the A- and B-capsids and in-situ portals of human cytomegalovirus. We show that scaffolds bind to the hydrophobic cavities formed by the dimerization and Johnson-fold domains of the major capsid proteins. We further show that 12 loop-helix-loop fragments-presumably from the scaffold domain-insert into the hydrophobic pocket of the portal crown domain. The portal also undergoes significant changes both positionally and conformationally as it accompanies DNA packaging. These findings unravel the mechanism by which the portal interacts with the scaffold to nucleate capsid assembly and further our understanding of scaffold expulsion and DNA incorporation.

Initiation of HIV-1 Gag lattice assembly is required for recognition of the viral genome packaging signal.

The encapsidation of HIV-1 gRNA into virions is enabled by the binding of the nucleocapsid (NC) domain of the HIV-1 Gag polyprotein to the structured viral RNA packaging signal (Ψ) at the 5' end of the viral genome. However, the subcellular location and oligomeric status of Gag during the initial Gag-Ψ encounter remain uncertain. Domains other than NC, such as capsid (CA), may therefore indirectly affect RNA recognition. To investigate the contribution of Gag domains to Ψ recognition in a cellular environment, we performed protein-protein crosslinking and protein-RNA crosslinking immunoprecipitation coupled with sequencing (CLIP-seq) experiments. We demonstrate that NC alone does not bind specifically to Ψ in living cells, whereas full-length Gag and a CANC subdomain bind to Ψ with high specificity. Perturbation of the Ψ RNA structure or NC zinc fingers affected CANC:Ψ binding specificity. Notably, CANC variants with substitutions that disrupt CA:CA dimer, trimer, or hexamer interfaces in the immature Gag lattice also affected RNA binding, and mutants that were unable to assemble a nascent Gag lattice were unable to specifically bind to Ψ. Artificially multimerized NC domains did not specifically bind Ψ. CA variants with substitutions in inositol phosphate coordinating residues that prevent CA hexamerization were also deficient in Ψ binding and second-site revertant mutants that restored CA assembly also restored specific binding to Ψ. Overall, these data indicate that the correct assembly of a nascent immature CA lattice is required for the specific interaction between Gag and Ψ in cells.

Understanding Retroviral Life Cycle and its Genomic RNA Packaging.

Members of the family Retroviridae are important animal and human pathogens. Being obligate parasites, their replication involves a series of steps during which the virus hijacks the cellular machinery. Additionally, many of the steps of retrovirus replication are unique among viruses, including reverse transcription, integration, and specific packaging of their genomic RNA (gRNA) as a dimer. Progress in retrovirology has helped identify several molecular mechanisms involved in each of these steps, but many are still unknown or remain controversial. This review summarizes our present understanding of the molecular mechanisms involved in various stages of retrovirus replication. Furthermore, it provides a comprehensive analysis of our current understanding of how different retroviruses package their gRNA into the assembling virions. RNA packaging in retroviruses holds a special interest because of the uniqueness of packaging a dimeric genome. Dimerization and packaging are highly regulated and interlinked events, critical for the virus to decide whether its unspliced RNA will be packaged as a "genome" or translated into proteins. Finally, some of the outstanding areas of exploration in the field of RNA packaging are highlighted, such as the role of epitranscriptomics, heterogeneity of transcript start sites, and the necessity of functional polyA sequences. An in-depth knowledge of mechanisms that interplay between viral and cellular factors during virus replication is critical in understanding not only the virus life cycle, but also its pathogenesis, and development of new antiretroviral compounds, vaccines, as well as retroviral-based vectors for human gene therapy.

The impact of mutations affecting highly conserved amino acids in the simian immunodeficiency virus nucleocapsid protein on virion assembly, genomic RNA packaging and viral infectivity.

The nucleocapsid (NC) domain of the retroviral Gag polyproteins mediates the incorporation of the viral genomic RNA into virions. Although SIV is widely used as a model for human immunodeficiency virus type 1 (HIV-1) infections, the SIV NC has been the subject of few studies which have provided discrepant data on the relative contribution of the two NC zinc finger motifs to genomic RNA encapsidation. Here, we demonstrate that mutations affecting the first cysteine in the distal zinc finger motif (C33S) or the N-terminal NC basic domain (R7A/K8A) drastically impair virion assembly and viral RNA binding. By contrast, amino acid substitutions targeting the first cysteine of the proximal zinc finger (C12S) or the basic region connecting both zinc fingers (R29A/R30A) allow substantial particle production and genomic RNA encapsidation. Our results help define the relative contribution of the SIV NC zinc finger motifs and basic regions to the NC biological properties.

Distinct Gag interaction properties of HIV-1 RNA 5' leader conformers reveal a mechanism for dimeric genome selection.

During HIV-1 assembly, two copies of viral genomic RNAs (gRNAs) are selectively packaged into new viral particles. This process is mediated by specific interactions between HIV-1 Gag and the packaging signals at the 5' leader (5'L) of viral gRNA. 5'L is able to adopt different conformations, which promotes either gRNA dimerization and packaging or Gag translation. Dimerization and packaging are coupled. Whether the selective packaging of the gRNA dimer is due to favorable interactions between Gag and 5'L in the packaging conformation is not known. Here, using RNAs mimicking the two 5'L conformers, we show that the 5'L conformation dramatically affects Gag-RNA interactions. Compared to the RNA in the translation conformation (5'LT), the RNA in the packaging conformation (5'LP) can bind more Gag molecules. Gag associates with 5'LP faster than it binds to 5'LT, whereas Gag dissociates from 5'LP more slowly. The Gag-5'LP complex is more stable at high salt concentrations. The NC-SP2-p6 region of Gag likely accounts for the faster association and slower dissociation kinetics for the Gag-5'LP interaction and for the higher stability. In summary, our data suggest that conformational changes play an important role in the selection of dimeric genomes, probably by affecting the binding kinetics and stability of the Gag-5'L complex.

CryoEM structure and assembly mechanism of a bacterial virus genome gatekeeper.

Numerous viruses package their dsDNA genome into preformed capsids through a portal gatekeeper that is subsequently closed. We report the structure of the DNA gatekeeper complex of bacteriophage SPP1 (gp612gp1512gp166) in the post-DNA packaging state at 2.7 Å resolution obtained by single particle cryo-electron microscopy. Comparison of the native SPP1 complex with assembly-naïve structures of individual components uncovered the complex program of conformational changes leading to its assembly. After DNA packaging, gp15 binds via its C-terminus to the gp6 oligomer positioning gp15 subunits for oligomerization. Gp15 refolds its inner loops creating an intersubunit ß-barrel that establishes different types of contacts with six gp16 subunits. Gp16 binding and oligomerization is accompanied by folding of helices that close the portal channel to keep the viral genome inside the capsid. This mechanism of assembly has broad functional and evolutionary implications for viruses of the prokaryotic tailed viruses-herpesviruses lineage.