Capsid Research Articles

Exploring the recombinant evolution and hosts of crucivirus based on novel oyster-associated viruses

“Crucivirus” represents a group of viruses with chimeric genomes, significant for viral evolution and recombination studies. Their capsid proteins share homology with the RNA virus tombusvirus, while their replicase-associated proteins are homologous to a class of single-stranded DNA viruses, namely CRESS DNA viruses. This study identifies seven novel crucivirus genomes from oysters cultivated along the coast of the South China Sea. Phylogenetic analysis reveals that five sequences form a distinct branch, which may indicate the presence of a new subclass within the crucivirus family. We analyzed crucivirus from multiple perspectives, including viral genomes, hallmark proteins, sequence similarity, and potential hosts. The results indicate that the crucivirus genomes and replicase-associated proteins (Rep) from oysters conform to the typical characteristics of crucivirus; Crucivirus Rep appears to have a direct parallel origin from multiple clades of CRESS DNA viruses, while only the S-domain of their capsid proteins shows some evolutionary relationship with tombusvirus. We found protein sequences in rotifers that are highly similar to the Cap three-dimensional structure of crucivirus, which may suggest host relevance. Overall, this study provides new insights into the classification, evolution, and host origins of crucivirus.

Marek's disease virus-1 unique gene LORF1 is involved in viral replication and MDV-1/Md5-induced atrophy of the bursa of Fabricius.

Marek's disease virus (MDV), an alphaherpesvirus, causes severe immunosuppression and T cell lymphomas in chickens, known as Marek's disease (MD), an economically important poultry disease primarily controlled by vaccination. Importantly, it also serves as a comparative model for studying herpesvirus-induced tumor formation in humans. MDV encodes more than 100 genes, most of which have unknown functions. MDV LORF1 is unique to serotype I MDV (MDV-1), lacking homologs in other herpesviruses, and has not been explored yet. To this end, an infectious bacterial artificial chromosome (BAC) harboring the complete genome of the MDV-1 very virulent strain Md5 was generated, and the rescued rMd5 maintained biological properties similar to the parental virus both in vitro and in vivo. Subsequently, rMd5ΔLORF1, a recombinant Md5 virus deficient in pLORF1 expression, was generated by a frameshift mutation in the LORF1 gene. Chickens infected with rMd5ΔLORF1 exhibited a lower mortality rate and delayed bursal atrophy than those infected with the parental rMd5 and the revertant virus (rMd5-reLORF1). Consistently, viral loads of rMd5ΔLORF1 were obviously lower than those of rMd5 or rMd5-reLORF1 in the bursa, but not in the spleen. Importantly, we found that pLORF1 deficiency impairs viral replication in bursal B cells. Furthermore, we showed that pLORF1 associated with the cellular membrane, interacted with MDV structural proteins, and exhibited punctate colocalization with tegument or capsid proteins in the cytoplasm. Taken together, this study demonstrates for the first time that the MDV-1 unique gene LORF1 is involved in MDV-induced bursal atrophy but not in tumor formation.

Effects of GS-CA1 on nuclear envelope-associated early HIV-1 infection steps

The novel HIV-1 drugs GS-CA1 and the recently approved lenacapavir (GS-6207) target the viral structural protein capsid (CA). However, their multiple mechanisms of action have not been fully characterized. Here, we investigated the effects of GS-CA1 on the early stages of HIV-1 infection, specifically the steps involving the nuclear envelope, in comparison to the antiviral cytokine IFN-β. Mass spectrometry data indicated that nuclear envelope proteins were only modestly affected by either GS-CA1 treatment or HIV-1 infection, but combining the two had a more significant impact, altering the levels of many proteins including proteasomal components. GS-CA1 induced a small but clear accumulation of HIV-1 capsid cores at nuclear pores, as seen by microscopy, whereas IFN-β caused a strong accumulation of HIV-1 cores at the nuclear envelope but not specifically at nuclear pores. These observations are consistent with GS-CA1 inhibiting the nuclear translocation of HIV-1 capsid cores through nuclear pores.

