The Effect of Tryptophan-to-Tyrosine Mutation at Position 61 of the Nonstructural Protein of Severe Fever with Thrombocytopenia Syndrome Virus on Viral Replication through Autophagosome Modulation.

In our prior investigations, we elucidated the role of the tryptophan-to-tyrosine substitution at the 61st position in the nonstructural protein NSsW61Y in diminishing the interaction between nonstructural proteins (NSs) and nucleoprotein (NP), impeding viral replication. In this study, we focused on the involvement of NSs in replication via the modulation of autophagosomes. Initially, we examined the impact of NP expression levels, a marker for replication, upon the infection of HeLa cells with severe fever thrombocytopenia syndrome virus (SFTSV), with or without the inhibition of NP binding. Western blot analysis revealed a reduction in NP levels in NSsW61Y-expressing conditions. Furthermore, the expression levels of the canonical autophagosome markers p62 and LC3 decreased in HeLa cells expressing NSsW61Y, revealing the involvement of individual viral proteins on autophagy. Subsequent experiments confirmed that NSsW61Y perturbs autophagy flux, as evidenced by reduced levels of LC3B and p62 upon treatment with chloroquine, an inhibitor of autophagosome-lysosome fusion. LysoTracker staining demonstrated a decrease in lysosomes in cells expressing the NS mutant compared to those expressing wild-type NS. We further explored the mTOR-associated regulatory pathway, a key regulator affected by NS mutant expression. The observed inhibition of replication could be linked to conformational changes in the NSs, impairing their binding to NP and altering mTOR regulation, a crucial upstream signaling component in autophagy. These findings illuminate the intricate interplay between NSsW61Y and the suppression of host autophagy machinery, which is crucial for the generation of autophagosomes to facilitate viral replication.

An mRNA-LNP-based Lassa virus vaccine induces protective immunity in mice.

The mammarenavirus Lassa virus (LASV) causes the life-threatening hemorrhagic fever disease, Lassa fever. The lack of licensed medical countermeasures against LASV underscores the urgent need for the development of novel LASV vaccines, which has been hampered by the requirement for a biosafety level 4 facility to handle live LASV. Here, we investigated the efficacy of mRNA-lipid nanoparticle (mRNA-LNP)-based vaccines expressing the LASV glycoprotein precursor (LASgpc) or nucleoprotein (LCMnp) of the prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), in mice. Two doses of LASgpc- or LCMnp-mRNA-LNP administered intravenously (i.v.) protected C57BL/6 mice from a lethal challenge with a recombinant (r) LCMV expressing a modified LASgpc (rLCMV/LASgpc2m) inoculated intracranially. Intramuscular (i.m.) immunization with two doses of LASgpc- or LCMnp-mRNA-LNP significantly reduced the viral load in C57BL/6 mice inoculated i.v. with rLCMV/LASgpc2m. High levels of viremia and lethality were observed in CBA mice inoculated i.v. with rLCMV/LASgpc2m, which were abrogated by i.m. immunization with two doses of LASgpc-mRNA-LNP. The protective efficacy of two i.m. doses of LCMnp-mRNA-LNP was confirmed in a lethal hemorrhagic disease model of FVB mice i.v. inoculated with wild-type rLCMV. In all conditions tested, negligible and high levels of LASgpc- and LCMnp-specific antibodies were detected in mRNA-LNP-immunized mice, respectively, but robust LASgpc- and LCMnp-specific CD8+ T cell responses were induced. Accordingly, plasma from LASgpc-mRNA-LNP-immunized mice did not exhibit neutralizing activity. Our findings and surrogate mouse models of LASV infection, which can be studied at a reduced biocontainment level, provide a critical foundation for the rapid development of mRNA-LNP-based LASV vaccines.IMPORTANCELassa virus (LASV) is a highly pathogenic mammarenavirus responsible for several hundred thousand infections annually in West African countries, causing a high number of lethal Lassa fever (LF) cases. Despite its significant impact on human health, clinically approved, safe, and effective medical countermeasures against LF are not available. The requirement of a biosafety level 4 facility to handle live LASV has been one of the main obstacles to the research and development of LASV countermeasures. Here, we report that two doses of mRNA-lipid nanoparticle-based vaccines expressing the LASV glycoprotein precursor (LASgpc) or nucleoprotein (LCMnp) of lymphocytic choriomeningitis virus (LCMV), a mammarenavirus genetically closely related to LASV, conferred protection to recombinant LCMV-based surrogate mouse models of lethal LASV infection. Notably, robust LASgpc- and LCMnp-specific CD8+ T cell responses were detected in mRNA-LNP-immunized mice, whereas no virus-neutralizing activity was observed.

