Biophysics-Guided Lead Discovery of HBV Capsid Assembly Modifiers.

Hepatitis B virus (HBV) is the leading cause of chronic liver pathologies worldwide. HBV nucleocapsid, a key structural component, is formed through the self-assembly of the capsid protein units. Therefore, interfering with the self-assembly process is a promising approach for the development of novel antiviral agents. Applied to HBV, this approach has led to several classes of capsid assembly modulators (CAMs). Here, we report structurally novel CAMs with moderate activity and low toxicity, discovered through a biophysics-guided approach combining docking, molecular dynamics simulations, and a series of assays with a particular emphasis on biophysical experiments. Several of the identified compounds induce the formation of aberrant capsids and inhibit HBV DNA replication in vitro, suggesting that they possess modest capsid assembly modulation effects. The synergistic computational and experimental approaches provided key insights that facilitated the identification of compounds with promising activities. The discovery of preclinical CAMs presents opportunities for subsequent optimization efforts, thereby opening new avenues for HBV inhibition.

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.

Functionality of IAV packaging signals depends on site-specific charges within the viral nucleoprotein.

The coordinated packaging of the segmented genome of the influenza A virus (IAV) into virions is an essential step of the viral life cycle. This process is controlled by the interaction of packaging signals present in all eight viral RNA (vRNA) segments and the viral nucleoprotein (NP), which binds vRNA via a positively charged binding groove. However, mechanistic models of how the packaging signals and NP work together to coordinate genome packaging are missing. Here, we studied genome packaging in influenza A/SC35M virus mutants that carry mutated packaging signals as well as specific amino acid substitutions at the highly conserved lysine (K) residues 184 and 229 in the RNA-binding groove of NP. Because these lysines are acetylated and thus neutrally charged in infected host cells, we replaced them with glutamine to mimic the acetylated, neutrally charged state or arginine to mimic the non-acetylated, positively charged state. Our analysis shows that the coordinated packaging of eight vRNAs is influenced by (i) the charge state of the replacing amino acid and (ii) its location within the RNA-binding groove. Accordingly, we propose that lysine acetylation induces different charge states within the RNA-binding groove of NP, thereby supporting the activity of specific packaging signals during coordinated genome packaging. IMPORTANCE: Influenza A viruses (IAVs) have a segmented viral RNA (vRNA) genome encapsidated by multiple copies of the viral nucleoprotein (NP) and organized into eight distinct viral ribonucleoprotein complexes. Although genome segmentation contributes significantly to viral evolution and adaptation, it requires a highly sophisticated genome-packaging mechanism. How eight distinct genome complexes are incorporated into the virion is poorly understood, but previous research suggests an essential role for both vRNA packaging signals and highly conserved NP amino acids. By demonstrating that the packaging process is controlled by charge-dependent interactions of highly conserved lysine residues in NP and vRNA packaging signals, our study provides new insights into the sophisticated packaging mechanism of IAVs.

Identification of clickable HIV-1 capsid-targeting probes for viral replication inhibition.

Of the targets for HIV-1 therapeutics, the capsid core is a relatively unexploited but alluring drug target due to its indispensable roles throughout virus replication. Because of this, we aimed to identify "clickable" covalent modifiers of the HIV-1 capsid protein (CA) for future functionalization. We screened a library of fluorosulfate compounds that can undergo sulfur(VI) fluoride exchange (SuFEx) reactions, and five compounds were identified as hits. These molecules were further characterized for antiviral effects. Several compounds impacted in vitro capsid assembly. One compound, BBS-103, covalently bound CA via a SuFEx reaction to Tyr145 and had antiviral activity in cell-based assays by perturbing virus production, but not uncoating. The covalent binding of compounds that target the HIV-1 capsid could aid in the future design of antiretroviral drugs or chemical probes that will help study aspects of HIV-1 replication.

A myristoyl switch at the plasma membrane triggers cleavage and oligomerization of Mason-Pfizer monkey virus matrix protein.