Molecular characterization and lineage analysis of canine astrovirus strains from dogs with gastrointestinal disease in Ecuador based on ORF-2 gene analysis

Canine Astrovirus (CaAstV) part of the Astroviridae family and genus Mamastrovirus, is a linear RNA virus with a genome of approximately 6.6 kb with three open reading frames (ORF): ORF1a and ORF1b, which code for the most conserved non-structural proteins, and ORF2, which code for the capsid protein, the most variable region of the genome. This pathogen has been linked to gastrointestinal infections, primarily causing symptoms such as vomiting, diarrhea, lethargy and severe dehydration, mainly in co-infection with other enteric viruses. In the present study, the presence of CaAstV was identified in dogs with gastrointestinal disease in Ecuador using RT-qPCR with hydrolysis probes, these samples have also tested positive for canine parvovirus type 2 (CPV-2) and canine coronavirus (CCoV). Positive samples were used for end-point RT-PCR amplification and sequencing of ORF-2 using Sanger technology. The sequences were subjected to phylogenetic analysis to determine lineages and possible recombination events. Of the 502 samples tested, 336 were found to be positive for CaAstV, 49.4% in co-infection with CPV-2, 1% in co-infection with CCoV, and 4% in simultaneous infection with all three viruses. The presence of 4 of the 5 previously reported CaAstV lineages were identified, and three possible recombinant strains were identified. Given the high frequency of CaAstV infections in dogs with gastroenteritis and its high genetic variability, it emphasizes the need to implement routine diagnostic measures that include this pathogen as one of the main causes of the disease and a risk agent in case of multiple infections.

Simplified MS2 phage-like particle production including a novel maturation protein internal fusion affinity tag

Phage-like particles (PLPs) are fabricated self-assembling nanoparticles derived from the structural elements of bacteriophages. These particles have biotechnological utility because of the ability to easily modify surface chemistry and compartmentalize nucleic acids or other materials. A consequential implementation of PLPs in diagnostics is as process controls in nucleic acid amplification tests, where control RNAs are packaged within the protein capsid and protected from degradation by RNases in the sample matrix. Key developments in PLP controls have enhanced the packing efficiency of RNAs into particles, reduced the complexity of their plasmid expression systems, and shifted purification from ultracentrifugation to affinity chromatography, producing progressively greater yields with higher purity. Expanding on prior improvements, this study establishes a revised set of plasmid vectors for Emesvirus zinderi (MS2) derived PLPs that streamline vector manipulations for rapid prototyping of new particles, provide validation of an alternative affinity tag for purification, and contributes a high-throughput low-volume spin column purification strategy. These advancements are combined with a novel internal fusion site in MS2 maturation protein A, a passive element of the MS2 capsid in prior PLP designs, that is capable of displaying polypeptides on the particles’ surface. The functionality of the chimeric maturation protein’s surface display is verified with an affinity tag fusion and subsequent purification. This advancement increases the number of available peptide display sites for the MS2 PLP platform with wide-ranging implications for future applications.

Tetravalent Virus-like Particles Engineered To Display Envelope Domain IIIs of Four Dengue Serotypes in Silkworm as Vaccine Candidates.

