Search

HIV-1 Vpr promotes efficient spread of HIV-1 from macrophages to T cells by transcriptionally downmodulating restriction factors that target HIV-1 Envelope protein (Env). Here we find that Vpr induces broad transcriptomic changes by targeting PU.1, a transcription factor necessary for expression of host innate immune response genes, including those that target Env. Consistent with this, we find silencing PU.1 in infected macrophages lacking Vpr rescues Env. Vpr downmodulates PU.1 through a proteasomal degradation pathway that depends on physical interactions with PU.1 and DCAF1, a component of the Cul4A E3 ubiquitin ligase. The capacity for Vpr to target PU.1 is highly conserved across primate lentiviruses. In addition to impacting infected cells, we find that Vpr suppresses expression of innate immune response genes in uninfected bystander cells, and that virion-associated Vpr can degrade PU.1. Together, we demonstrate Vpr counteracts PU.1 in macrophages to blunt antiviral immune responses and promote viral spread.

The compartmentalization of eukaryotic cells presents considerable challenges to the herpesvirus life cycle. The herpesvirus tegument, a bulky proteinaceous aggregate sandwiched between herpesviruses' capsid and envelope, is uniquely evolved to address these challenges, yet tegument structure and organization remain poorly characterized. We use deep-learning-enhanced cryogenic electron microscopy to investigate the tegument of human cytomegalovirus virions and noninfectious enveloped particles (NIEPs; a genome packaging-aborted state), revealing a portal-biased tegumentation scheme. We resolve atomic structures of portal vertex-associated tegument (PVAT) and identify multiple configurations of PVAT arising from layered reorganization of pUL77, pUL48 (large tegument protein), and pUL47 (inner tegument protein) assemblies. Analyses show that pUL77 seals the last-packaged viral genome end through electrostatic interactions, pUL77 and pUL48 harbor a head-linker-capsid-binding motif conducive to PVAT reconfiguration, and pUL47/48 dimers form 45-nm-long filaments extending from the portal vertex. These results provide a structural framework for understanding how herpesvirus tegument facilitates and evolves during processes spanning viral genome packaging to delivery.

Chemical pesticide delivery is a fundamental aspect of agriculture. However, the extensive use of pesticides severely endangers the ecosystem because they accumulate on crops, in soil, as well as in drinking and groundwater. New frontiers in nano-engineering have opened the door for precision agriculture. We introduced Tobacco mild green mosaic virus (TMGMV) as a viable delivery platform with a high aspect ratio and favorable soil mobility. In this work, we assess the use of TMGMV as a chemical nanocarrier for agriculturally relevant cargo. While plant viruses are usually portrayed as rigid/solid structures, these are "dynamic materials," and they "breathe" in solution in response to careful adjustment of pH or bathing media [e.g., addition of solvent such as dimethyl sulfoxide (DMSO)]. Through this process, coat proteins (CPs) partially dissociate leading to swelling of the nucleoprotein complexes-allowing for the infusion of active ingredients (AI), such as pesticides [e.g., fluopyram (FLP), clothianidin (CTD), rifampicin (RIF), and ivermectin (IVM)] into the macromolecular structure. We developed a "breathing" method that facilitates inter-coat protein cargo loading, resulting in up to  ~ 1000 AIs per virion. This is of significance since in the agricultural setting, there is a need to develop nanoparticle delivery strategies where the AI is not chemically altered, consequently avoiding the need for regulatory and registration processes of new compounds. This work highlights the potential of TMGMV as a pesticide nanocarrier in precision farming applications; the developed methods likely would be applicable to other protein-based nanoparticle systems.

Bovine adenovirus (BAdV)-3 genome encodes a 26 kDa core protein designated as protein VII, which localizes to the nucleus/nucleolus. The requirement of a protein VII-complementing cell line for the replication of VII-deleted BAdV-3 suggests that protein VII is required for the production of infectious progeny virions. An analysis of the BAV.VIId+ virus (only phenotypically positive for protein VII) detected no noticeable differences in the expression and incorporation of viral proteins in the virions. Moreover, protein VII does not appear to be essential for the formation of mature BAV.VIId+. However, protein VII appeared to be required for the efficient assembly of mature BAV.VIId- virions. An analysis of the BAV.VIId- virus (genotypically and phenotypically negative for protein VII) in non-complementing cells detected the inefficient release of virions from endosomes, which affected the expression of viral proteins or DNA replication. Moreover, the absence of protein VII altered the proteolytic cleavage of protein VI of BAV.VIId-. Our results suggest that BAdV-3 protein VII appears to be required for efficient production of mature virions. Moreover, the absence of protein VII produces non-infectious BAdV-3 by altering the release of BAdV-3 from endosomes/vesicles. [ABSTRACT FROM AUTHOR]

Endolysins produced by bacteriophages hydrolyze host cell wall peptidoglycan to release newly assembled virions. D29 mycobacteriophage specifically infects mycobacteria including the pathogenic Mycobacterium tuberculosis. D29 encodes LysA endolysin, which hydrolyzes mycobacterial cell wall peptidoglycan. We previously showed that LysA harbors two catalytic domains (N‐terminal domain [NTD] and lysozyme‐like domain [LD]) and a C‐terminal cell wall binding domain (CTD). While the importance of LD and CTD in mycobacteriophage biology has been examined in great detail, NTD has largely remained unexplored. Here, to address NTD's significance in D29 physiology, we generated NTD‐deficient D29 (D29∆NTD) by deleting the NTD‐coding region from D29 genome using CRISPY‐BRED. We show that D29∆NTD is viable, but has a longer latent period, and a remarkably reduced burst size and plaque size. A large number of phages were found to be trapped in the host during the D29∆NTD‐mediated cell lysis event. Such poor release of progeny phages during host cell lysis strongly suggests that NTD‐deficient LysA produced by D29∆NTD, despite having catalytically‐active LD, is unable to efficiently lyse host bacteria. We thus conclude that LysA NTD is essential for optimal release of progeny virions, thereby playing an extremely vital role in phage physiology and phage propagation in the environment. [ABSTRACT FROM AUTHOR]

Enteroviruses (EVs and RVs) are prevalent worldwide and cause various diseases in humans, of which the VP1-pocket is a target of antivirals, with a lipid molecule as a pocket factor to stabilize the virion. However, the characterization of the structure of the VP1-pocket in EVs is poor. Here, we compared the published capsid crystals of EVs and RVs and proposed a structural framework for the VP1-pocket: Frame 1–4, which is located at the CD loop, GH loop, and C-terminus, presenting with an outward opening appearance or not. The non-outward viral strains—CVB3, Echo 11, RV-A81, and RV-B70—are more thermally stable, with a breakpoint temperature (B.T.) of 51~62 °C for genome releasing, which is 4~10 °C higher than its outward temperature of 41~47 °C, and infectivity preservation when treated at 50 °C for 3 min. Its outward versus non-outward opening is correlated significantly with the B.T. for genome release (r = −0.90; p = 0.0004) and infectivity (r = −0.82, p = 0.0039). The energy of Frames 1, 2, and 4, including Van der Waals attractive and repulsive interactions and hydrogen bonds, showed significant correlations with the B.T. (r = −0.67, 0.75, and −0.8; p = 0.034, 0.013, and 0.006, respectively). These characters of the VP1-pocket could be predictors for virion thermostability and aid in the development of vaccines or antivirals. [ABSTRACT FROM AUTHOR]