For most retroviruses, including HIV, association with the plasma membrane (PM) promotes the assembly of immature particles, which occurs simultaneously with budding and maturation. In these viruses, maturation is initiated by oligomerization of polyprotein precursors. In contrast, several retroviruses, such as Mason-Pfizer monkey virus (M-PMV), assemble in the cytoplasm into immature particles that are transported across the PM. Therefore, protease activation and specific cleavage must not occur until the pre-assembled particle interacts with the PM. This interaction is triggered by a bipartite signal consisting of a cluster of basic residues in the matrix (MA) domain of Gag polyprotein and a myristoyl moiety N-terminally attached to MA. Here, we provide evidence that myristoyl exposure from the MA core and its insertion into the PM occurs in M-PMV. By a combination of experimental methods, we show that this results in a structural change at the C-terminus of MA allowing efficient cleavage of MA from the downstream region of Gag. This suggests that, in addition to the known effect of the myristoyl switch of HIV-1 MA on the multimerization state of Gag and particle assembly, the myristoyl switch may have a regulatory role in initiating sequential cleavage of M-PMV Gag in immature particles.

Human cytomegalovirus degrades DMXL1 to inhibit autophagy, lysosomal acidification, and viral assembly.

Human cytomegalovirus (HCMV) is an important human pathogen that regulates host immunity and hijacks host compartments, including lysosomes, to assemble virions. We combined a quantitative proteomic analysis of HCMV infection with a database of proteins involved in vacuolar acidification, revealing Dmx-like protein-1 (DMXL1) as the only protein that acidifies vacuoles yet is degraded by HCMV. Systematic comparison of viral deletion mutants reveals the uncharacterized 7 kDa US33A protein as necessary and sufficient for DMXL1 degradation, which occurs via recruitment of the E3 ubiquitin ligase Kip1 ubiquitination-promoting complex (KPC). US33A-mediated DMXL1 degradation inhibits lysosome acidification and autophagic cargo degradation. Formation of the virion assembly compartment, which requires lysosomes, occurs significantly later with US33A-expressing virus infection, with reduced viral replication. These data thus identify a viral strategy for cellular remodeling, with the potential to employ US33A in therapies for viral infection or rheumatic conditions, in which inhibition of lysosome acidification can attenuate disease.

Cooperative Membrane Binding of HIV-1 Matrix Proteins.

The HIV-1 assembly process begins with a newly synthesized Gag polyprotein being targeted to the inner leaflet of the plasma membrane of the infected cells to form immature viral particles. Gag-membrane interactions are mediated through the myristoylated (Myr) N-terminal matrix (MA) domain of Gag, which eventually multimerize on the membrane to form trimers and higher order oligomers. The study of the structure and dynamics of peripheral membrane proteins like MA has been challenging for both experimental and computational studies due to the complex transient dynamics of protein-membrane interactions. Although the roles of anionic phospholipids (PIP2, PS) and the Myr group in the membrane targeting and stable membrane binding of MA are now well-established, the cooperative interactions between the MA monomers and MA-membrane remain elusive in the context of viral assembly and release. Our present study focuses on the membrane binding dynamics of a higher order oligomeric structure of MA protein (a dimer of trimers), which has not been explored before. Employing time-lagged independent component analysis (tICA) to our microsecond-long trajectories, we investigate conformational changes of the matrix protein induced by membrane binding. Interestingly, the Myr switch of an MA monomer correlates with the conformational switch of adjacent monomers in the same trimer. Together, our findings suggest complex protein dynamics during the formation of the immature HIV-1 lattice; while MA trimerization facilitates Myr insertion, MA trimer-trimer interactions in the immature lattice can hinder the same.

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.

Mechanism of Viral DNA Packaging in Phage T4 Using Single-Molecule Fluorescence Approaches.