SFTSV nucleoprotein mediates DNA sensor cGAS degradation to suppress cGAS-dependent antiviral responses.

Cyclic GMP-AMP synthase (cGAS) is an important DNA pattern recognition receptor that senses double-stranded DNA derived from invading pathogens or self DNA in cytoplasm, leading to an antiviral interferon response. A tick-borne Bunyavirus, severe fever with thrombocytopenia syndrome virus (SFTSV), is an RNA virus that causes a severe emerging viral hemorrhagic fever in Asia with a high case fatality rate of up to 30%. However, it is unclear whether cGAS interacts with SFTSV infection. In this study, we found that SFTSV infection upregulated cGAS RNA transcription and protein expression, indicating that cGAS is an important innate immune response against SFTSV infection. The mechanism of cGAS recognizing SFTSV is by cGAS interacting with misplaced mitochondrial DNA in the cytoplasm. Depletion of mitochondrial DNA significantly inhibited cGAS activation under SFTSV infection. Strikingly, we found that SFTSV nucleoprotein (N) induced cGAS degradation in a dose-dependent manner. Mechanically, N interacted with the 161-382 domain of cGAS and linked the cGAS to LC3. The cGAS-N-LC3 trimer was targeted to N-induced autophagy, and the cGAS was degraded in autolysosome. Taken together, our study discovered a novel antagonistic mechanism of RNA viruses, SFTSV is able to suppress the cGAS-dependent antiviral innate immune responses through N-hijacking cGAS into N-induced autophagy. Our results indicated that SFTSV N is an important virulence factor of SFTSV in mediating host antiviral immune responses. IMPORTANCE: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne RNA virus that is widespread in East and Southeast Asian countries with a high fatality rate of up to 30%. Up to now, many cytoplasmic pattern recognition receptors, such as RIG-I, MDA5, and SAFA, have been reported to recognize SFTSV genomic RNA and trigger interferon-dependent antiviral responses. However, current knowledge is not clear whether SFTSV can be recognized by DNA sensor cyclic GMP-AMP synthase (cGAS). Our study demonstrated that cGAS could recognize SFTSV infection via ectopic mitochondrial DNA, and the activated cGAS-stimulator of interferon genes signaling pathway could significantly inhibit SFTSV replication. Importantly, we further uncovered a novel mechanism of SFTSV to inhibit innate immune responses by the degradation of cGAS. cGAS was degraded in N-induced autophagy. Collectively, this study illustrated a novel virulence factor of SFTSV to suppress innate immune responses through autophagy-dependent cGAS degradation.

Impact of Ebola virus nucleoprotein on VP40 virus-like particle production: a computational approach.

Ebola virus (EBOV) matrix protein VP40 can assemble and bud as virus-like particles (VLPs) when expressed alone in mammalian cells. Nucleoprotein (NP) could be recruited to VLPs as inclusion body (IB) when co-expressed, and increase VLP production. However, the mechanism behind it remains unclear. Here, we use a computational approach to study NP-VP40 interactions. Our simulations indicate that NP may enhance VLP production through stabilizing VP40 filaments and accelerating the VLP budding step. Further, both the relative timing and amount of NP expression compared to VP40 are important for the effective production of IB-containing VLPs. We predict that relative NP/VP40 expression ratio and time are important for efficient production of IB-containing VLPs. We conclude that disrupting the expression timing and amount of NP and VP40 could provide new avenues to treat EBOV infection. This work provides quantitative insights into EBOV proteins interactions and how virion generation and drug efficacy could be influenced.