Spiroplasma virus 4 (SpV4) is a bacteriophage of the Microviridae, which packages circular ssDNA within non-enveloped T = 1 icosahedral capsids. It infects spiroplasmas, which are known pathogens of honeybees. Here, the structure of the SpV4 virion is determined using cryo-electron microscopy to a resolution of 2.5 Å. A striking feature of the SpV4 capsid is the mushroom-like protrusions at the 3-fold axes, which is common among all members of the subfamily Gokushovirinae. While the function of the protrusion is currently unknown, this feature varies widely in this subfamily and is therefore possibly an adaptation for host recognition. Furthermore, on the interior of the SpV4 capsid, the location of DNA-binding protein VP8 was identified and shown to have low structural conservation to the capsids of other viruses in the family. The structural characterization of SpV4 will aid future studies analyzing the virus–host interaction, to understand disease mechanisms at a molecular level. Furthermore, the structural comparisons in this study, including a low-resolution structure of the chlamydia phage 2, provide an overview of the structural repertoire of the viruses in this family that infect various bacterial hosts, which in turn infect a wide range of animals and plants. [ABSTRACT FROM AUTHOR]

Plus, minus, and double-strand RNA viruses are all found in nature. We use computational models to study the relative success of these strategies. We consider translation, replication, and virion assembly inside one cell, and transmission of virions between cells. For viruses which do not incorporate a polymerase in the capsid, transmission of only plus strands is the default strategy because virions containing minus strands are not infectious. Packaging only plus strands has a significant advantage if the number of RNA strands produced per cell is larger than the number of capsids. In this case, by not packaging minus strands, the virus produces more plus-strand virions. Therefore, plus-strand viruses are selected at low multiplicity of infection. However, at high multiplicity of infection, it is preferable to package both strands because the additional minus virions produced are helpful when there are multiple infections per cell. The fact that plus-strand viruses are widespread while viruses that package both strands are not seen in nature suggests that RNA strands are indeed produced in excess over capsids, and that the multiplicity of infection is not sufficiently high to favor the production of both kinds of virions. For double-strand viruses, we show that it is advantageous to produce only plus strands from the double strand within the cell, as is observed in real viruses. The reason for the success of minus-strand viruses is more puzzling initially. For viruses that incorporate a polymerase in the virion, minus virions are infectious. However, this is not sufficient to explain the success of minus-strand viruses, because in this case, viruses that package both strands outcompete those that package only minus or only plus. Real minus-strand viruses make use of replicable strands that are coated by a nucleoprotein, and separate translatable plus strands that are uncoated. Here we show that when there are distinct replicable and translatable strands, minus-strand viruses are selected. [ABSTRACT FROM AUTHOR]

Viruses are obligate parasites that depend on the cellular machinery for their propagation. Several viruses also incorporate cellular proteins that facilitate viral spread. Defining these cellular proteins is critical to decipher viral life cycles and delineate novel therapeutic strategies. While numerous studies have explored the importance of host proteins in coronavirus spread, information about their presence in mature virions is limited. In this study, we developed a protocol to highly enrich mature HCoV-OC43 virions and characterize them by proteomics. Recognizing that cells release extracellular vesicles whose content is modulated by viruses, and given our ability to separate virions from these vesicles, we also analyzed their protein content in both uninfected and infected cells. We uncovered 69 unique cellular proteins associated with virions including 31 high-confidence hits. These proteins primarily regulate RNA metabolism, enzymatic activities, vesicular transport, cell adhesion, metabolite interconversion, and translation. We further discovered that the virus had a profound impact on exosome composition, incorporating 47 novel cellular proteins (11 high confidence) and excluding 92 others (61 high confidence) in virus-associated extracellular vesicles compared to uninfected cells. Moreover, a dsiRNA screen revealed that 11 of 18 select targets significantly impacted viral yields, including proteins found in virions or extracellular vesicles. Overall, this study provides new and important insights into the incorporation of numerous host proteins into HCoV-OC43 virions, their biological significance, and the ability of the virus to modulate extracellular vesicles. IMPORTANCE In recent years, coronaviruses have dominated global attention, making it crucial to develop methods to control them and prevent future pandemics. Besides viral proteins, host proteins play a significant role in viral propagation and offer potential therapeutic targets. Targeting host proteins is advantageous because they are less likely to mutate and develop resistance compared to viral proteins, a common issue with many antiviral treatments. In this study, we examined the protein content of the less virulent biosafety level 2 HCoV-OC43 virus as a stand-in for the more virulent SARS-CoV-2. Our findings reveal that several cellular proteins incorporated into the virion regulate viral spread. In addition, we report that the virus extensively modulates the content of extracellular vesicles, enhancing viral dissemination. This underscores the critical interplay between the virus, host proteins, and extracellular vesicles. [ABSTRACT FROM AUTHOR]

Bunyaviruses are enveloped negative or ambisense single-stranded RNA viruses with a genome divided into several segments. The canonical view depicts each viral particle packaging one copy of each genomic segment in one polarity named the viral strand. Several opposing observations revealed nonequal ratios of the segments, uneven number of segments per virion, and even packaging of viral complementary strands. Unfortunately, these observations result from studies often addressing other questions, on distinct viral species, and not using accurate quantitative methods. Hence, what RNA segments and strands are packaged as the genome of any bunyavirus remains largely ambiguous. We addressed this issue by first investigating the virion size distribution and RNA content in populations of the tomato spotted wilt virus (TSWV) using microscopy and tomography. These revealed heterogeneity in viral particle volume and amount of RNA content, with a surprising lack of correlation between the two. Then, the ratios of all genomic segments and strands were established using RNA sequencing and qRT-PCR. Within virions, both plus and minus strands (but no mRNA) are packaged for each of the three L, M, and S segments, in reproducible nonequimolar proportions determined by those in total cell extracts. These results show that virions differ in their genomic content but together build up a highly reproducible genetic composition of the viral population. This resembles the genome formula described for multipartite viruses, with which some species of the order Bunyavirales may share some aspects of the way of life, particularly emerging properties at a supravirion scale.

The majority of rod-shaped and some filamentous plant viruses encode a cysteine-rich protein (CRP) that functions in viral virulence; however, the roles of these CRPs in viral infection remain largely unknown. Here, we used barley stripe mosaic virus (BSMV) as a model to investigate the essential role of its CRP in virus morphogenesis. The CRP protein γb directly interacts with BSMV coat protein (CP), the mutations either on the His-85 site in γb predicted to generate a potential CCCH motif or on the His-13 site in CP exposed to the surface of the virions abolish the zinc-binding activity and their interaction. Immunogold-labeling assays show that γb binds to the surface of rod-shaped BSMV virions in a Zn2+-dependent manner, which enhances the RNA binding activity of CP and facilitates virion assembly and stability, suggesting that the Zn2+-dependent physical association of γb with the virion is crucial for BSMV morphogenesis. Intriguingly, the tightly binding of diverse CRPs to their rod-shaped virions is a general feature employed by the members in the families Virgaviridae (excluding the genus Tobamovirus) and Benyviridae. Together, these results reveal a hitherto unknown role of CRPs in the assembly and stability of virus particles, and expand our understanding of the molecular mechanism underlying virus morphogenesis. Author summary: The symmetrical structures of plant RNA viruses are usually composed of numerous identical coat protein (CP) subunits encapsidated one molecule of viral genomic RNA. Interestingly, a large number of rod-shaped and filamentous plant viruses encode a cysteine-rich protein (CRP), which usually function as viral suppressors of RNA silencing and pathogenicity determinants during viral infection. However, their roles in viral life cycle remain poorly understood. Here, our study reveals the essential role of CRPs in virus morphogenesis. The cysteine-rich γb protein binds to the surface of rod-shaped barley stripe mosaic virus (BSMV) virion by interacting with CP in a Zn2+-dependent manner, thereby facilitating virion assembly and stability. Both His-85 site in γb predicted to generate a potential CCCH motif and His-13 site in CP exposed to the surface of the virions are key amino acids in γb-CP interaction and the zinc-binding activity. Attractively, the phenomenon of CRPs being tightly bound to their rod-shaped virions occurs in a broad range of distantly related viruses, implying a conserved role for the CRPs in orchestrating virus morphogenesis. [ABSTRACT FROM AUTHOR]