In all tailed phages, the packaging of the double-stranded genome into the head by a terminase motor complex is an essential step in virion formation. Despite extensive research, there are still major gaps in the understanding of this highly dynamic process and the mechanisms responsible for DNA translocation. Over the last fifteen years, single-molecule fluorescence technologies have been applied to study viral nucleic acid packaging using the robust and flexible T4 in vitro packaging system in conjunction with genetic, biochemical, and structural analyses. In this review, we discuss the novel findings from these studies, including that the T4 genome was determined to be packaged as an elongated loop via the colocalization of dye-labeled DNA termini above the portal structure. Packaging efficiency of the TerL motor was shown to be inherently linked to substrate structure, with packaging stalling at DNA branches. The latter led to the design of multiple experiments whose results all support a proposed torsional compression translocation model to explain substrate packaging. Evidence of substrate compression was derived from FRET and/or smFRET measurements of stalled versus resolvase released dye-labeled Y-DNAs and other dye-labeled substrates relative to motor components. Additionally, active in vivo T4 TerS fluorescent fusion proteins facilitated the application of advanced super-resolution optical microscopy toward the visualization of the initiation of packaging. The formation of twin TerS ring complexes, each expected to be ~15 nm in diameter, supports a double protein ring-DNA synapsis model for the control of packaging initiation, a model that may help explain the variety of ring structures reported among pac site phages. The examination of the dynamics of the T4 packaging motor at the single-molecule level in these studies demonstrates the value of state-of-the-art fluorescent tools for future studies of complex viral replication mechanisms.

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.

HIV-1 RNA genome packaging: it's G-rated.

A member of the Retroviridae, human immunodeficiency virus type 1 (HIV-1), uses the RNA genome packaged into nascent virions to transfer genetic information to its progeny. The genome packaging step is a highly regulated and extremely efficient process as a vast majority of virus particles contain two copies of full-length unspliced HIV-1 RNA that form a dimer. Thus, during virus assembly HIV-1 can identify and selectively encapsidate HIV-1 unspliced RNA from an abundant pool of cellular RNAs and various spliced HIV-1 RNAs. Several "G" features facilitate the packaging of a dimeric RNA genome. The viral polyprotein Gag orchestrates virus assembly and mediates RNA genome packaging. During this process, Gag preferentially binds unpaired guanosines within the highly structured 5' untranslated region (UTR) of HIV-1 RNA. In addition, the HIV-1 unspliced RNA provides a scaffold that promotes Gag:Gag interactions and virus assembly, thereby ensuring its packaging. Intriguingly, recent studies have shown that the use of different guanosines at the junction of U3 and R as transcription start sites results in HIV-1 unspliced RNA species with 99.9% identical sequences but dramatically distinct 5' UTR conformations. Consequently, one species of unspliced RNA is preferentially packaged over other nearly identical RNAs. These studies reveal how conformations affect the functions of HIV-1 RNA elements and the complex regulation of HIV-1 replication. In this review, we summarize cis- and trans-acting elements critical for HIV-1 RNA packaging, locations of Gag:RNA interactions that mediate genome encapsidation, and the effects of transcription start sites on the structure and packaging of HIV-1 RNA.

SRPK2 Mediates HBV Core Protein Phosphorylation and Capsid Assembly via Docking Interaction.

Members of the serine-arginine protein kinase (SRPK) family, SRPK1 and SRPK2, phosphorylate the hepatitis B core protein (Cp) and are crucial for pregenomic RNA encapsidation during viral nucleocapsid assembly. Among them, SRPK2 exhibits higher kinase activity toward Cp. In this study, we identified Cp sites that are phosphorylated by SRPK2 and demonstrated that the kinase utilizes an SRPK-specific docking groove to interact with and regulate the phosphorylation of the C-terminal arginine rich domain of Cp. We determined that direct interaction between the docking groove of SRPK2 and unphosphorylated Cp inhibited premature viral capsid assembly in vitro, whereas the phosphorylation of the viral protein reactivated the process. Pull-down assays together with the new cryo-electron microscopy structure of the HBV capsid in complex with SRPK2 revealed that the kinases decorate the surface of the viral capsid by interacting with the C-terminal domain of Cp, underscoring the importance of the docking interaction in regulating capsid assembly and pregenome packaging. Moreover, SRPK2-knockout in HepG2 cells suppressed Cp phosphorylation, indicating that SRPK2 is an important cellular kinase for HBV life cycle.

Hierarchical assembly is more robust than egalitarian assembly in synthetic capsids.