Dengue virus (DENV) causes dengue fever, the leading mosquito-borne viral disease affecting millions globally. Licensed vaccines have their restrictions, and the development of vaccines is in progress to overcome the limitations. In this study, we expressed two types of virus-like particles (VLPs) and four DENV serotype antigens, 1EDIII-4EDIII (tetEDIII), in silkworm larvae and engineered them into tetravalent VLPs (tetVLPs) displaying tetEDIII. Canine parvovirus-like particles (CPV-LPs) were self-assembled in vivo from viral protein VP2 of CPV (CPV-VP2) as heterologous VLPs; dengue virus capsid-like particles (DENV C-LPs) from capsid protein of DENV serotype 2 (DENV-C2) as homologous VLPs. The tetEDIII was displayed on the surface of CPV-LPs and DENV C-LPs through in vitro SpyTag/SpyCatcher (SpT/SpC) covalent ligation. The EDIII display of CPV-LP is better than that of DENV C-LP. Both tetEDIII-displaying tetravalent CPV-LPs (tetCPV-LPs) and tetravalent DENV C-LPs (tetDENV C-LPs) elicited neutralizing antibodies in BALB/c mice assayed through the single-round infectious particles (SRIP) method. The immunogenicity of tetDENV C-LPs for anti-IgG EDIIIs was higher than that of tetCPV-LPs for serotypes 1 and 3. The neutralization activity of tetDENV C-LPs was higher than that of tetCPV-LPs for D1-SRIP, while tetCPV-LPs were higher than that of tetDENV C-LPs for D2- and D4-SRIP. These results suggest that homologous tetDENV C-LPs and heterologous tetCPV-LPs can be suitable vaccine candidates for further evaluation. This result is the first report to display a tetEDIII on the surface of the DENV C-LPs and the CPV-LPs by in vitro bioconjugation.

Bacteriophages RCF and 1-6bf can control the growth of avian pathogenic Escherichia coli.

Escherichia coli (E. coli) is a widely distributed pathogenic bacterium that poses a substantial hazard to poultry, leading to the development of a severe systemic disease known as colibacillosis. Colibacillosis is involved in multimillion-dollar losses to the poultry industry each year worldwide. Avian pathogenic E. coli is also involved in causing meningitis and urinary tract infections in humans. This creates a significant risk to public health. The increasing incidence of multidrug-resistant illnesses and the failure of antibiotics in human and veterinary medicine have led to a pressing demand for alternate approaches. This study investigates the possibility of bacteriophages as an acceptable substitute for antibiotics in managing E. coli infections in poultry. In the current study, two novel phages targeting E. coli (EP1) strain were isolated from sewage water and thoroughly characterized in vitro. Transmission electron microscopy reveals that Rcf and 1-6bf belong to the "Podoviridae" and "Caudovirales". Rcf has an icosahedral capsid of 18 nm with a tail size of 5 nm, while 1-6bf has an elongated head capsid of 93 nm and a short non-contractile tail of 8 nm with tail fibers for attachment. RCF and 1-6bf have genome sizes of 38 kb and 77 kb, with GC content of 50.98 % and 42.1 % respectively. Notably, phage 1-6bf displayed remarkable tolerance to high temperatures, retaining lytic activity at 95°C. Both phages effectively controlled host bacterial growth for up to 12 h post-infection. Rcf and 1-6bf produce clear plaques with a latent period of 10 min and 5 min with a burst size of 85 and 220 PFU/cell respectively.

Prognostic Significance of the Detection of Human Papilloma Virus L1 Protein in Smears of Cervical Intraepithelial Neoplasia Grade 3 in Pregnant Women.

Cervical intraepithelial neoplasia (CIN) III/high-grade squamous lesions (HSIL) remains a significant challenge during pregnancy. Current data on the course of disease are contradictory, with cases of progression to cervical cancer (CC) during pregnancy being observed. Evidence suggests that the expression of L1 capsid protein is associated with a favorable prognosis in non-pregnant women. The aim of this study was to evaluate L1 expression in pregnant women with CIN III/HSIL. Between 2008 and 2021, the conventional PAP-smears from pregnant women were retrospectively analyzed for the expression of L1. Only women with histologically confirmed CIN III/HSIL during pregnancy were included. A total of 161 women were included in this study; among them, 32 women (19.9%) had regressive disease postpartum. The majority of women (n=123, 76.4%) had persistent disease. In six cases, invasive CC was histologically proven postpartum (3.7%). In 113 women (70.2%) the PAP-Smears were L1- and 29.2% (n=48) of women were L1+. The rates of regression for L1+ were higher than for L1-, 25% vs. 17.7%, respectively. Rates for persistence were similar, at 75% and 78%, respectively. All cases of progression to CC were L1 negative during pregnancy. In pregnant women, the rates of regression were higher in L1+ CIN III/HSIL cases. All women who progressed to CC were L1-. Therefore, detecting L1 expression could serve as a valuable test to rule out progression to CC in pregnant women. This approach may provide reassurance to women, allowing them to continue their pregnancies with reduced fear and anxiety about CIN III/HSIL during pregnancy.