The maturation of HIV-1 virions is a crucial process in viral replication. Although T-cells are a primary source of virus production, much of our understanding of virion maturation comes from studies using the HEK293T human embryonic kidney cell line. Notably, there is a lack of comparative analyses between T-cells and HEK293T cells in terms of virion maturation efficiency in existing literature. We previously developed an advanced virion visualization system based on the FRET principle, enabling the effective distinction between immature and mature virions via fluorescence microscopy. In this study, we utilized pseudotyped, single-round infectious viruses tagged with FRET labels (HIV-1 Gag-iFRET∆Env) derived from Jurkat (a human T-lymphocyte cell line) and HEK293T cells to evaluate their virion maturation rates. HEK293T-derived virions demonstrated a maturity rate of 81.79%, consistent with other studies and our previous findings. However, virions originating from Jurkat cells demonstrated a significantly reduced maturation rate of 68.67% (p < 0.0001). Correspondingly, viruses produced from Jurkat cells exhibited significantly reduced infectivity compared to those derived from HEK293T cells, with the relative infectivity measured at 65.3%. This finding is consistent with the observed relative maturation rate of viruses produced by Jurkat cells. These findings suggest that initiation of virion maturation directly correlates with viral infectivity. Our observation highlights the dynamic nature of virus–host interactions and their implications for virion production and infectivity. [ABSTRACT FROM AUTHOR]

Objective: Fourth-generation HIV Ag/Ab Combo assay is used for HIV screening of blood for transfusion in developing countries, however, the sensitivity of the assay is questionable during the acute phase of HIV infection. Thus, the study aimed to determine the effect of combining centrifugation with HIV-1 virion lysis on the sensitivity of the fourth-generation HIV Ag/Ab combo assay. Results: When the 50 HIV-1 antibody-negative samples were run on the fourth-generation HIV Ag/Ab combo assay, 8 (16%) were positive following centrifugation, 13 (26%) were positive following lysis while 25 (50%) were positive after combining centrifugation with HIV-1 virion lysis. [ABSTRACT FROM AUTHOR]

A modified method for culturing, concentrating, and purifying phage ϕ24B preparations was developed. In particular, a new lysogenic phage-producing strain lacking flagella was used, induction conditions were optimized, and purification in a sucrose gradient followed by concentration by deposition on a Freon 113 cushion were used. Using this method, a preparation of the Stx-converting bacteriophage ϕ24B was obtained, suitable for CryoEM direct analysis. Based on CryoEM data for this phage, the first primary three-dimensional reconstruction of its virions was performed. The structure of the phage ϕ24B tail is described. It was shown that the adsorption apparatus of this virus is represented by six thin lateral fibrils, and an axial fibril located at the end of the tail. This arrangement of the tail structure is consistent with the previously proposed hypothesis based on analysis of the receptor binding proteins (RBPs) of this bacteriophage. [ABSTRACT FROM AUTHOR]

This paper is concerned with threshold dynamics of a degenerate diffusive HBV infection model with DNA-containing capsids in heterogeneous environment. We firstly address the existence of global solutions, uniform and ultimate boundedness of solutions, asymptotic smoothness of semiflows and existence of a connected global attractor for the diffusive model. Then, we identify the basic reproduction number R 0 and establish a threshold-type result for the disease eradication or uniform persistence when R 0 ≤ 1 or R 0 > 1 , respectively. Especially, when R 0 > 1 and the diffusion rate of capsids or the diffusion rate of virions is zero, we further show that the model admits a unique infection steady state which is globally attractive. Our results indicate that the pathogen can be eliminated by limiting the mobility of the capsids or virions. [ABSTRACT FROM AUTHOR]

Hepatitis E virus (HEV) infection is typically a self-limiting, acute illness that spreads through the gastrointestinal tract but replicates in the liver. However, chronic infections are possible in immunocompromised individuals. The HEV virion has two shapes: exosome-like membrane-associated quasi-enveloped virions (eHEV) found in circulating blood or in the supernatant of infected cell cultures and non-enveloped virions ("naked") found in infected hosts' feces and bile to mediate inter-host transmission. Although HEV is mainly spread via enteric routes, it is unclear how it penetrates the gut wall to reach the portal bloodstream. Both virion types are infectious, but they infect cells in different ways. To develop personalized treatment/prevention strategies and reduce HEV impact on public health, it is necessary to decipher the entry mechanism for both virion types using robust cell culture and animal models. The contemporary knowledge of the cell entry mechanism for these two HEV virions as possible therapeutic target candidates is summarized in this narrative review. [ABSTRACT FROM AUTHOR]

Virus‐glycan interactions play a crucial role in the infection process of many viruses. NMR spectroscopy has emerged as a powerful tool for studying these interactions at the molecular level. In this article, we review several published papers and reports that have highlighted the application of NMR spectroscopy in understanding the complex questions of how viruses engage with and bind to receptor glycans. The use of saturation transfer difference (STD) NMR spectroscopy has demonstrated itself as highly advantageous in investigating the interaction between glycans and intact virions or virus‐like particles (VLPs). The broad NMR signal linewidth of virions and VLPs allows efficient saturation without affecting the glycan signals. The advantage of this approach is that the viral capsid environment in protein organization and function is not ignored and therefore provides a more biologically relevant model for exploring the interactions between the virus and the host cell glycans. We will review some examples of using NMR spectroscopy to study influenza cell tropism, rotaviruses, and noroviruses. [ABSTRACT FROM AUTHOR]

Host restriction factor SERINC5 (SER5) incorporates into the HIV-1 membrane and inhibits infectivity by a poorly understood mechanism. Recently, SER5 was found to exhibit scramblase-like activity leading to the externalization of phosphatidylserine (PS) on the viral surface, which has been proposed to be responsible for SER5's antiviral activity. This and other reports that document modulation of HIV-1 infectivity by viral lipid composition prompted us to investigate the role of PS in regulating SER5-mediated HIV-1 restriction. First, we show that the level of SER5 incorporation into virions correlates with an increase in PS levels in the outer leaflet of the viral membrane. We developed an assay to estimate the PS distribution across the viral membrane and found that SER5, but not SER2, which lacks antiviral activity, abrogates PS asymmetry by externalizing this lipid. Second, SER5 incorporation diminished the infectivity of pseudoviruses produced from cells lacking a flippase subunit CDC50a and, therefore, exhibited a higher baseline level of surface-accessible PS. Finally, exogenous manipulation of the viral PS levels utilizing methyl-alpha-cyclodextrin revealed a lack of correlation between external PS and virion infectivity. Taken together, our study implies that the increased PS exposure to SER5-containing virions itself is not directly linked to HIV-1 restriction. [ABSTRACT FROM AUTHOR]