Self-assembly of complex and functional materials remains a grand challenge in soft material science. Efficient assembly depends on a delicate balance between thermodynamic and kinetic effects, requiring fine-tuning affinities and concentrations of subunits. By contrast, we introduce an assembly paradigm that allows large error-tolerance in the subunit affinity and helps avoid kinetic traps. Our combined experimental and computational approach uses a model system of triangular subunits programmed to assemble into T = 3 icosahedral capsids comprising 60 units. The experimental platform uses DNA origami to create monodisperse colloids whose three-dimensional geometry is controlled to nanometer precision, with two distinct bonds whose affinities are controlled to kBT precision, quantified in situ by static light scattering. The computational model uses a coarse-grained representation of subunits, short-ranged potentials, and Langevin dynamics. Experimental observations and modeling reveal that when the bond affinities are unequal, two distinct hierarchical assembly pathways occur, in which the subunits first form dimers in one case and pentamers in another. These hierarchical pathways produce complete capsids faster and are more robust against affinity variation than egalitarian pathways, in which all binding sites have equal strengths. This finding suggests that hierarchical assembly may be a general engineering principle for optimizing self-assembly of complex target structures.

Development of Small-Molecule Anti-HIV-1 Agents Targeting HIV-1 Capsid Proteins.

The capsid of human immunodeficiency virus type 1 (HIV-1) forms a conical structure by assembling oligomers of capsid (CA) proteins and is a virion shell that encapsulates viral RNA. The inhibition of the CA function could be an appropriate target for suppression of HIV-1 replication because the CA proteins are highly conserved among many strains of HIV-1, and the drug targeting CA, lenacapavir, has been clinically developed by Gilead Sciences, Inc. Interface hydrophobic interactions between two CA molecules via the Trp184 and Met185 residues in the CA sequence are indispensable for conformational stabilization of the CA multimer. Our continuous studies found two types of small molecules with different scaffolds, MKN-1 and MKN-3, designed by in silico screening as a dipeptide mimic of Trp184 and Met185 have significant anti-HIV-1 activity. In the present study, MKN-1 derivatives have been designed and synthesized. Their structure-activity relationship studies found some compounds having potent anti-HIV activity. The present results should be useful in the design of novel CA-targeting molecules with anti-HIV activity.

Proteomic analysis of SARS-CoV-2 particles unveils a key role of G3BP proteins in viral assembly.

Considerable progress has been made in understanding the molecular host-virus battlefield during SARS-CoV-2 infection. Nevertheless, the assembly and egress of newly formed virions are less understood. To identify host proteins involved in viral morphogenesis, we characterize the proteome of SARS-CoV-2 virions produced from A549-ACE2 and Calu-3 cells, isolated via ultracentrifugation on sucrose cushion or by ACE-2 affinity capture. Bioinformatic analysis unveils 92 SARS-CoV-2 virion-associated host factors, providing a valuable resource to better understand the molecular environment of virion production. We reveal that G3BP1 and G3BP2 (G3BP1/2), two major stress granule nucleators, are embedded within virions and unexpectedly favor virion production. Furthermore, we show that G3BP1/2 participate in the formation of cytoplasmic membrane vesicles, that are likely virion assembly sites, consistent with a proviral role of G3BP1/2 in SARS-CoV-2 dissemination. Altogether, these findings provide new insights into host factors required for SARS-CoV-2 assembly with potential implications for future therapeutic targeting.

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.

BGLF4 kinase regulates the formation of the EBV cytoplasmic assembly compartment and the recruitment of cellular IQGAP1 for virion release.