Cross-protective efficacy and safety of an adenovirus-based universal influenza vaccine expressing nucleoprotein, hemagglutinin, and the ectodomain of matrix protein 2.

It is necessary to develop universal vaccines that act broadly and continuously to combat regular seasonal epidemics of influenza and rare pandemics. The aim of this study was to find the optimal dose regimen for the efficacy and safety of a mixture of previously developed recombinant adenovirus-based vaccines that expressed influenza nucleoprotein, hemagglutinin, and ectodomain of matrix protein 2 (rAd/NP and rAd/HA-M2e). The vaccine efficacy and safety were measured in the immunized mice with the mixture of rAd/NP and rAd/HA-M2e intranasally or intramuscularly. The minimum dose that would be efficacious in a single intranasal administration of the vaccine mixture and cross-protective efficacy against various influenza strains were examined. In addition, the immune responses that may affect the cross-protective efficacy were measured. We found that intranasal administration is an optimal route for 107 pfu of vaccine mixture, which is effective against pre-existing immunity against adenovirus. In a study to find the minimum dose with vaccine efficacy, the 106 pfu of vaccine mixture showed higher antibody titers to the nucleoprotein than did the same dose of rAd/NP alone in the serum of immunized mice. The 106 pfu of vaccine mixture overcame the morbidity and mortality of mice against the lethal dose of pH1N1, H3N2, and H5N1 influenza infections. No noticeable side effects were observed in single and repeated toxicity studies. We found that the mucosal administration of adenovirus-based universal influenza vaccine has both efficacy and safety, and can provide cross-protection against various influenza infections even at doses lower than those previously known to be effective.

The W195 Residue of the Newcastle Disease Virus V Protein Is Critical for Multiple Aspects of Viral Self-Regulation through Interactions between V and Nucleoproteins.

The transcription and replication of the Newcastle disease virus (NDV) strictly rely on the viral ribonucleoprotein (RNP) complex, which is composed of viral NP, P, L and RNA. However, it is not known whether other viral non-RNP proteins participate in this process for viral self-regulation. In this study, we used a minigenome (MG) system to identify the regulatory role of the viral non-RNP proteins V, M, W, F and HN. Among them, V significantly reduced MG-encoded reporter activity compared with the other proteins and inhibited the synthesis of viral mRNA and cRNA. Further, V interacted with NP. A mutation in residue W195 of V diminished V-NP interaction and inhibited inclusion body (IB) formation in NP-P-L-cotransfected cells. Furthermore, a reverse-genetics system for the highly virulent strain F48E9 was established. The mutant rF48E9-VW195R increased viral replication and apparently enhanced IB formation. In vivo experiments demonstrated that rF48E9-VW195R decreased virulence and retarded time of death. Overall, the results indicate that the V-NP interaction of the W195 mutant V decreased, which regulated viral RNA synthesis, IB formation, viral replication and pathogenicity. This study provides insight into the self-regulation of non-RNP proteins in paramyxoviruses.

Geographic Disparities in Domestic Pig Population Exposure to Ebola Viruses, Guinea, 2017-2019.

Although pigs are naturally susceptible to Reston virus and experimentally to Ebola virus (EBOV), their role in Orthoebolavirus ecology remains unknown. We tested 888 serum samples collected from pigs in Guinea during 2017-2019 (between the 2013-16 epidemic and its resurgence in 2021) by indirect ELISA against the EBOV nucleoprotein. We identified 2 hotspots of possible pig exposure by IgG titer levels: the northern coast had 48.7% of positive serum samples (37/76), and Forest Guinea, bordering Sierra Leone and Liberia, where the virus emerged and reemerged, had 50% of positive serum samples (98/196). The multitarget Luminex approach confirms ELISA results against Ebola nucleoprotein and highlights cross-reactivities to glycoprotein of EBOV, Reston virus, and Bundibugyo virus. Those results are consistent with previous observations of the circulation of Orthoebolavirus species in pig farming regions in Sierra Leone and Ghana, suggesting potential risk for Ebola virus disease in humans, especially in Forest Guinea.