UV light at 222 nm inactivates SARS-CoV-2 virions at intensities appearing to be benign to skin and eyes. It can disinfect air without requiring protection of persons from UV as is needed at longer UV wavelengths. The lower risk of damage from 222-nm UV results from enhanced absorption of this UV in the outermost parts of eyes and skin. Microbes can be partially shielded from 250- to 285-nm UV when in host particles. Does the higher absorption at 222-nm increase the shielding of microbes within particles, and if so, to what extent? We use literature values to approximate the compositions and optical properties of SARS-CoV-2 virions and four types of fluids from the respiratory tract and mouth. We model virions within spherical host particles (homogeneous except for the virions), calculate the UV absorbed by the virions, and use these values to calculate survival fractions of virions in particles (Sp). We find that shielding is relatively small for particles generated in quiet breathing (typically 1.5-µm or smaller). For larger particles, shielding increases as the host-particle diameter increases. Shielding is greater at 222- than at 254-nm for all pairs of UV-fluence and particle-diameters calculated. The higher eye-safety thresholds at 222-nm allow use of larger fluences and shorter inactivation times. Because shielding increases as host-particle size increases, and because significant fractions of airborne disease-transmitting particles have diameter >1.5-µm, it appears that the sizes and optical properties of host particles must be considered in designing and verifying UV inactivation systems for such particles. [ABSTRACT FROM AUTHOR]

Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA virus etiologically associated with multiple malignancies. Both latency and sporadic lytic reactivation contribute to KSHV-associated malignancies, however, the specific roles of many KSHV lytic gene products in KSHV replication remains elusive. In this study, we report that ablation of ORF55, a late gene encoding a tegument protein, does not impact KSHV lytic reactivation but significantly reduces the production of progeny virions. We found that cysteine 10 and 11 (C10 and C11) of pORF55 are palmitoylated, and the palmytoilation is essential for its Golgi localization and secondary envelope formation. Palmitoylation-defective pORF55 mutants are unstable and undergo proteasomal degradation. Notably, introduction of a putative Golgi localization sequence to these palmitoylation-defective pORF55 mutants restores Golgi localization and fully reinstates KSHV progeny virion production. Together, our study provides new insight into the critical role of pORF55 palmitoylation in KSHV progeny virion production and offers potential therapeutic targets for the treatment of related malignancies. Author summary: Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus associated with multiple human malignancies. Nonetheless, the roles of numerous viral proteins in the viral life cycle remain inadequately characterized. Employing CRISPR knockout screening, we identified the viral tegument protein pORF55 as pivotal in the production of infectious progeny virions. We found that pORF55 is palmitoylated at cysteine 10 and 11, which is required for its Golgi localization. Palmitoylation-deficient mutants of pORF55 are unstable and fail to support secondary envelopment formation, a critical step for viral assembly and egress. Interestingly, we found that forced restoration of the Golgi localization of the palmitoylation-deficient pORF55 mutants completely reinstates the infectious progeny virion production. Hence, our study underscores the central role of Golgi localization resulting from pORF55 palmitoylation. Our study not only elucidates the role of pORF55 in viral replication, but also suggests targeting its palmitoylation as a potential therapeutic strategy for curtailing viral replication and treating related pathogenesis. [ABSTRACT FROM AUTHOR]

The globally distributed marine alga Emiliania huxleyi has cooling effect on the Earth's climate. The population density of E. huxleyi is restricted by Nucleocytoviricota viruses, including E. huxleyi virus 201 (EhV-201). Despite the impact of E. huxleyi viruses on the climate, there is limited information about their structure and replication. Here, we show that the dsDNA genome inside the EhV-201 virion is protected by an inner membrane, capsid, and outer membrane. EhV-201 virions infect E. huxleyi by using fivefold vertices to bind to and fuse the virus' inner membrane with the cell plasma membrane. Progeny virions assemble in the cytoplasm at the surface of endoplasmic reticulum-derived membrane segments. Genome packaging initiates synchronously with the capsid assembly and completes through an aperture in the forming capsid. The genome-filled capsids acquire an outer membrane by budding into intracellular vesicles. EhV-201 infection induces a loss of surface protective layers from E. huxleyi cells, which enables the continuous release of virions by exocytosis. [ABSTRACT FROM AUTHOR]

The coat protein (CP) is an important structural protein that plays many functional roles during the viral cycle. In this study, the CP of pepper mild mottle virus (PMMoV) was genetically fused to GFP using the foot-and-mouth disease virus peptide 2A linker peptide and the construct (PMMoV-GFP2A) was shown to be infectious. The systemic spread of the virus was monitored by its fluorescence in infected plants. Electron microscopy and immunocolloidal gold labelling confirmed that PMMoV-GFP2A forms rod-shaped particles on which GFP is displayed. Studies of tissue ultrastructure and virion self-assembly confirmed that PMMoV-GFP2A could be used to monitor the real-time dynamic changes of CP location during virus infection. Aggregations of GFP-tagged virions appeared as fluorescent plaques in confocal laser microscopy. Altogether, PMMoV-GFP2A is a useful tool for studying the spatial and temporal changes of PMMoV CP during viral infection. [ABSTRACT FROM AUTHOR]

Vertical transmission has been described following monkeypox virus (MPXV) infection in pregnant women. The presence of MPXV has been reported in the placenta from infected women, but whether pathogens colonize placenta remains unexplored. We identify trophoblasts as a target cell for MPXV replication. In a pan‐microscopy approach, we decipher the specific infectious cycle of MPXV and inner cellular structures in trophoblasts. We identified the formation of a specialized region for viral morphogenesis and replication in placental cells. We also reported infection‐induced cellular remodeling. We found that MPXV stimulates cytoskeleton reorganization with intercellular extensions for MPXV cell spreading specifically to trophoblastic cells. Altogether, the specific infectious cycle of MPXV in trophoblast cells and these protrusions that were structurally and morphologically similar to filopodia reveal new insights into the infection of MPXV. [ABSTRACT FROM AUTHOR]

ABSTRACT Escherichia coli is a commensal inhabitant of the mammalian gut microbiota, frequently associated with various gastrointestinal diseases. There is increasing interest in comprehending the variety of bacteriophages (phages) that target this bacterium, as such insights could pave the way for their potential use in therapeutic applications. Here, we report the isolation and characterization of four newly identified E. coli infecting tailed phages (W70, A7-1, A5-4, and A73) that were found to constitute a novel genus, Septuagintavirus, within the subfamily Vequintavirinae. Genomes of these phages ranged from 137 kbp to 145 kbp, with a GC content of 41 mol%. They possess a maximum nucleotide similarity of 30% with phages of the closest phylogenetic genus, Certrevirus, while displaying limited homology to other genera of the Vequintavirinae family. Host range analysis showed that these phages have limited activity against a panel of E. coli strains, infecting 6 out of 16 tested isolates, regardless of their phylotype. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was performed on the virion of phage W70, allowing the identification of 28 structural proteins, 19 of which were shared with phages of other genera of Vequintavirinae family. The greatest diversity was identified with proteins forming tail fiber structures, likely indicating the adaptation of virions of each phage genus of this subfamily for the recognition of their target receptor on host cells. The findings of this study provide greater insights into the phages of the subfamily Vequintavirinae, contributing to the pool of knowledge currently known about these phages.IMPORTANCEEscherichia coli is a well-known bacterium that inhabits diverse ecological niches, including the mammalian gut microbiota. Certain strains are associated with gastrointestinal diseases, and there is a growing interest in using bacteriophages, viruses that infect bacteria, to combat bacterial infections. Here, we describe the isolation and characterization of four novel E. coli bacteriophages that constitute a new genus, Septuagintavirus, within the subfamily Vequintavirinae. We conducted mass spectrometry on virions of a representative phage of this novel clade and compared it to other phages within the subfamily. Our analysis shows that virion structure is highly conserved among all phages, except for proteins related to tail fiber structures implicated in the host range. These findings provide greater insights into the phages of the subfamily Vequintavirinae, contributing to the existing pool of knowledge about these phages.