Truncation-Enhanced Aptamer Binding Affinity and Its Potential as a Sensor for Macrobrachium rosenbergii Nodavirus Detection.

White tail disease in Macrobrachium rosenbergii is caused by M. rosenbergii nodavirus (MrNV) infection, resulting in up to 100% mortality in larvae and post-larvae stages, severely impacting aquaculture production. Existing genome-based detection methods for MrNV are costly and time-consuming, highlighting the need for rapid and cost-effective diagnostic tests. This study evaluated the effects of truncating selected aptamer on its binding affinity to the MrNV capsid protein. The previously isolated and identified aptamer through magnetic-capture SELEX and Next Generation Sequencing demonstrated high binding affinity to the MrNV capsid protein. Truncation at the primer overhang was found to improve binding affinity, reducing the dissociation constant from 347 nM to 30.1 nM. The calculated limit of detection for the truncated aptamer decreased from 5.64 nM to 1.7 nM, while the limit of quantification decreased from 17.1 nM to 5.16 nM. These reductions indicate that the truncated aptamer has higher sensitivity compared to the full-length aptamer. In tests with MrNV-infected M. rosenbergii samples, both the enzyme-linked aptamer assay and the gold nanoparticle aptasensor assay showed consistent results when 0.5 μg of total protein lysate was used. This indicates that the prawn protein concentration interferes with the detection of the viral protein. These findings suggest the potential application of the truncated aptamer as a sensor in the development of a practical aptamer-based diagnostic kit. For instance, an aptamer-based lateral flow assay test kit could provide a user-friendly, cost-effective solution that eliminates the need for sophisticated instrumentation for diagnosis or data interpretation, making it ideal for detecting MrNV infection in M. rosenbergii aquaculture.

575. Safety and Immunogenicity of mRNA-1403, a Multivalent Norovirus mRNA Vaccine, in Healthy Adults: Interim Results of a Phase 1/2, Randomized, Observer-Blind, Placebo-Controlled, Dose-Ranging Trial