Alternative translation contributes to the generation of a cytoplasmic subpopulation of the Junín virus nucleoprotein that inhibits caspase activation and innate immunity.

The highly pathogenic arenavirus, Junín virus (JUNV), expresses three truncated alternative isoforms of its nucleoprotein (NP), i.e., NP53kD, NP47kD, and NP40kD. While both NP47kD and NP40kD have been previously shown to be products of caspase cleavage, here, we show that expression of the third isoform NP53kD is due to alternative in-frame translation from M80. Based on this information, we were able to generate recombinant JUNVs lacking each of these isoforms. Infection with these mutants revealed that, while all three isoforms contribute to the efficient control of caspase activation, NP40kD plays the predominant role. In contrast to full-length NP (i.e., NP65kD), which is localized to inclusion bodies, where viral RNA synthesis takes place, the loss of portions of the N-terminal coiled-coil region in these isoforms leads to a diffuse cytoplasmic distribution and a loss of function in viral RNA synthesis. Nonetheless, NP53kD, NP47kD, and NP40kD all retain robust interferon antagonistic and 3'-5' exonuclease activities. We suggest that the altered localization of these NP isoforms allows them to be more efficiently targeted by activated caspases for cleavage as decoy substrates, and to be better positioned to degrade viral double-stranded (ds)RNA species that accumulate in the cytoplasm during virus infection and/or interact with cytosolic RNA sensors, thereby limiting dsRNA-mediated innate immune responses. Taken together, this work provides insight into the mechanism by which JUNV leverages apoptosis during infection to generate biologically distinct pools of NP and contributes to our understanding of the expression and biological relevance of alternative protein isoforms during virus infection.IMPORTANCEA limited coding capacity means that RNA viruses need strategies to diversify their proteome. The nucleoprotein (NP) of the highly pathogenic arenavirus Junín virus (JUNV) produces three N-terminally truncated isoforms: two (NP47kD and NP40kD) are known to be produced by caspase cleavage, while, here, we show that NP53kD is produced by alternative translation initiation. Recombinant JUNVs lacking individual NP isoforms revealed that all three isoforms contribute to inhibiting caspase activation during infection, but cleavage to generate NP40kD makes the biggest contribution. Importantly, all three isoforms retain their ability to digest double-stranded (ds)RNA and inhibit interferon promoter activation but have a diffuse cytoplasmic distribution. Given the cytoplasmic localization of both aberrant viral dsRNAs, as well as dsRNA sensors and many other cellular components of innate immune activation pathways, we suggest that the generation of NP isoforms not only contributes to evasion of apoptosis but also robust control of the antiviral response.

Chromatin accessibility complex subunit 1 enhances tumor growth by regulating the oncogenic transcription of YAP in breast and cervical cancer.

Background: As a component of chromatin remodeling complex, chromatin accessibility complex subunit 1 (CHRAC1) is critical in transcription and DNA replication. However, the significance of CHRAC1 in cancer progression has not been investigated extensively. This research aimed to determine the function of CHRAC1 in breast and cervical cancer and elucidate the molecular mechanism. Methods: The Bio-ID method was used to identify the interactome of transcriptional activator Yes-associated protein (YAP) and the binding between YAP and CHRAC1 was verified by immunofluorescence. CCK8, colony formation and subcutaneous xenograft assays were conducted to explore the function of CHRAC1 in cancer cell proliferation. RNA-seq analysis and RT-PCR were used to analyze the transcription program change after CHRAC1 ablation. The diagnostic value of CHRAC1 was analyzed by TCGA database and further validated by immunohistochemistry staining. Results: In the current study, we found that the chromatin remodeler CHRAC1 was a potential YAP interactor. CHRAC1 depletion suppressed breast and cervical cancer cell proliferation and tumor growth. The potential mechanism may be that CHRAC1 interacts with YAP to facilitate oncogenic transcription of YAP target genes in Hippo pathway, thereby promoting tumorigenesis. CHRAC1 was elevated in cervical and breast cancer biopsies and the upregulation correlated with shorter survival, poor pathological stages and metastasis of cancer patients. Moreover, CHRAC1 expression was statistically associated with YAP in breast and cervical cancer biopsies. Conclusions: These findings highlight that CHRAC1 contributes to cancer progression through regulating the oncogenic transcription of YAP, which makes it a potential therapeutic target for cancer treatment.