Alkaline exonucleases (AE) are present in several large DNA viruses including bacteriophage λ and herpesviruses, where they play roles in viral DNA processing during genome replication. Given the genetic conservation of AEs across viruses infecting different kingdoms of life, these enzymes likely assume central roles in the lifecycles of viruses where they have yet to be well characterized. Here, we applied a structure-guided functional analysis of the bifunctional AE in the oncogenic human gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV), called SOX. In addition to identifying a preferred DNA substrate preference for SOX, we define key residues important for DNA binding and DNA processing, and how SOX activity on DNA partially overlaps with its functionally separable cleavage of mRNA. By engineering these SOX mutants into KSHV, we reveal roles for its DNase activity in viral gene expression and infectious virion production. Our results provide mechanistic insight into gammaherpesviral AE activity as well as areas of functional conservation between this mammalian virus AE and its distant relative in phage λ.

Many plant virus-like particles (VLPs) utilized in nanotechnology are 30-nm icosahedrons. To expand the VLP platforms, we produced VLPs of Cytoplasmic type citrus leprosis virus (CiLV-C) in Nicotiana benthamiana. We were interested in CiLV-C because of its unique bacilliform shape (60-70 nm × 110-120 nm). The CiLV-C capsid protein (p29) gene was transferred to the pTRBO expression vector transiently expressed in leaves. Stable VLPs were formed, as confirmed by agarose gel electrophoresis, transmission electron microscopy and size exclusion chromatography. Interestingly, the morphology of the VLPs (15.8 ± 1.3 nm icosahedral particles) differed from that of the native bacilliform particles indicating that the assembly of native virions is influenced by other viral proteins and/or the packaged viral genome. The smaller CiLV-C VLPs will also be useful for structure-function studies to compare with the 30-nm icosahedrons of other VLPs.

Precise, scalable, and quantitative evaluation of whole slide images is crucial in neuropathology. We release a deep learning model for rapid object detection and precise information on the identification, locality, and counts of cored plaques and cerebral amyloid angiopathy (CAA). We trained this object detector using a repurposed image-tile dataset without any human-drawn bounding boxes. We evaluated the detector on a new manually-annotated dataset of whole slide images (WSIs) from three institutions, four staining procedures, and four human experts. The detector matched the cohort of neuropathology experts, achieving 0.64 (model) vs. 0.64 (cohort) average precision (AP) for cored plaques and 0.75 vs. 0.51 AP for CAAs at a 0.5 IOU threshold. It provided count and locality predictions that approximately correlated with gold-standard human CERAD-like WSI scoring (p = 0.07 ± 0.10). The openly-available model can quickly score WSIs in minutes without a GPU on a standard workstation.

Efforts to develop vaccine and immunotherapeutic countermeasures against the COVID-19 pandemic focus on targeting the trimeric spike (S) proteins of SARS-CoV-2. Vaccines and therapeutic design strategies must impart the characteristics of virion S from historical and emerging variants onto practical constructs such as soluble, stabilized trimers. The virus spike is a heterotrimer of two subunits: S1, which includes the receptor binding domain (RBD) that binds the cell surface receptor ACE2, and S2, which mediates membrane fusion. Previous studies suggest that the antigenic, structural, and functional characteristics of virion S may differ from current soluble surrogates. For example, it was reported that certain anti-glycan, HIV-1 neutralizing monoclonal antibodies bind soluble SARS-CoV-2 S but do not neutralize SARS-CoV-2 virions. In this study, we used single-molecule fluorescence correlation spectroscopy (FCS) under physiologically relevant conditions to examine the reactivity of broadly neutralizing and non-neutralizing anti-S human monoclonal antibodies (mAbs) isolated in 2020. Binding efficiency was assessed by FCS with soluble S trimers, pseudoviruses and inactivated wild-type virions representing variants emerging from 2020 to date. Anti-glycan mAbs were tested and compared. We find that both anti-S specific and anti-glycan mAbs exhibit variable but efficient binding to a range of stabilized, soluble trimers. Across mAbs, the efficiencies of soluble S binding were positively correlated with reactivity against inactivated virions but not pseudoviruses. Binding efficiencies with pseudoviruses were generally lower than with soluble S or inactivated virions. Among neutralizing mAbs, potency did not correlate with binding efficiencies on any target. No neutralizing activity was detected with anti-glycan antibodies. Notably, the virion S released from membranes by detergent treatment gained more efficient reactivity with anti-glycan, HIV-neutralizing antibodies but lost reactivity with all anti-S mAbs. Collectively, the FCS binding data suggest that virion surfaces present appreciable amounts of both functional and nonfunctional trimers, with neutralizing anti-S favoring the former structures and non-neutralizing anti-glycan mAbs binding the latter. S released from solubilized virions represents a nonfunctional structure bound by anti-glycan mAbs, while engineered soluble trimers present a composite structure that is broadly reactive with both mAb types. The detection of disparate antigenicity and immunoreactivity profiles in engineered and virion-associated S highlight the value of single-virus analyses in designing future antiviral strategies against SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Prevention and early diagnosis are the best and most effective ways for defeating HIV. There is still no vaccine, but treatments with antiretroviral drugs are now available which, in many cases, allow the infection to become chronic. However, research has highlighted side effects of these drugs and the fact that a flare-up of the infection occurs if the therapy is stopped. In recent years, the presence of virus reserves located in various parts of the body, including the brain, has been hypothesized. The possibility of controlling the infection of healthy cells and of interrupting the proliferation of virions inside the brain has been studied, proposing optimal control strategies. [ABSTRACT FROM AUTHOR]

Lymphocytic choriomeningitis virus (LCMV) is a bisegmented negativesense RNA virus classified within the Arenaviridae family of the Bunyavirales order. LCMV is associated with fatal disease in immunocompromized populations, and as the prototypical arenavirus, acts as a model for the many serious human pathogens within this group. Here, we examined the dependence of LCMV multiplication on cellular trafficking components using a recombinant LCMV expressing enhanced green fluorescent protein in conjunction with a curated siRNA library. The screen revealed a requirement for subunits of both the coat protein 1 (COPI) coatomer and adapter protein 4 (AP-4) complexes. By rescuing a recombinant LCMV harboring a FLAG-tagged glycoprotein (GP-1) envelope spike (rLCMV-GP1-FLAG), we showed infection resulted in marked co-localization of individual COPI and AP-4 components with both LCMV nucleoprotein (NP) and GP-1, consistent with their involvement in viral processes. To further investigate the role of both COPI and AP-4 complexes during LCMV infection, we utilized the ARF-I inhibitor brefeldin A (BFA) that prevents complex formation. Within a single 12-h cycle of virus multiplication, BFA pre-treatment caused no significant change in LCMV-specific RNA synthesis, alongside no significant change in LCMV NP expression, as measured by BFA time-of-addition experiments. In contrast, BFA addition resulted in a significant drop in released virus titers, approaching 50-fold over the same 12-h period, rising to over 600-fold over 24 h. Taken together, these findings suggest COPI and AP-4 complexes are important host cell factors required for the formation and release of infectious LCMV. [ABSTRACT FROM AUTHOR]