Abstract Background Norovirus (NoV) is a leading cause of acute gastroenteritis worldwide and is associated with significant disease burden across all ages. Currently, there is no licensed NoV vaccine. NoV’s high genetic diversity coupled with genotype-specific immune responses indicate that a multivalent approach may be required to generate a broadly protective vaccine. A trivalent mRNA-based NoV vaccine candidate (mRNA-1403), consisting of mRNAs encoding for the major capsid protein (VP1) of 3 globally prevalent NoV genotypes (GII.4, GI.3 and GII.3) formulated in a lipid nanoparticle, is in development. Methods In this ongoing Phase 1/2, randomized, placebo-controlled, observer-blind, dose-ranging study, safety and immunogenicity of mRNA-1403 are being evaluated in healthy adults 18 to 80 years of age (NCT05992935). In Phase 1, healthy younger (18-49 years) and older (60-80 years) adults were randomized to receive 1 of 4 dose levels of mRNA-1403 given as 2 doses, 1 dose level of mRNA-1403 given as 1 dose, or Placebo. In Phase 2, 3 dose levels of mRNA-1403 given as a single-dose schedule were selected for further evaluation in a larger sample size of healthy adults 18-80 years of age. Results In Phase 1, 335 healthy adults received 1 or 2 doses of mRNA-1403 or Placebo. mRNA-1403 was well-tolerated across all dose levels with no safety concerns identified through 8 months of follow-up from dose 1. A single injection of mRNA-1403 elicited robust levels of serum histo-blood group antigen (HBGA)-blocking antibodies and binding antibodies against vaccine-matched NoV genotypes at 1 month post-dose across all dose levels tested. In Phase 2, 616 healthy adults received a single injection of 1 of 3 selected dose levels of mRNA-1403 or Placebo. Data in this larger sample size of healthy younger and older adults through 1 month post-dose confirmed that a single-dose schedule of all 3 dose levels of mRNA-1403 was well-tolerated, safe and immunogenic. Conclusion Available data from this ongoing Phase 1/2 study show that mRNA-1403 was generally safe and well-tolerated, and induced antigen-specific immune responses at all dose levels in both younger and older healthy adults. These results informed dose selection for a Phase 3 efficacy study in adults. Disclosures Till Schoofs, MD, Moderna, Inc: Employee|Moderna, Inc: Stocks/Bonds (Public Company) Brooke A. Bollman, PhD, Moderna, Inc: Employee|Moderna, Inc: Stocks/Bonds (Public Company) Kevin Mancini, MS, Moderna, Inc: Employee|Moderna, Inc: Stocks/Bonds (Public Company) Wenlin Yuan, MS, PhD, Moderna, Inc: Employee|Moderna, Inc: Stocks/Bonds (Public Company) Lauren Bailey, PhD, Moderna, Inc: Employee|Moderna, Inc: Stocks/Bonds (Public Company) Tin Bartholomew, MA, Moderna, Inc: Employee|Moderna, Inc: Stocks/Bonds (Public Company) Esther Levine, MD, Moderna, Inc: Employee|Moderna, Inc: Stocks/Bonds (Public Company) Katherine B. Carlson, PhD, MPH, Moderna, Inc: Employee|Moderna, Inc: Stocks/Bonds (Public Company) Alexander Rumyantsev, MD, PhD, Moderna, Inc: Employee|Moderna, Inc: Stocks/Bonds (Public Company) Meklit Workneh, MD, MPH, Moderna, Inc: Employee|Moderna, Inc: Stocks/Bonds (Public Company) Doran Fink, MD, PhD, Moderna, Inc: Employee|Moderna, Inc: Stocks/Bonds (Public Company) Jaap Oostendorp, PhD, Moderna, Inc: Employee|Moderna, Inc: Stocks/Bonds (Public Company)

P-606. A Single-round Infectious Virus with Codon-deoptimized Genome: A Powerful Tool for Regulating Pathogenicity of WT/Live-Attenuated Viruses

Abstract Background Coxsackievirus B3 (CVB3) is a ssRNA(+) virus of the Picornaviridae family associated with myocarditis and type I diabetes. Prevention of mother-to-child transmission is important, especially since the outcomes of neonatal infection can be fatal. However, vaccines have not yet been developed. In this study, we revealed that CVB3 codon-deoptimized in the nonstructural proteins’ region (CVB3cd) can be used as live-attenuated vaccines (LAVs). Furthermore, we showed that in co-infection experiments, a CVB3cd-based single-round infectious virus induces interference, significantly reducing the pathogenicity of viruses. Methods CVB3cd were designed to be enriched in CpG-containing rare codons in the 3CD region of the viral genome. Viral proliferation was evaluated based on plaque size and growth curve; and their pathogenicity in mice was determined by weight loss and survival after infection. Defective viral particles (DVPs) were generated by packaging WT or CVB3cd replicons with a trans-expressed viral capsid protein. To evaluate the effect of viral interference with DVPs, WT or CVB3cd were co-administered with the DVPs, and their pathogenicity in mice was monitored. Results A decrease in viral replication was observed in correlation with the number of deoptimized codons, in addition to an increase in the survival rate of animals infected with the codon-deoptimized viruses. Among vaccination methods, intramuscular injection of the attenuated virus emerged as the least pathogenic option, effectively inducing neutralizing antibodies and providing protection against challenge by the WT virus. In addition, simultaneous infection of CVB3 with DVPs revealed that CVB3cd replicon-based DVP (DVPcd), reduced the pathogenicity of CVB3 in mice and improved the safety of LAVs without compromising immunogenicity. Conclusion Our data suggest that viral pathogenicity can be modulated by a combination of codon-deoptimization, route of administration, and viral interference. This approach could be a powerful strategy to develop effective and safe LAVs in a short period of time. Moreover, DVPcd could be used as therapeutic tools against viruses due to their efficient virus-interfering effects. Disclosures Anju Miyamori, Master of Medicine, The research foundation for microbial diseases of Osaka university: Grant/Research Support Hirotaka Ebina, Doctor of Medicine, The research foundation for microbial diseases of Osaka university: Grant/Research Support

