The HIV-1 Tat protein is regulated by post-translational modifications, particularly ubiquitination, but the full spectrum of deubiquitinating enzymes (DUBs) controlling HIV-1 transcription remains incompletely understood. We developed a NanoBRET-based screening system using a library of 120 DUB expression vectors and identified USP45 as a novel Tat-interacting DUB. Functional characterization revealed that USP45 specifically deubiquitinates Tat at lysine 19, targeting both K48-linked and K63-linked ubiquitin chains. Notably, USP45 overexpression suppressed Tat-dependent transcriptional activation and HIV-1 viral particle production, while USP45 knockdown enhanced both processes. Transcriptional profiling in latently infected J-Lat 8.4 cells using digital PCR revealed that USP45 primarily inhibits the initial stages of viral transcription, thereby contributing to the maintenance of the latent state by restricting downstream transcriptional processes. Furthermore, USP45 expression is induced by interferons, identifying it as an interferon-stimulated gene. These findings establish that USP45 functions as a host restriction factor that negatively regulates HIV-1 transcription by promoting Tat deubiquitination at lysine 19, representing a promising therapeutic target for controlling HIV-1 latency.

Hepatitis B virus (HBV) infection remains a major global health challenge. While sodium taurocholate co-transporting polypeptide (NTCP) is the primary receptor for HBV entry, the molecular mechanisms regulating NTCP-mediated viral entry remain incompletely understood. Here, we identified CD46 as a crucial regulatory factor for NTCP membrane expression. We found that CD46 interacted with NTCP in cis at the plasma membrane through proximity-based labeling screening. The depletion of CD46 significantly reduced cell-surface NTCP levels and HBV infection in hepatocytes. Anti-CD46 monoclonal antibodies, particularly clone E4.3, inhibited HBV infection by triggering NTCP internalization from the plasma membrane to intracellular vesicles. The antiviral effect of CD46 antibodies was also confirmed in primary human hepatocytes. Our study reveals a previously unknown mechanism regulating NTCP-mediated HBV entry and suggests CD46 as a potential therapeutic target for HBV infection.

Mumps virus (MuV) genotype G is widely represented among circulating strains, but the evolutionary patterns of the hemagglutinin–neuraminidase (HN) gene remain incompletely understood. In this study, we analyzed publicly available full-length genotype G HN sequences using phylogenetic, phylodynamic, codon-based selection, and structure-guided epitope prediction approaches. The genotype G HN sequences were categorized into Clade 1, an operationally defined Diverse group, and Clade 2. Clade 1, which was composed mainly of Japanese strains, showed a relatively structured pattern over time. In contrast, Clade 2 showed more recent diversification and an overall increase in relative genetic diversity, although this phylodynamic pattern was sensitive to sampling structure. The Diverse group was phylogenetically heterogeneous, and its Bayesian skyline estimates were not used for biological interpretation because repeated analyses showed unstable posterior behavior. Root-to-tip regression supported temporal structure in the complete dataset, with the strongest signal in Clade 2. Bayesian molecular dating estimated the time to the most recent common ancestor of the sampled genotype G HN sequences at approximately 1932, and the mean evolutionary rate was 4.925 × 10−4 substitutions/site/year. Although the HN protein is a major surface antigen and a target of neutralizing antibodies, codon-based analyses showed no robust evidence of positive selection using multiple methods. Instead, many codon sites were inferred to be under purifying selection, suggesting that genotype G HN evolution is largely constrained by the need to maintain protein function. Predicted B-cell epitope regions were broadly similar among representative genotype G strains. Overall, these findings indicate that genotype G HN lineages have followed distinct evolutionary patterns, while the HN gene remains mainly shaped by purifying selection. These findings may help improve our understanding of MuV genotype G HN gene evolution and support future molecular surveillance.