HSV-1 major tegument protein VP22 is present in multiple subcellular locations in the late stages of productive viral infection. We initially performed a detailed time course experiment and observed that VP22 was detected in nuclear and nuclear matrix fractions as early as 4 hpi. The goal was to determine the fate of virion-derived incoming VP22, and we report the following: (i) VP22 was detected in nuclear matrix fractions 1 hpi. (ii) In the presence of cycloheximide (CHX), VP22 was present in the nuclear matrix 1–6 hpi, demonstrating the stability of the protein. (iii) The nuclear matrix targeting of VP22 occurred in infected Vero, HEp-2, and human mammary epithelial (HME) cells and following synchronized infection. Based on these results, we conclude that (iv) VP22 targets the nuclear matrix and chromatin upon entry into cells during productive HSV-1 infection. [ABSTRACT FROM AUTHOR]

HIV particles in the blood largely originate from activated lymphocytes and can overshadow variants which may be expressed from other cell types. Investigations of virus persistence must be able to distinguish cells refractory to viral clearance that serve as reservoirs. To investigate additional cell types that may be associated with in vivo HIV expression we developed a virus particle immunomagnetic capture method targeting several markers of cellular origin that become embedded within virion envelopes during budding. We evaluated the ability of markers to better distinguish cell lineage source subpopulations by assessing combinations of different antibodies with cell-sorted in vitro culture and clinical specimens. Various deductive algorithms were designed to discriminate source cell lineages and subsets. From the particle capture algorithms, we identified distinct variants expressed within individuals that were associated with disparate cellular markers. Among the variants uncovered were minority-level viruses with drug resistance mutations undetected by sequencing and often were associated with markers indicative of myeloid lineage (CD3-/CD10-/CD16+ or /CD14+, and CD3-/CD16-/CD14-/CD11c+ or /HLA-DR+) cell sources. The diverse HIV genetic sequences expressed from different cell types within individuals, further supported by the appearance of distinct drug-resistant variants, highlights the complexity of HIV reservoirs in vivo which must be considered for HIV cure strategies. This approach could also be helpful in examining in vivo host cell origins and genetic diversity in infections involving other families of budding viruses. [ABSTRACT FROM AUTHOR]

Bacteria have evolved diverse antiviral defence mechanisms to protect themselves against phage infection. Phages integrated into bacterial chromosomes, known as prophages, also encode defences that protect the bacterial hosts in which they reside. Here, we identify a type of anti-phage defence that interferes with the virion assembly pathway of invading phages. The protein that mediates this defence, which we call Tab (for 'Tail assembly blocker'), is constitutively expressed from a Pseudomonas aeruginosa prophage. Tab allows the invading phage replication cycle to proceed, but blocks assembly of the phage tail, thus preventing formation of infectious virions. While the infected cell dies through the activity of the replicating phage lysis proteins, there is no release of infectious phage progeny, and the bacterial community is thereby protected from a phage epidemic. Prophages expressing Tab are not inhibited during their own lytic cycle because they express a counter-defence protein that interferes with Tab function. Thus, our work reveals an anti-phage defence that operates by blocking virion assembly, thereby both preventing formation of phage progeny and allowing destruction of the infected cell due to expression of phage lysis genes. Bacteria have evolved various defence mechanisms to protect themselves against viral infection. Here, Patel et al. identify a type of antiviral defence that blocks virion assembly, thus preventing formation of infectious virions and allowing destruction of the infected cell. [ABSTRACT FROM AUTHOR]

We present the first complete stochastic model of vesicular stomatitis virus (VSV) intracellular replication. Previous models developed to capture VSV's intracellular replication have either been ODE-based or have not represented the complete replicative cycle, limiting our ability to understand the impact of the stochastic nature of early cellular infections on virion production between cells and how these dynamics change in response to mutations. Our model accurately predicts changes in mean virion production in gene-shuffled VSV variants and can capture the distribution of the number of viruses produced. This model has allowed us to enhance our understanding of intercellular variability in virion production, which appears to be influenced by the duration of the early phase of infection, and variation between variants, arising from balancing the time the genome spends in the active state, the speed of incorporating new genomes into virions, and the production of viral components. Being a stochastic model, we can also assess other effects of mutations beyond just the mean number of virions produced, including the probability of aborted infections and the standard deviation of the number of virions produced. Our model provides a biologically interpretable framework for studying the stochastic nature of VSV replication, shedding light on the mechanisms underlying variation in virion production. In the future, this model could enable the design of more complex viral phenotypes when attenuating VSV, moving beyond solely considering the mean number of virions produced. Author summary: This study presents the first complete stochastic model of vesicular stomatitis virus (VSV) replication. Our model captures the dynamic process of VSV's replication within host cells, accounting for the stochastic nature of early cellular infections and how these dynamics change in response to mutations. By accurately predicting changes in mean virion production and the distribution of viruses in gene-shuffled VSV variants, our model enhances our understanding of viral replication and the variation we see in virion production. Importantly, our findings shed light on the mechanisms underlying the production of VSV virions, revealing the influence of factors such as the duration of the early infection phase and the interplay between the genome's ability to switch into an inactive state and viral protein production. We go beyond assessing the mean number of virions produced and examine other effects of mutations, including the probability of aborted infections and the variability in virion production. This stochastic model provides a valuable framework for studying the complex nature of viral replication, contributing to our understanding of single-cell viral dynamics and variability. Ultimately, this knowledge could pave the way for designing more effective strategies to attenuate VSV. [ABSTRACT FROM AUTHOR]

The human family of APOBEC3 enzymes are primarily studied as single-stranded DNA deoxycytidine deaminases that act as host restriction factors for a number of viruses and retroelements. The deamination of deoxycytidine to deoxyuridine causes inactivating mutations in target DNA and the nucleic acid binding ability may also cause deamination independent restriction. There are seven APOBEC3 enzymes in humans, named A-H, excluding E, each of which has restriction activity against a subset of viruses or retroelements. There are primarily four, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H that have been found to restrict replication of HIV-1, however their restriction activity varies and they have primarily been studied individually despite co-expression in the cells that HIV-1 infects. It is known that APOBEC3F hetero-oligomerizes with APOBEC3G and APOBEC3H and that this influences host restriction outcomes during HIV-1 infection in tissue culture. Here, we examined if APOBEC3F interacts with APOBEC3D and the functional outcomes. We found that APOBEC3D mRNA expression was similar to or higher than APOBEC3F mRNA in multiple donors, suggesting that the proteins would be co-expressed, allowing for interactions to occur. We determined that APOBEC3F and APOBEC3D interacted primarily through an RNA intermediate; however, this interaction resulted in APOBEC3D competitively excluding APOBEC3F from virions. Although HIV-1 restriction still occurred when APOBEC3F and APOBEC3D were co-expressed, it was due to primarily APOBEC3D-mediated deamination-independent restriction. The APOBEC3D-mediated exclusion of APOBEC3F from HIV-1 encapsidation could be recapitulated in vitro through RNA capture experiments in which APOBEC3D decreased or abrogated the ability of APOBEC3F to bind to HIV-1 protease or 5'UTR RNA, respectively. Overall, the data suggest that there are mechanisms at the protein level that segregate APOBEC3s into different virus particles. [ABSTRACT FROM AUTHOR]

Lung infection by influenza A virus (IAV) is a major cause of global mortality from lung injury, a disease defined by widespread dysfunction of the lung's air-blood barrier. Endocytosis of IAV virions by the alveolar epithelium - the cells that determine barrier function - is central to barrier loss mechanisms. Here, we address the current understanding of the mechanistic steps that lead to endocytosis in the alveolar epithelium, with an eye to how the unique structure of lung alveoli shapes endocytic mechanisms. We highlight where future studies of alveolar interactions with IAV virions may lead to new therapeutic approaches for IAV-induced lung injury. [ABSTRACT FROM AUTHOR]