C-28 linker length modulates the activity of second-generation HIV-1 maturation inhibitors

Maturation inhibitors (MIs) block HIV-1 maturation by preventing the cleavage of the capsid protein and spacer peptide 1 (CA-SP1). Bevirimat (BVM), a first-in-class MI, displayed sub-optimal efficacy in clinical trials due to presence of SP1:V7A polymorphism in the Gag protein.This polymorphism is inherently present in HIV-1 subtype C and conferred resistance to BVM. Second generation BVM analogs with modifications at C-28 position gained potent activity against HIV-1 subtype C. In this study, we have evaluated the efficacy of nine second-generation MIs (BVM analogs) with varying length of C28 carbon linker against HIV-1 subtype B and C. Increasing the length of carbon linker decreased their activity against both subtypes. These MIs were also active against integrase inhibitor-resistant viruses and protease inhibitor-resistant viruses. Our data has provided vital information that in addition to the nature of the functional group at C28 position of the MI, the length of linker contributes significantly to its activity. The shorter the length, the better the activity of MIs. These results will further pave way for design of novel and potent MIs.

The primary mechanism for highly potent inhibition of HIV-1 maturation by lenacapavir.

Lenacapavir (LEN) is a highly potent, long-acting antiretroviral medication for treating people infected with muti-drug-resistant HIV-1 phenotypes. The inhibitor targets multifaceted functions of the viral capsid protein (CA) during HIV-1 replication. Previous studies have mainly focused on elucidating LEN's mode of action during viral ingress. Additionally, the inhibitor has been shown to interfere with mature capsid assembly during viral egress. However, the mechanism for how LEN affects HIV-1 maturation is unknown. Here, we show that pharmacologically relevant LEN concentrations do not impair proteolytic processing of Gag in virions. Instead, we have elucidated the primary mechanism for highly potent inhibition of HIV-1 maturation by sub-stoichiometric LEN:CA ratios. The inhibitor exerts opposing effects on formation of CA pentamers versus hexamers, the key capsomere intermediates in mature capsid assembly. LEN impairs formation of pentamers, whereas it induces assembly of hexameric lattices by imposing an opened CA conformation and stabilizing a dimeric form of CA. Consequently, LEN treatment results in morphologically atypical virus particles containing malformed, hyper-stable CA assemblies, which fail to infect target cells. Moreover, we have uncovered an inverse correlation between inhibitor potency and CA levels in cell culture assays, which accounts for LEN's ability to potently (with picomolar EC50 values) inhibit HIV-1 maturation at clinically relevant drug concentrations.

Adenoviral Therapy for Cervical Cancer: From Targeted Modification to Immunotherapy.