Profiling of newly synthesized proteins (NSPs) provides access to dynamic changes in protein production that accompany acute cellular responses. Bioorthogonal noncanonical amino acid tagging (BONCAT)-based approaches enable selective labeling of NSPs; however, their broader application remains constrained by labor-intensive enrichment workflows and limited sensitivity for direct peptide-level analysis. Here, we developed a workflow termed "Phos-tag Click Tip" by integrating a phosphorylated variant of bicyclononyne (pBCN) with Phos-tag affinity purification to selectively capture azidohomoalanine (AHA)-labeled peptides for newly synthesized proteome analysis (NSProteomics). This approach overcomes key limitations of conventional proteomics and BONCAT-based strategies by enabling efficient enrichment and sensitive detection of NSP-derived peptides. Using this workflow, we performed comprehensive NSP profiling of host cells during influenza A virus infection. We identified dynamic changes in distinct NSP profiles associated with viral replication, host restriction, and immune responses, many of which were not readily detected with conventional whole-cell- or phospho-proteomic analyses. Overall, the Phos-tag Click Tip workflow provides a complementary approach for stimulus-responsive NSP profiling, offering functionally relevant insights into host-virus interactions and cellular response mechanisms.

Bioinformatics has transformed modern virology by linking genomic variation to epidemiology, protein structure, and public health action. This review integrates core analytical frameworks-sequence alignment and genome annotation; maximum-likelihood and Bayesian phylogenetic/phylodynamic inference; codon-based selection and recombination analyses; and AI-assisted structural prediction combined with deep mutational scanning (DMS)-to convert viral sequences into mechanistic and predictive insight. We emphasize how global surveillance ecosystems (GISRS, GISAID, and Nextstrain) and sustained regional programs reveal genotype turnover, antigenic drift, and seasonality in RSV, HPIV, norovirus, and SARS-CoV-2, enabling near real-time lineage tracking and vaccine-strain deliberation. Mapping positively selected residues and recombination breakpoints onto three-dimensional protein structures clarifies immune escape in key surface glycoproteins (e.g., RSV F/G, HPIV HN/F, norovirus VP1) and strengthens genotype-phenotype interpretation. Comparative reinfection patterns-lifelong immunity in measles versus recurrent RSV/HPIV infections-illustrate how evolutionary rate and antigenic constraint shape population immunity and control strategies. Despite major advances, progress remains constrained by geographic sampling bias, incomplete metadata, uneven computational capacity, and uncertainties in molecular clocks, recombination inference, and machine-learning predictions. The field is now moving toward predictive virology, integrating AI-enabled structural modeling, mutational fitness landscapes, and clinical-immunological metadata within real-time analytical platforms to anticipate immune-escape trajectories. Prioritizing pediatric respiratory pathogens alongside influenza and coronaviruses, and reinforcing equitable data-sharing and governance, will be essential for globally inclusive, forward-looking viral surveillance and intervention.

Optogenetic dimerization modules are widely used to regulate protein function, yet their behaviour within folded enzymes remains incompletely understood. Here, we engineered vesicular stomatitis virus by inserting an intramolecularly tethered light-inducible dimerizer into the viral RNA-dependent RNA polymerase [large (L) protein], enabling assessment of its effects on early viral transcription and viral replication. Blue light illumination enhanced viral replication, but reduced temperature produced a similar increase in viral replication even in the absence of light. In addition, a thermally stabilized dimerizer variant supported robust replication under dark conditions. These observations suggest that early viral transcription and viral replication are influenced by the structural and biophysical properties of the inserted dimerizer and their temperature dependence, rather than by light alone. Structural modelling suggested that permissive insertion sites are located within or adjacent to regions with low predicted Local Distance Difference Test (pLDDT) scores, whereas non-permissive insertions showed greater predicted local structural deviations, particularly in the capping domain. Although these analyses are based on predicted structures, the observed structural differences are consistent with the functional differences among insertion sites. Together, our results show that intramolecular optogenetic modules enable light- and temperature-dependent modulation of early viral transcription and replication. While the structural analyses are based on predicted models, they provide a useful framework for identifying permissive insertion sites and generate testable hypotheses regarding how the structural and biophysical properties of the inserted dimerizer may influence polymerase function.