Analysis of highly frequent point mutations in glycoprotein C, glycoprotein N, and nucleoprotein of CCHFV.

Crimean-Congo hemorrhagic fever virus (CCHFV) is classified among top 10 priority pathogens by World Health Organization. CCHFV belongs to Bunyaviridae family and negative sense ssRNA genome composed of three RNA segments: L, M, and S. RNA viruses show higher mutation rate as compared to DNA viruses. To gain deeper understanding of impact of point mutations in CCHFV M and S segment, mutation profiling, homology modeling, and molecular dynamic (MD) simulation were performed. Structural glycoproteins (glycoprotein C [Gc] and glycoprotein N [Gn]) of CCHFV are important for host-virus interaction and genome packaging, whereas CCHFV nucleoprotein (NP) is crucial for viral replication. Hence, current study is focused on evaluation of eight mutations in structural glycoproteins (Gc: 7 and Gn: 1) of M segment and seven mutations in NP of S segment. All these mutations were highly frequent, with mutation frequency between 0.81 and 1.0 and found to be persistent in the recent strains of CCHFV. Solubility analysis predicted that selected point mutations reduce solubility of Gc protein and increase solubility of Gn and NP proteins. MD simulation study deciphered that A1046V and G1158E in Gc protein, I778T in Gn protein, and H195R in NP protein displayed large deviation and fluctuation, and affected intramolecular interactions. In conclusion, we observed that point mutations could impact structure, stability, and host-virus interaction of protein, and might lead to evolution of new strains for better survival and drug resistance.

Tetrameric UvrD Helicase Is Located at the E. Coli Replisome due to Frequent Replication Blocks.

DNA replication in all organisms must overcome nucleoprotein blocks to complete genome duplication. Accessory replicative helicases in Escherichia coli, Rep and UvrD, help remove these blocks and aid the re-initiation of replication. Mechanistic details of Rep function have emerged from recent live cell studies; however, the division of UvrD functions between its activities in DNA repair and role as an accessory helicase remain unclear in live cells. By integrating super-resolved single-molecule fluorescence microscopy with biochemical analysis, we find that UvrD self-associates into tetrameric assemblies and, unlike Rep, is not recruited to a specific replisome protein despite being found at approximately 80% of replication forks. Instead, its colocation with forks is likely due to the very high frequency of replication blocks composed of DNA-bound proteins, including RNA polymerase and factors involved in repairing DNA damage. Deleting rep and DNA repair factor genes mutS and uvrA, and inhibiting transcription through RNA polymerase mutation and antibiotic inhibition, indicates that the level of UvrD at the fork is dependent on UvrD's function. Our findings show that UvrD is recruited to sites of nucleoprotein blocks via different mechanisms to Rep and plays a multi-faceted role in ensuring successful DNA replication.

Bovine nucleoprotein transitions genes and protein abundance as valuable markers of sperm motility and the correlation with fertility.