Members of the serine incorporator (SERINC) protein family exert broad antiviral activity, and many viruses encode SERINC antagonists to circumvent these restrictions. Significant new insight was recently gained into the mechanisms that mediate restriction and antagonism. In this review, we summarize our current understanding of the mode of action and relevance of SERINC proteins in HIV-1 infection. Particular focus will be placed on recent findings that provided important new mechanistic insights into the restriction of HIV-1 virion infectivity, including the discovery of SERINC's lipid scramblase activity and its antagonism by the HIV-1 pathogenesis factor Nef. We also discuss the identification and implications of several additional antiviral activities by which SERINC proteins enhance pro-inflammatory signaling and reduce viral gene expression in myeloid cells. SERINC proteins emerge as versatile and multifunctional regulators of cell-intrinsic immunity against HIV-1 infection. [ABSTRACT FROM AUTHOR]

Species-specific differences in acidic nuclear phosphoprotein 32 family member A (ANP32A) determine the restriction of avian-signature polymerase in mammalian cells. Mutations that evade this restriction, such as PB2-E627K, are frequently acquired when avian influenza A viruses jump from avian hosts to mammalian hosts. However, the mechanism underlying this adaptation process is still unclear. Here, we report that host factor ANP32 proteins can be incorporated into influenza viral particles through combination with the viral RNA polymerase (vPol) and then transferred into targeted cells where they support virus replication. The packaging of the ANP32 proteins into influenza viruses is dependent on their affinity with the vPol. Avian ANP32A (avANP32A) delivered by avian influenza A virions primes early viral replication in mammalian cells, thereby favoring the downstream interspecies transmission event by increasing the total amount of virus carrying adaptive mutations. Our study clarifies one role of avANP32A where it is used by avian influenza virus to help counteract the restriction barrier in mammals. [ABSTRACT FROM AUTHOR]

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. IMPORTANCE EBV 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. [ABSTRACT FROM AUTHOR]

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. Here, the authors uncover that, among 92 identified host factors associated with SARS-CoV-2 virions, G3BP1 and G3BP2 are present at the assembly site, incorporated within virions, and unexpectedly promote the production of SARS-CoV-2 virions. [ABSTRACT FROM AUTHOR]

BST2/Tetherin is a restriction factor with broad antiviral activity against enveloped viruses, including coronaviruses. Specifically, BST2 traps nascent particles to membrane compartments, preventing their release and spread. In turn, viruses have evolved multiple mechanisms to counteract BST2. Here, we examined the interactions between BST2 and SARS-CoV-2. Our study shows that BST2 reduces SARS-CoV-2 virion release. However, the virus uses the Spike (S) protein to downregulate BST2. This requires a physical interaction between S and BST2, which routes BST2 for lysosomal degradation in a Clathtin- and ubiquitination-dependent manner. By surveying different SARS-CoV-2 variants of concern (Alpha-Omicron), we found that Omicron is more efficient at counteracting BST2, and that mutations in S account for its enhanced anti-BST2 activity. Mapping analyses revealed that several surfaces in the extracellular region of BST2 are required for an interaction with the Spike, and that the Omicron variant has changed its patterns of association with BST2 to improve its counteraction. Therefore, our study suggests that, besides enhancing receptor binding and evasion of neutralizing antibodies, mutations accumulated in the Spike afford more efficient counteraction of BST2, which highlights that BST2 antagonism is important for SARS-CoV-2 infectivity and spread. Author summary: BST2/Tetherin is a potent antiviral factor that prevents the egress of multiple enveloped viruses. In turn, viruses have evolved mechanisms to circumvent this block. Here, we found that SARS-CoV-2 primarily uses the Spike protein to promote the lysosomal degradation of BST2, thus, removing it from sites of virion assembly and facilitating virus release. When analyzing several SARS-CoV-2 variants of concern, we found that Omicron is more efficient at counteracting BST2, to the point where its replication is barely impacted by this restriction factor. Subsequent studies identified mutations accumulated in the Omicron Spike as responsible for improving an interaction between Spike and BST2. However, the surfaces in BST2 required for this association differ between Wuhan and Omicron. Remarkably, increased Spike-BST2 binding is associated with enhanced BST2 downregulation. Therefore, these observations suggest that, in addition to enhancing receptor binding and immune evasion, mutations in the Spike afford more efficient counteraction of BST2, highlighting that BST2 antagonism is important for SARS-CoV-2 infectivity. [ABSTRACT FROM AUTHOR]

Thermus thermophilus bacteriophage P23-45 encodes a giant 5,002-residue tail tape measure protein (TMP) that defines the length of its extraordinarily long tail. Here, we show that the N-terminal portion of P23-45 TMP is an unusual RNA polymerase (RNAP) homologous to cellular RNAPs. The TMP-fused virion RNAP transcribes pre-early phage genes, including a gene that encodes another, non-virion RNAP, that transcribes early and some middle phage genes. We report the crystal structures of both P23-45 RNAPs. The non-virion RNAP has a crab-claw-like architecture. By contrast, the virion RNAP adopts a unique flat structure without a clamp. Structure and sequence comparisons of the P23-45 RNAPs with other RNAPs suggest that, despite the extensive functional differences, the two P23-45 RNAPs originate from an ancient gene duplication in an ancestral phage. Our findings demonstrate striking adaptability of RNAPs that can be attained within a single virus species. The authors describe the structure and function of two evolutionarily diverged RNA polymerases of a thermophilic phage. One of the polymerases is fused to the phage tape measure protein, a virion component dictating the length of the phage tail. [ABSTRACT FROM AUTHOR]

Background: Controlling novel coronavirus pandemic infection (COVID-19) is a global challenge, and highly sensitive testing is essential for effective control. The saliva is a promising sample for high-sensitivity testing because it is easier to collect than nasopharyngeal swab samples and allows large-volume testing. Results: We developed a simple SARS-CoV-2 concentration method from saliva samples that can be completed in less than 60 min. We performed a spike test using 12 ml of saliva samples obtained from healthy volunteer people, and the developed method performance was evaluated by comparison using a combination of automatic nucleic acid extraction followed by RT-qPCR detection. In saliva spike tests using a 10-fold dilution series of SARS-CoV-2, the developed method was consistently 100-fold more sensitive than the conventional method. Conclusions: The developed method can improve the analytical sensitivity of the SARS-CoV-2 test using saliva and speed up and save labor in screening tests by pooling many samples. Furthermore, the developed method has the potential to contribute to the highly sensitive detection of various human and animal viral pathogens from the saliva and various clinical samples.

Many eukaryotic viruses assemble mature particles within distinct subcellular compartments, but bacteriophages are generally assumed to assemble randomly throughout the host cell cytoplasm. Here, we show that viral particles of Pseudomonas nucleus-forming jumbo phage PhiPA3 assemble into a unique structure inside cells we term phage bouquets. We show that after capsids complete DNA packaging at the surface of the phage nucleus, tails assemble and attach to capsids, and these particles accumulate over time in a spherical pattern, with tails oriented inward and the heads outward to form bouquets at specific subcellular locations. Bouquets localize at the same fixed distance from the phage nucleus even when it is mispositioned, suggesting an active mechanism for positioning. These results mark the discovery of a pathway for organizing mature viral particles inside bacteria and demonstrate that nucleus-forming jumbo phages, like most eukaryotic viruses, are highly spatially organized during all stages of their lytic cycle.