In Japan, many healthcare workers received up to seven doses of SARS-CoV-2 mRNA vaccines by April 2024. While repeated vaccination aims to strengthen immunity, concerns about immune imprinting and IgG subclass switching, particularly to IgG4, have arisen. This study examined 19 healthcare workers who completed seven doses. Serum and PBMCs collected at five or three time points respectively to measure were analyzed for IgG subclasses, neutralizing antibodies and cellular responses. IgG1-3 levels declined after the second dose but recovered after the third, whereas IgG4 increased post-second dose and remained stable. Neutralizing antibodies against Omicron XBB.1.5 were undetectable after two doses but appeared after the third, despite all being Wuhan strain vaccines, and rose further after the sixth and seventh doses with updated formulations. Cellular immunity remained stable throughout. These findings indicate repeated mRNA vaccination enhances humoral immunity without progressive IgG4 elevation or additional cellular response boosts.

In this study, we investigated the long-term evolutionary dynamics of human norovirus GII.17[P17] using the RNA-dependent RNA polymerase (RdRp) region and the VP1 capsid gene, integrating phylogenetics, time-scaled inference, phylodynamics, and structure-based analyses. Maximum-likelihood phylogenies of both genomic regions consistently resolved four major clades (Clades 1-4). VP1 patristic-distance distributions indicated higher within-clade diversity in the phylogenetically basal Clades 1 and 3, whereas Clades 2 and 4 showed lower diversity, consistent with recent demographic expansion. Similarity-plot analysis identified pronounced variability in the VP1 P2 domain, while the S and P1 domains remained comparatively conserved, supporting P2 as the primary hotspot of diversification. Bayesian time-scaled analyses estimated the most recent common ancestor around 1993 (VP1) and 2000 (RdRp) and revealed two major lineages (Clade 1/2 and Clade 3/4), with the split between Clades 3 and 4 occurring around 2016-2017. Bayesian skyline plots showed a marked increase in effective population size after 2013, and substitution-rate estimates indicated faster evolution in VP1 than in RdRp, with higher VP1 rates in the Clade 3/4 lineage than in Clade 1/2. Capsid dimer modeling further mapped high-confidence conformational B-cell epitopes and positively selected residues predominantly to the distal surface of P2, with broadly conserved spatial patterns across clades. Compared with the Clade 1 reference (Kawasaki323), Clade 2 accumulated numerous P2 substitutions, whereas Clades 3 and 4 retained fewer changes and remained closer to Clade 1 at the amino-acid level. Together, these results suggest lineage turnover within GII.17[P17] driven by constrained diversification at the P2 surface, potentially contributing to the recent predominance of the Clade 3/4 lineage.