After Epstein-Barr virus (EBV) genome replication and encapsidation in the nucleus, nucleocapsids are translocated into the cytoplasm for subsequent tegumentation and maturation. The EBV BGLF4 kinase, which induces partial disassembly of the nuclear lamina, and the nuclear egress complex BFRF1/BFLF2 coordinately facilitate the nuclear egress of nucleocapsids. Here, we demonstrate that within EBV reactivated epithelial cells, viral capsids, tegument proteins, and glycoproteins are clustered in the juxtanuclear concave region, accompanied by redistributed cytoplasmic organelles and the cytoskeleton regulator IQ-domain GTPase-activation protein 1 (IQGAP1), close to the microtubule-organizing center (MTOC). The assembly compartment (AC) structure was diminished in BGLF4-knockdown TW01-EBV cells and BGLF4-knockout bacmid-carrying TW01 cells, suggesting that the formation of AC structure is BGLF4-dependent. Notably, glycoprotein gp350/220 was observed by confocal imaging to be distributed in the perinuclear concave region and surrounded by the endoplasmic reticulum (ER) membrane marker calnexin, indicating that the AC may be located within a globular structure derived from ER membranes, adjacent to the outer nuclear membrane. Moreover, the viral capsid protein BcLF1 and tegument protein BBLF1 were co-localized with IQGAP1 near the cytoplasmic membrane in the late stage of replication. Knockdown of IQGAP1 did not affect the AC formation but decreased virion release from both TW01-EBV and Akata+ cells, suggesting IQGAP1-mediated trafficking regulates EBV virion release. The data presented here show that BGLF4 is required for cytoskeletal rearrangement, coordination with the redistribution of cytoplasmic organelles and IQGAP1 for virus maturation, and subsequent IQGAP1-dependent virion release.IMPORTANCEEBV genome is replicated and encapsidated in the nucleus, and the resultant nucleocapsids are translocated to the cytoplasm for subsequent virion maturation. We show that a cytoplasmic AC, containing viral proteins, markers of the endoplasmic reticulum, Golgi, and endosomes, is formed in the juxtanuclear region of epithelial and B cells during EBV reactivation. The viral BGLF4 kinase contributes to the formation of the AC. The cellular protein IQGAP1 is also recruited to the AC and partially co-localizes with the virus capsid protein BcLF1 and tegument protein BBLF1 in EBV-reactivated cells, dependent on the BGLF4-induced cytoskeletal rearrangement. In addition, virion release was attenuated in IQGAP1-knockdown epithelial and B cells after reactivation, suggesting that IQGAP1-mediated trafficking may regulate the efficiency of virus maturation and release.

Antibody inhibition of influenza A virus assembly and release.

Antibodies are frontline defenders against influenza virus infection, providing protection through multiple complementary mechanisms. Although a subset of monoclonal antibodies (mAbs) has been shown to restrict replication at the level of virus assembly and release, it remains unclear how potent and pervasive this mechanism of protection is, due in part to the challenge of separating this effect from other aspects of antibody function. To address this question, we developed imaging-based assays to determine how effectively a broad range of mAbs against the IAV surface proteins can specifically restrict viral egress. We find that classically neutralizing antibodies against hemagglutinin are broadly multifunctional, inhibiting virus assembly and release at concentrations 1-20-fold higher than the concentrations at which they inhibit viral entry. These antibodies are also capable of altering the morphological features of shed virions, reducing the proportion of filamentous particles. We find that antibodies against neuraminidase and M2 also restrict viral egress and that inhibition by anti-neuraminidase mAbs is only partly attributable to a loss in enzymatic activity. In all cases, antigen crosslinking-either on the surface of the infected cell, between the viral and cell membrane, or both-plays a critical role in inhibition, and we are able to distinguish between these modes experimentally and through a structure-based computational model. Together, these results provide a framework for dissecting antibody multifunctionality that could help guide the development of improved therapeutic antibodies or vaccines and that can be extended to other viral families and antibody isotypes.IMPORTANCEAntibodies against influenza A virus provide multifaceted protection against infection. Although sensitive and quantitative assays are widely used to measure inhibition of viral attachment and entry, the ability of diverse antibodies to inhibit viral egress is less clear. We address this challenge by developing an imaging-based approach to measure antibody inhibition of virus release across a panel of monoclonal antibodies targeting the influenza A virus surface proteins. Using this approach, we find that inhibition of viral egress is common and can have similar potency to the ability of an antibody to inhibit viral entry. Insights into this understudied aspect of antibody function may help guide the development of improved countermeasures.

Interconnection of cellular autophagy and endosomal vesicle trafficking and its role in hepatitis B virus replication and release.

Hepatitis B virus (HBV) produces and releases various particle types, including complete virions, subviral particles with envelope proteins, and naked capsids. Recent studies demonstrate that HBV exploits distinct intracellular membrane trafficking pathways, including the endosomal vesicle trafficking and autophagy pathway, to assemble and release viral and subviral particles. Herein, we summarize the findings about the distinct roles of autophagy and endosomal membrane trafficking and the interaction of both pathways in HBV replication, assembly, and release.