Bovine nucleoprotein transitions (TNPs), specifically TNP1 and TNP2, are essential molecules in sperm nucleus rich in arginine and lysine. These molecules act in the phase between histone expulsion and before incorporation of protamine in the spermatid nucleus. Therefore, this study aimed to analyze genes and protein abundance of TNP1 and TNP2 in sperm to determine the potential as motility markers and correlation with fertility in the field. An objective evaluation method, CASA-Sperm Vision, was used to separate 22 bulls into two groups (mg-A and mg-B) based on their increasing motility. Sperm quality parameters were also examined including velocity, mitochondrial membrane potential (MMP) by the JC-1 method, head defects using William staining, and DNA fragmentation by Halomax. TNPs genes abundance was performed using the RT-qPCR method, and the protein abundance was examined with the EIA approach. The fertility rate was also analyzed based on the conception rate generated from each bull in the field, with the data obtained from iSIKHNAS. The results showed that TNPs genes and protein abundance were significantly higher (P < 0.05) in mg-A compared to mg-B, followed by various sperm quality parameters and fertility rates (P < 0.05). Positive correlations were found in TNPs genes and protein abundance with motility, velocity, MMP, and fertility (P < 0.01). Meanwhile, a negative correlation (P < 0.01) was found between head defects and DNA fragmentation. These results showed the potential of TNPs as sperm motility markers and bull fertility.

ICTV Virus Taxonomy Profile: Discoviridae 2023.

Discoviridae is a family of negative-sense RNA viruses with genomes of 6.2-9.7 kb that have been associated with fungi and stramenopiles. The discovirid genome consists of three monocistronic RNA segments with open reading frames (ORFs) that encode a nucleoprotein (NP), a nonstructural protein (Ns), and a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Discoviridae, which is available at ictv.global/report/discoviridae.

ICTV Virus Taxonomy Profile: Leishbuviridae 2023.

Leishbuviridae is a family of negative-sense RNA viruses with genomes of about 8.0 kb that have been found in protists. The leishbuvirid genome consists of three monocistronic RNA segments with open reading frames (ORFs) that encode a nucleoprotein (NP), a glycoprotein (GP), and a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Leishbuviridae, which is available at ictv.global/report/leishbuviridae.

ICTV Virus Taxonomy Profile: Jingchuvirales 2023.

Jingchuvirales is an order of negative-sense RNA viruses with genomes of 9.1-15.3 kb that have been associated with arachnids, barnacles, crustaceans, insects, fish and reptiles in Africa, Asia, Australia, Europe, North America and South America. The jingchuviral genome has two to four open reading frames (ORFs) that encode a glycoprotein (GP), a nucleoprotein (NP), a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain, and/or proteins of unknown function. Viruses in the order are only known from their genome sequences. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the order Jingchuvirales and on the families Aliusviridae, Chuviridae, Crepuscuviridae, Myriaviridae and Natareviridae, which are available at ictv.global/report/jingchuvirales, ictv.global/report/aliusviridae, ictv.global/report/chuviridae, ictv.global/report/crepuscuviridae, ictv.global/report/myriaviridae and ictv.global/report/natareviridae, respectively.

ICTV Virus Taxonomy Profile: Tulasviridae 2023.

Tulasviridae is a family of ambisense RNA viruses with genomes of about 12.2 kb that have been found in fungi. The tulasvirid genome is nonsegmented and contains three open reading frames (ORFs) that encode a nucleoprotein (NP), a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain, and a protein of unknown function (X). This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Tulasviridae, which is available at ictv.global/report/tulasviridae.

Genetic analyses of rabies virus glycoprotein and nucleoprotein gene sequences reveal the emergence of multiple lineages in animals in Arkhangai province, a central region of Mongolia.

Genetic characterizations of rabies viruses circulating in carnivore and non-carnivore animals were investigated for the first time in Arkhangai province, a central region of Mongolia. Also, glycoprotein gene of the rabies virus was sequenced for the first time in Mongolia. The nucleotide sequences of the glycoprotein and nucleoprotein genes were analysed, revealing the presence of multiple lineages in this area. Of particular concern are the lineages identified in carnivores, which might emerge to spread throughout Mongolia, further facilitating transboundary transmission to neighbouring countries, including China and Russia.