ABSTRACT Hepatitis B virus (HBV) infection remains a major global health burden, and HBV X protein (HBx) plays a central role in modulating host pathways that influence viral replication. We previously reported that the oxidative stress sensor Kelch-like ECH-associated protein 1 (Keap1) recognizes HBx to activate the NF-E2-related factor 2 (Nrf2) signaling pathway to suppress HBV replication. Although canonical K48-linked ubiquitylation is known to control Nrf2 turnover, the contribution of non-canonical ubiquitin linkages to Nrf2 regulation during HBV infection remains unclear. Here, we investigated the role of HECT, UBA, and WWE domain-containing E3 ubiquitin ligase 1 (HUWE1) in the regulation of Nrf2 in the context of HBV replication. Cell-based ubiquitylation assays demonstrated that HUWE1 knockdown reduced HBx-mediated K6-linked polyubiquitylation of Nrf2, while overexpression of wild-type HUWE1, but not the catalytically inactive HUWE1(C4341A) mutant, enhanced it. Coimmunoprecipitation and proximity ligation assays demonstrated that HUWE1 interacts with HBx in the cytoplasm and binds Nrf2 only in the presence of HBx, suggesting that HBx promotes the interaction between HUWE1 and Nrf2. Cycloheximide chase assays demonstrated that HUWE1 knockdown destabilized Nrf2 in HBx-expressing cells. Furthermore, depletion or pharmacological inhibition of HUWE1 increased intracellular HBV RNA and pgRNA levels as well as extracellular HBV DNA and HBsAg levels in HBV-infected cells. Collectively, these results support a model in which HUWE1 mediates HBx-dependent K6-linked polyubiquitylation and stabilization of Nrf2 to restrict HBV replication. This study expands current understanding of non-canonical ubiquitin signaling in HBV-host interactions. DATA SUMMARY All data are presented in the main figures. The data that support the findings of this study is available at bioRxiv ( https://doi.org/10.64898/2026.04.20.719611 ). Raw sequencing data, microscopy images, materials, and sequence information are available upon request. Correspondence and requests for materials should be addressed to Professor Ikuo Shoji. IMPACT STATEMENT Hepatitis B virus (HBV) chronically infects approximately 254 million people worldwide, yet host mechanisms that restrict viral replication remain incompletely understood. The Keap1/ Nrf2 signaling pathway is a central defense against oxidative stress. Under basal conditions, Nrf2 is targeted for degradation via Keap1/Cullin3-mediated K48-linked polyubiquitylation. Here, we provide evidence that the E3 ubiquitin ligase HUWE1 contributes to HBx-dependent K6-linked polyubiquitylation and stabilization of Nrf2. Our findings support a model in which non-canonical ubiquitin signaling helps shape the HBV-host interactions and contributes to suppression of viral replication. This study extends current understanding of the ubiquitin code in HBV infection and highlights HUWE1 as a candidate component of an anti-HBV regulatory pathway.

Human metapneumovirus genotype B (HMPV-B) is an important respiratory pathogen, requiring detailed elucidation of the evolutionary and antigenic features of its fusion (F) gene. Using 500 sequences collected between 1982 and 2024, we investigated the molecular evolution, phylodynamics, and structural epitope landscape of the HMPV-B F gene. Time-scaled phylogeny dated the divergence of sublineages B1 and B2 to around 1937, and Bayesian Skyline Plot analysis showed that these sublineages exhibited distinct demographic trajectories over time. The F gene evolved at a rate of 1.01 × 10−3 substitutions/site/year; however, amino acid variation remained limited, consistent with pervasive purifying selection, with 39% of codons under strong negative selection and little consensus evidence for positive selection. Conformational B-cell epitope prediction demonstrated a high degree of conservation across neutralizing antibody binding regions (sites Ø and I–V), and amino acid substitutions occurring within these sites were not predicted to substantially alter epitope architecture. Together, these findings indicate that the HMPV-B F gene evolves under strong evolutionary constraint while maintaining stable antigenic features, supporting the potential for antibody-based strategies that target neutralizing antibody binding regions of the F protein.

mRNA vaccines have demonstrated broad effectiveness against coronavirus disease 2019 (COVID-19); nonetheless, substantial interindividual variability in vaccine-induced immunity limits our ability to predict protective efficacy at the individual level, particularly against severe manifestations of the disease. Cellular immune activity offers valuable insights into protection levels, yet assay complexity limits its large-scale evaluation. In this study, we aimed to evaluate the persistence of cellular immunity following COVID-19 mRNA vaccination and to assess its relationship with breakthrough infection severity. We used the streamlined, high-throughput ex vivo activation of genes in leukocytes (EAGL) assay to assess T-cell responses in blood samples from vaccinated Japanese donors by tracking immunity at intervals post-vaccination. Vaccine-induced cellular immunity persisted longer than the antibody titers. Notably, lower antibody titers tended to precede infection, whereas cellular immune responses did not correlate with the occurrence of breakthrough infection in our cohort. However, among breakthrough cases, asymptomatic donors showed a trend toward higher Th1/CD8+ T-cell cytokine responses (IFNG, IL2, TNFSF2) than those with mild symptoms, although these differences did not reach statistical significance. Transcriptome analysis identified CXCL9 as a potential novel marker for disease outcome, with reduced expression correlating with persistent symptoms. Interestingly, in individuals ≥55 years old, Th1/CD8+ T cell cytokine expression decreased significantly and CXCL9 expression fell below the threshold associated with persistent symptoms. By contrast, we did not observe a consistent age-related decline in humoral measures. Overall, our findings suggest that cellular immune readouts, particularly IFNG in combination with CXCL9, may serve as exploratory correlates of vaccine-mediated symptom attenuation. Furthermore, the EAGL assay constitutes an efficient and scalable platform for high-throughput assessment of vaccine-induced cellular immunity, offering potential as a predictive tool for individual-level vaccine efficacy in mitigating symptoms.