The polymerase complex of nonsegmented negative-strand RNA viruses primarily consists of a large (L) protein and a phosphoprotein (P). L is a multifunctional enzyme carrying out RNA-dependent RNA polymerization and all other steps associated with transcription and replication, while P is the nonenzymatic cofactor, regulating the function and conformation of L. The structure of a purified vesicular stomatitis virus (VSV) polymerase complex containing L and associated P segments has been determined; however, the location and manner of the attachments of L and P within each virion are unknown, limiting our mechanistic understanding of VSV RNA replication and transcription and hindering engineering efforts of this widely used anticancer and vaccine vector. Here, we have used cryo-electron tomography to visualize the VSV virion, revealing the attachment of the ring-shaped L molecules to VSV nucleocapsid proteins (N) throughout the cavity of the bullet-shaped nucleocapsid. Subtomogram averaging and three-dimensional classification of regions containing N and the matrix protein (M) have yielded the in situ structure of the polymerase complex. On average, ∼55 polymerase complexes are packaged in each virion. The capping domain of L interacts with two neighboring N molecules through flexible attachments. P, which exists as a dimer, bridges separate N molecules and the connector and C-terminal domains of L. Our data provide the structural basis for recruitment of L to N by P in virus assembly and for flexible attachments between L and N, which allow a quick response of L in primary transcription upon cell entry.

Purpose: the article presents the result of an attempt to assess the possibility of using scanning electron microscopy and energy dispersive spectroscopy (SEM/EDS) to identify silica nanoparticles (SiO2), which, due to the size of individual particles (200 nm), can be used as coronavirus markers in simulation tests. Methodology: SEM/EDS evaluation was performed using three different media types; namely membrane filters, sponge filters and graphite discs. Result: SEM/EDS studies, consisting in determining the morphology and grain size of SiO2 markers and X-ray microanalysis of their elemental composition, proved that this technique can be successfully used to identify markers. Originality: due to their particle size, ease of handling with various types of surfaces, and biological and physicochemical neutrality, silica markers can act as coronavirus substitutes in experimental studies. [ABSTRACT FROM AUTHOR]

Coat proteins (CP) of the potato virus A virions (PVA) contain partially disordered N-terminal domains, which are necessary for performing vital functions of the virus. Comparative analysis of the structures of coat proteins (CPs) in the intact PVA virions and in the virus particles lacking N-terminal 32 amino acids (PVAΔ32) was carried out in this work based on the tritium planigraphy data. Using atomic-resolution structure of the potato virus Y potyvirus (PVY) protein, which is a homolog of the CP PVA, the available CP surfaces in the PVY virion were calculated and the areas of intersubunit/interhelix contacts were determined. For this purpose, the approach of Lee and Richards [Lee, B., and Richards, F. M. (1971) J. Mol. Biol., 55, 379-400] was used. Comparison of incorporation profiles of the tritium label in the intact and trypsin-degraded PVA∆32 revealed position of the ΔN-peptide shielding the surface domain (a.a. 66-73, 141-146) and the interhelix zone (a.a. 161-175) of the PVA CP. Presence of the channels/cavities was found in the virion, which turned out to be partially permeable to tritium atoms. Upon removal of the ∆N-peptide, decrease in the label incorporation within the virion (a.a. 184-200) was also observed, indicating possible structural transition leading to the virion compactization. Based on the obtained data, we can conclude that part of the surface ∆N-peptide is inserted between the coils of the virion helix thus increasing the helix pitch and providing greater flexibility of the virion, which is important for intercellular transport of the viruses in the plants. [ABSTRACT FROM AUTHOR]

Viral endocytosis involves elastic cell deformation, driven by chemical adhesion energy, and depends on physical interactions between the virion and cell membrane. These interactions are not easy to quantify experimentally. Hence, this study aimed to develop a mathematical model of the interactions of HIV particles with host cells and explore the effects of mechanical and morphological parameters during full virion engulfment. The invagination force and engulfment energy were described as viscoelastic and linear-elastic functions of radius and elastic modulus of virion and cell, ligand-receptor energy density and engulfment depth. The influence of changes in the virion-cell contact geometry representing different immune cells and ultrastructural membrane features and the decrease in virion radius and shedding of gp120 proteins during maturation on invagination force and engulfment energy was investigated. A low invagination force and high ligand-receptor energy are associated with high virion entry ability. The required invagination force was the same for immune cells of different sizes but lower for a local convex geometry of the cell membrane at the virion length scale. This suggests that localized membrane features of immune cells play a role in viral entry ability. The available engulfment energy decreased during virion maturation, indicating the involvement of additional biological or biochemical changes in viral entry. The developed mathematical model offers potential for the mechanobiological assessment of the invagination of enveloped viruses towards improving the prevention and treatment of viral infections. [ABSTRACT FROM AUTHOR]

Cyanophages affect the abundance, diversity, metabolism, and evolution of picocyanobacteria in marine ecosystems. Here we report an estuarine Synechococcus phage, S-CREM2, which represents a novel viral genus and leads to the establishment of a new T4-like cyanophage clade named cluster C. S-CREM2 possesses the longest tail (-418 nm) among isolated cyanomyoviruses and encodes six tail-related proteins that are exclusively homologous to those predicted in the cluster C cyanophages. Furthermore, S-CREM2 may carry three regulatory proteins in the virion, which may play a crucial role in optimizing the host intracellular environment for viral replication at the initial stage of infection. The cluster C cyanophages lack auxiliary metabolic genes (AMGs) that are commonly found in cyanophages of the T4-like clusters A and B and encode unique AMGs like an S-type phycobilin lyase gene. A variation in the composition of tRNA and cis-regulatory RNA genes was observed between the marine and freshwater phage strains in cluster C, reflecting their different modes of coping with hosts and habitats. The cluster C cyanophages are widespread in estuarine and coastal regions and exhibit equivalent or even higher relative abundance compared to those of clusters A and B cyanophages in certain estuarine regions. The isolation of cyanophage S-CREM2 provides new insights into the phage- host interactions mediated by both newly discovered AMGs and virion-associated proteins and emphasizes the ecological significance of cluster C cyanophages in estuarine environments. [ABSTRACT FROM AUTHOR]

Human T-cell tropic virus type 1 (HTLV-1) is known to be mainly transmitted by cell-to-cell contact due to the lower infectivity of the cell-free virion. However, the reasons why cell-free HTLV-1 infection is poor remain unknown. In this study, we found that the retrovirus pseudotyped with HTLV-1 viral envelope glycoprotein (Env) was infectious when human immunodeficiency virus type 1 (HIV-1) was used to produce the virus. We found that the incorporation of HTLV-1 Env into virus-like particles (VLPs) was low when HTLV-1 Gag was used to produce VLPs, whereas VLPs produced using HIV-1 Gag efficiently incorporated HTLV-1 Env. The production of VLPs using Gag chimeras between HTLV-1 and HIV-1 Gag and deletion mutants of HIV-1 Gag showed that the p6 domain of HIV-1 Gag was responsible for the efficient incorporation of HTLV-1 Env into the VLPs. Further mutagenic analyses of the p6 domain of HIV-1 Gag revealed that the PTAP motif in the p6 domain of HIV-1 Gag facilitates the incorporation of HTLV-1 Env into VLPs. Since the PTAP motif is known to interact with tumor susceptibility gene 101 (TSG101) during the budding process, we evaluated the effect of TSG101 knockdown on the incorporation of HTLV-1 Env into VLPs. We found that TSG101 knockdown suppressed the incorporation of HTLV-1 Env into VLPs and decreased the infectivity of cell-free HIV-1 pseudotyped with HTLV-1 Env. Our results suggest that the interaction of TSG101 with the PTAP motif of the retroviral L domain is involved not only in the budding process but also in the efficient incorporation of HTLV-1 Env into the cell-free virus. [ABSTRACT FROM AUTHOR]