Cervical cancer is a significant global health threat, ranking as the fourth most common malignancy among women and resulting in over 300,000 deaths annually. Although screening and vaccination initiatives have led to a decline in incidence rates, treatment options for advanced or recurrent cervical cancer remain inadequate, often proving ineffective and costly. In this context, adenoviral therapy has emerged as a promising strategy to enhance therapeutic outcomes. Adenoviruses are non-enveloped viruses that can efficiently infect a wide range of cells, including tumor cells, while exhibiting a favorable safety profile, making them suitable candidates for clinical applications. Adenoviral vectors possess the unique ability to package large segments of therapeutic genes, allowing for diverse treatment approaches, including oncolytic virotherapy, which selectively targets and destroys tumor cells while stimulating robust immune responses. By engineering adenoviruses to express tumor suppressor genes such as p53, researchers can restore critical apoptotic pathways often disrupted in cervical cancer. Furthermore, genetic modifications to capsid proteins can enhance the targeting of tumor cells and reduce the immunogenicity associated with these viral vectors. Additionally, adenoviral vectors can serve as delivery systems for therapeutic vaccines against HPV oncogenes E6 and E7, promoting effective immune responses and potentially preventing disease progression. The combination of adenoviral therapy with immune checkpoint inhibitors offers a novel approach to overcoming the immunosuppressive tumor microenvironment, enhancing overall antitumor immunity. Overall, this review highlights the significant advancements in adenoviral therapy for cervical cancer, emphasizing the need for further research to optimize these strategies and translate preclinical successes into effective clinical applications. By harnessing the full potential of adenoviral vectors, we can improve treatment options for patients who have cervical cancer, paving the way for more personalized and effective therapeutic interventions.

Oxidative Stress Enhances Rubella Virus Infection in Immortalized Human First-Trimester Trophoblasts

Rubella infection (RuV) during early pregnancy is a known cause of congenital rubella syndrome (CRS). However, the mechanisms by which the virus crosses the placenta and infects the fetus are not fully understood. It has been known that various kinds of cell stresses can occur during the placenta formation. Previously, we demonstrated that low-glucose-induced endoplasmic reticulum stress could drastically enhance RuV infection in immortalized human first-trimester trophoblast cells. In this study, we investigated the roles of oxidative stress in RuV infection in these cells. Oxidative stress was induced in Swan.71 cells by culturing them in medium containing hydrogen peroxide (H2O2) in various concentrations and durations (50 µM or 100 µM for 24 h, or 150 µM for 1 h). RuV infection with a clinical strain was performed 24 h post-treatment, and capsid proteins were visualized at 24 and 48 h post-infection (hpi) using flow cytometry (FCM) and fluorescence microscopy (IF), respectively. The findings demonstrated that oxidative stress significantly enhanced RuV infection, as evidenced by FCM analysis, showing a twofold increase in infection rate, and confirmed by IF assay. Additionally, significantly increased intracellular viral replication was observed at 3 dpi. These findings suggest that oxidative stress during early pregnancy may promote the maternal-to-fetal transmission of rubella, contributing to the development of CRS.

Structural insights into HIV-2 CA lattice formation and FG-pocket binding revealed by single-particle cryo-EM.

One of the striking features of human immunodeficiency virus (HIV) is the capsid, a fullerene cone comprised of pleomorphic capsid protein (CA) that shields the viral genome and recruits cofactors. Despite significant advances in understanding the mechanisms of HIV-1 CA assembly and host factor interactions, HIV-2 CA assembly remains poorly understood. By templating the assembly of HIV-2 CA on functionalized liposomes, we report high-resolution structures of the HIV-2 CA lattice, including both CA hexamers and pentamers, alone and with peptides of host phenylalanine-glycine (FG)-motif proteins Nup153 and CPSF6. While the overall fold and mode of FG-peptide binding is conserved with HIV-1, this study reveals distinctive features of the HIV-2 CA lattice, including differing structural character at regions of host factor interactions and divergence in the mechanism of formation of CA hexamers and pentamers. This study extends our understanding of HIV capsids and highlights an approach facilitating the study of lentiviral capsid biology.

Interplay between the cyclophilin homology domain of RANBP2 and MX2 regulates HIV-1 capsid dependencies on nucleoporins.

Human immunodeficiency virus 1 (HIV-1) entry into the nucleus is an essential step in viral replication that involves complex interactions between the viral capsid (CA) and multiple cellular proteins, including nucleoporins (Nups) such as RANBP2. Nups also mediate the function of the antiviral protein myxovirus resistance 2 (MX2); however, determining the precise role of Nups in HIV infection has proved challenging due to the complex nature of the nuclear pore complex (NPC) and significant pleiotropic effects elicited by Nup depletion. We have used precise gene editing to assess the role of the cyclophilin domain of RANBP2 in HIV-1 infection and MX2 activity. We find that this domain affects viral infection, nucleoporin requirements, MX2 sensitivity, and integration targeting in a CA-specific manner, providing detailed insights into how RANBP2 contributes to HIV-1 infection.