Background: Rapid and high-throughput diagnostic methods are essential for controlling the spread of infectious diseases, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Lateral flow immunoassay (LFIA) strips provide a cost-effective and user-friendly platform for point-of-care testing. However, the sensitivity of conventional LFIA kits is often limited by the performance of their detection probes. This study reports a highly sensitive LFIA strip for detecting the SARS-CoV-2 nucleocapsid (NP) protein using platinum-decorated poly(2-vinylpyridine) nanoparticles (Pt-P2VPs) as probes. Methods: Monoclonal antibodies against SARS-CoV-2 NP were conjugated with Pt-P2VPs and incorporated into LFIA strips. The test line was coated with anti-SARS-CoV-2 NP monoclonal antibody, and the control line with goat anti-mouse IgG. Recombinant proteins, viral strains, and nasopharyngeal swab specimens from patients were used to evaluate assay performance, with reverse transcription polymerase chain reaction (RT-PCR) as the reference standard. Diagnostic accuracy was assessed using nonparametric statistical tests. Results: Pt-P2VP-based LFIA strips enabled sensitive detection of recombinant NP and inactivated SARS-CoV-2, with minimal cross-reactivity. In 200 clinical specimens (100 PCR-negative and 100 PCR-positive), the assay achieved 74% sensitivity and 100% specificity, with strong correlation to viral RNA load. Compared with conventional LFIA kits, Pt-P2VP strips demonstrated superior sensitivity at lower viral loads. Conclusions: Pt-P2VPs represent a promising probe material for enhancing LFIA performance and may facilitate the development of rapid, sensitive, and scalable immunoassays for infectious disease diagnostics in biomedical applications.

OBJECTIVES:The SARS-CoV-2 pandemic has resulted in millions of deaths worldwide. However, the risk of transmission from COVID-19 corpses remain unclear, posing challenges for forensic medicine in establishing effective infection control measures during autopsies. This study aimed to investigate the presence of infectious SARS-CoV-2 in corpses and identify factors affecting viral infectivity. METHODS:External examinations of 76 corpses with COVID-19 were performed, and nasopharyngeal, perioral, hand swabs, serum, cerebrospinal fluid (CSF), and urine samples were collected. Viral RNA was quantified by using digital PCR, and infectious viruses were assessed via isolation. Multivariate logistic regression analysis was used to identify factors associated with viral infectivity. RESULTS:Infectious viruses were isolated from the nasopharynx (51%), perioral region (10.3%), and hands (1.8%). Common predictive factors for nasopharyngeal and perioral viral infectivity were a higher viral load and shorter time from symptom onset. Detectable viral RNA in serum was also associated with nasopharyngeal infectivity. No infectious viruses were detected in serum, CSF, or urine samples. CONCLUSIONS:Infectious SARS-CoV-2 was detected in the nasopharynx and on the surface of COVID-19 corpses. Viral infectivity correlated with viral load and time from symptom onset, highlighting the importance of strict infection control when handling COVID-19 corpses.