Assessment of hybrid population immunity to SARS-CoV-2 following breakthrough infections of distinct SARS-CoV-2 variants by the detection of antibodies to nucleoprotein.

Immunity induced by vaccination and infection, referred to as hybrid immunity, provides better protection against SARS-CoV-2 infections compared to immunity induced by vaccinations alone. To assess the development of hybrid immunity we investigated the induction of Nucleoprotein-specific antibodies in PCR-confirmed infections by Delta or Omicron in vaccinated individuals (n = 520). Eighty-two percent of the participants with a breakthrough infection reached N-seropositivity. N-seropositivity was accompanied by Spike S1 antibody boosting, and independent of vaccination status or virus variant. Following the infection relatively more antibodies to the infecting virus variant were detected. In conclusion, these data show that hybrid immunity through breakthrough infections is hallmarked by Nucleoprotein antibodies and broadening of the Spike antibody repertoire. Exposure to future SARS-CoV-2 variants may therefore continue to maintain and broaden vaccine-induced population immunity.

ABTB1 facilitates the replication of influenza A virus by counteracting TRIM4-mediated degradation of viral NP protein.

Influenza A viruses (IAVs) continue to cause tremendous economic losses to the global animal industry and respiratory diseases and deaths among humans. The nuclear import of the vRNP complex, composed of polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), nucleoprotein (NP), and viral RNA, is essential for the efficient replication of IAV. Host factors involved in this process can be targeted for the development of countermeasures against IAV infection. Here, we found that Ankyrin Repeat and BTB Domain Containing 1 (ABTB1) promotes the replication of IAV, and positively regulates the nuclear import of the vRNP complex. ABTB1 did not interact directly with NP, indicating that ABTB1 plays an indirect role in facilitating the nuclear import of the vRNP complex. Immunoprecipitation and mass spectrometry revealed that Tripartite Motif Containing 4 (TRIM4) interacts with ABTB1. We found that TRIM4 relies on its E3 ubiquitin ligase activity to inhibit the replication of IAV by targeting and degrading NP within the incoming vRNP complex as well as the newly synthesized NP. ABTB1 interacted with TRIM4, leading to TRIM4 degradation through the proteasome system. Notably, ABTB1-mediated degradation of TRIM4 blocked the effect of TRIM4 on NP stability, and largely counteracted the inhibitory effect of TRIM4 on IAV replication. Our findings define a novel role for ABTB1 in aiding the nuclear import of the vRNP complex of IAV by counteracting the destabilizing effect of TRIM4 on the viral NP protein.

Mechanical tomography of an archaeal lemon-shaped virus reveals membrane-like fluidity of the capsid and liquid nucleoprotein cargo.

Archaeal lemon-shaped viruses have unique helical capsids composed of highly hydrophobic protein strands which can slide past each other resulting in remarkable morphological reorganization. Here, using atomic force microscopy, we explore the biomechanical properties of the lemon-shaped virions of Sulfolobus monocaudavirus 1 (SMV1), a double-stranded DNA virus which infects hyperthermophilic (~80 °C) and acidophilic (pH ~ 2) archaea. Our results reveal that SMV1 virions are extremely soft and withstand repeated extensive deformations, reaching remarkable strains of 80% during multiple cycles of consecutive mechanical assaults, yet showing scarce traces of disruption. SMV1 virions can reversibly collapse wall-to-wall, reducing their volume by ~90%. Beyond revealing the exceptional malleability of the SMV1 protein shell, our data also suggest a fluid-like nucleoprotein cargo which can flow inside the capsid, resisting and accommodating mechanical deformations without further alteration. Our experiments suggest a packing fraction of the virus core to be as low as 11%, with the amount of the accessory proteins almost four times exceeding that of the viral genome. Our findings indicate that SMV1 protein capsid displays biomechanical properties of lipid membranes, which is not found among protein capsids of other viruses. The remarkable malleability and fluidity of the SMV1 virions are likely necessary for the structural transformations during the infection and adaptation to extreme environmental conditions.