DDX21 Promotes PCV3 Replication by Binding to Cap Protein and Inhibiting Interferon Responses

Porcine circovirus type 3 (PCV3) is an emerging pathogen that causes porcine dermatitis, nephropathy syndrome-like symptoms, multisystemic inflammation, and reproductive failure. The PCV3 capsid (Cap) protein interacts with DDX21, which functions mainly through controlling interferon (IFN)-β levels. However, how the interaction between DDX21 and PCV3 Cap regulates viral replication remains unknown. In the present study, upon shRNA-mediated DDX21 depletion in PK-15 cells, we observed impaired PCV3 proliferation via a lentivirus-delivered system, as indicated by reduced replicase (Rep) protein levels and viral titers. Furthermore, DDX21 negatively regulated IFN-β and interferon-stimulated gene (ISG) levels, promoting PCV3 replication. Mechanistically, PCV3 Cap co-localized and interacted with DDX21, and the nuclear localization signal (NLS) of PCV3 Cap and 763GSRSNRFQNK772 at the C-terminal domain (CTD) of DDX21 were indispensable to the interaction. Moreover, PCV3 infection prevented the repression of DDX21 to facilitate its pro-viral activity. Taken together, these results show that DDX21 promotes PCV3 replication by binding to the PCV3 Cap protein and prohibiting IFN-β response, which provides important insight on the prevention and control of PCV3 infection.

Novel cycloviruses identified by mining human blood metagenomic data show close relationship to those from animals

The family Circoviridae includes the genera Circovirus and Cyclovirus. Cycloviruses have been found in serum samples from chronic HBV, HCV, or HIV-infected individuals as well as asymptomatic blood donors. However, research on cycloviruses is relatively limited. We used viral metagenomics to mine, analyze, and visualize the human blood virome, successfully identifying three new genomes, each encoding Rep and Capsid proteins. These proteins are crucial for viral replication and host-cell interaction: the Rep protein is involved in initiating viral genome replication, while the Capsid protein plays a key role in the assembly of new virions and the virus's ability to interact with host immune systems. Distance matrix and phylogenetic analyses show that these cycloviruses share high sequence similarity with viruses found in both humans and animals across different regions of Africa. This finding not only confirms the presence of previously uncharacterized cycloviruses in human blood, but also provides insight into their potential role in host transmission and their ecological significance. Further research is needed to explore the functional roles of these cycloviruses in viral pathogenesis, particularly how they may influence host immunity and contribute to chronic infections. Additionally, studies investigating the host range and mechanisms of cross-species transmission will be essential to understanding the broader implications of cycloviruses in human and animal health.

Disassembly of Virus-Like Particles and the Stabilizing Role of the Nucleic Acid Cargo.

In many simple viruses and virus-like particles, the protein capsid self-assembles around a nucleic-acid genome. Although the assembly process has been studied in detail, relatively little is known about how the capsid disassembles, a potentially important step for infection (in viruses) or cargo delivery (in virus-like particles). We investigate capsid disassembly using a coarse-grained molecular dynamics model of a T = 1 dodecahedral capsid and an RNA-like polymer. We alter the interactions between the subunits of the capsid as well as the ionic strength of the solution to induce partial or complete disassembly of self-assembled particles. We find that disassembly follows nucleation-and-growth kinetics, where the nucleation barrier is related to the interaction strengths as well as to the conformation of the polymer. In particular, we find that polymer segments that interact with adjacent subunits reinforce the subunit-subunit contacts. These results have implications for the design of virus-like particles for applications such as drug delivery. A cargo designed with reinforcement in mind might be used to control the stability of such particles and mediate disassembly.