The emergence of SARS-CoV-2 variants poses ongoing challenges to vaccine efficacy. We evaluated neutralizing antibody responses against JN.1 and its derivatives (KP.3, KP.3.1.1, LB.1, and XEC) in healthcare workers who received seven doses of BNT162b2, including the XBB.1.5 monovalent vaccine. In COVID-19-naïve individuals, KP.3.1.1 and LB.1 showed substantial immune escape, whereas previously infected individuals maintained neutralization activity against all variants. We also demonstrated that JN.1-based immunization induces robust cross-neutralizing activity against emerging variants. A single amino acid deletion at position 31 in the spike protein may be associated with enhanced immune evasion. These findings support the potential effectiveness of JN.1-based vaccines while highlighting the need for continued surveillance and vaccine optimization.

Seasonal influenza is prevalent every winter, and influenza vaccines are used to safeguard public health. As the influenza vaccines are produced in embryonated hen eggs using vaccine viruses recommended by the World Health Organization each year based on the epidemic situation, it is important, from the perspective of public health, to evaluate the reactivity of the vaccines against circulating viruses in humans. This study was designed to determine whether evaluating influenza vaccine efficacy using mouse sera could help predict efficacy in humans. The split hemagglutinin (HA) vaccines, produced using vaccine viruses for the 2023/2024 season, were inoculated into ddY and BALB/c mice, and their neutralizing reactivity to circulating influenza viruses was evaluated using their antisera. The titers of antibodies against these viruses in the antisera from humans immunized with the split HA vaccine were also measured. The vaccines induced the production of functional vaccine-specific antibodies dose-dependently. In the case of circulating viruses, the neutralizing antibodies in the sera of immunized individuals were able to react to most the test viruses, but they were less reactive to some viruses. The titers of neutralizing antibodies against these circulating strains in the antisera from humans immunized with the split HA vaccine for the 2023/2024 season were similar to those in the mouse antisera. Furthermore, intraperitoneal inoculation in ddY mice induced antibody production with higher neutralizing titers than subcutaneous inoculation in BALB/c mice. Taken together, the immunization protocol using naive mice could be a suitable method for predicting vaccine efficacy in humans.

Despite widespread vaccination programs, mumps outbreaks persist even in highly vaccinated populations, raising concerns about current vaccine effectiveness against circulating strains. Existing serological methods exhibit limitations in practical implementation and antigenic specificity. To address these challenges, we developed a rapid neutralization assay using HiBiT-tagged mumps virus-like particles (hiMuV-VLPs) composed entirely of authentic viral structural proteins. Transmission electron microscopy confirmed these hiMuV-VLPs morphologically resembled native mumps virions. The VLPs demonstrated efficient cellular entry, quantifiable via luminescent signals generated by HiBiT-LgBiT complementation. Validation against conventional plaque reduction neutralization tests (PRNT) using human sera revealed the biological relevance and practicability of our assay. A key innovation was the successful incorporation of hemagglutinin-neuraminidase (HN) proteins from multiple mumps virus genotypes into the hiMuV-VLP platform, enabling assessment of strain-specific neutralizing antibody responses. This system represents a valuable tool for large-scale seroepidemiological surveillance, evaluation of vaccine-induced immunity against heterologous strains, and prediction of population susceptibility to emerging mumps virus variants.

Post-acute sequelae of SARS-CoV-2 infection (PASC), commonly known as “Long COVID”, represents a significant clinical challenge characterized by persistent symptoms following acute COVID-19 infection. We conducted a comprehensive retrospective cohort study to identify serum autoantibody biomarkers associated with PASC. Initial screening using a protein bead array comprising approximately 20,000 human proteins identified several candidate PASC-associated autoantibodies. Subsequent validation by enzyme-linked immunosorbent assay (ELISA) in an expanded cohort—consisting of PASC patients, non-PASC COVID-19 convalescents, and pre-pandemic healthy controls—revealed two promising biomarkers: autoantibodies targeting PITX2 and FBXO2. PITX2 autoantibodies demonstrated high accuracy in distinguishing PASC patients from both non-PASC convalescents (area under the curve [AUC] = 0.891) and healthy controls (AUC = 0.866), while FBXO2 autoantibodies showed moderate accuracy (AUC = 0.762 and 0.786, respectively). Notably, the levels of these autoantibodies were associated with several PASC symptoms, including fever, dyspnea, palpitations, loss of appetite, and brain fog. The identification of PITX2 and FBXO2 autoantibodies as biomarkers not only enhances our understanding of PASC pathophysiology but also provides promising candidates for further investigation.

A class of unstructured peptide segments termed disordered regions plays a crucial role in the regulation of protein structure and function. Although the N-terminal region of the matrix (MA) domain of the HIV-1 Gag precursor protein is composed of an unstructured peptide, the mechanisms underlying the regulation of the unstructured state relating to control of viral phenotypes remain unclarified. We examined, in association, the structural, evolutionary, and biological roles of the MA N-terminal region via mutagenesis. Molecular dynamics simulation of a full-length Gag dimer model suggested that an amino acid residue at position 9 in the MA N-terminal region (MA-9) participates in the Gag dimerization. Information entropy analysis indicated that the MA-9 residue is variable in nature, but the hydrophobic amino acid substitution is evolutionarily maladaptive. Disordered region prediction study suggested that single hydrophobic amino acid substitutions at MA-9 reduce the disordered state of the MA N-terminal region. Consistently, NMR analysis indicated that such substitution reduces motional dynamics of the MA N-terminal region and alters the conformation of the MA domain. A site-directed mutagenesis study showed that hydrophobic amino acid substitutions at the MA-9 residue impair, to different degrees, the elementary and overall processes of virus particle formation in the cells. Importantly, the level of the virus particle formation was positively correlated with the level of disorder of the MA N-terminal region. These results indicate that the maintenance of structural disorder and dynamics of the Gag N-terminal segment is regulated by the MA-9 residue and critical for maintaining the optimal production of HIV-1 particles.IMPORTANCEA class of unstructured peptide segments termed disordered regions plays crucial roles in the regulation of protein structure and function. Although HIV-1 Gag precursor protein has multiple disordered elements, molecular mechanisms underlying regulation of unstructured state and viral phenotypes largely remain elusive. In this study, by analyzing in association the structural, evolutionary, and virological roles of the disordered N-terminal region of the HIV-1 Gag protein, we show that an amino acid residue at position 9 of the Gag is able to modulate the N-terminal disordered state, and the level of disorder of the Gag N-terminal region is positively correlated with the level of virus particle formation. Our findings gain new insights into molecular mechanisms of regulation of Gag structure and highlight the importance of a previously unappreciated survival strategy of HIV-1-namely, preservation of the Gag N-terminal disorder.

HIV-2 viral protein X (Vpx) plays a pivotal role in antagonizing the host restriction factors, including SAMHD1 and components of the HUSH complex, to facilitate viral replication. However, the regulatory mechanisms controlling Vpx stability remain unclear. In this study, we identify the von Hippel-Lindau (VHL) tumor suppressor as a novel E3 ubiquitin ligase that specifically targets Vpx for proteasomal degradation. Mechanistically, we demonstrate that VHL-mediated degradation depends on the oxygen-dependent hydroxylation of Vpx at proline residue 41 (Pro41), a modification catalyzed by prolyl hydroxylase domain-containing protein 3 (PHD3). Using an integrated approach combining crosslinking mass spectrometry and molecular modeling analyses, we elucidate the structural architecture of the PHD3-Vpx complex, revealing the spatial orientation of the catalytic domain of PHD3 required for Pro41 hydroxylation. Furthermore, we establish the physiological significance of this pathway in human macrophages, where pharmacological inhibition or genetic ablation of VHL or PHD3 enhances HIV-2 infection by facilitating Vpx-mediated SAMHD1 degradation. Collectively, our findings unveil a previously unrecognized oxygen-sensitive regulatory mechanism influencing HIV-2 infection and suggest novel therapeutic strategies targeting Vpx stability through modulation of its prolyl hydroxylation status.