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Hepatitis B virus (HBV) core particle is critical for the transport and replication of the viral DNA genome. By characterizing HBV core particles in different subcellular compartments, we found that when the HBV core protein was expressed by itself, it formed core particles with a uniform and fast mobility on a non-denaturing agarose gel. However, when the core protein was expressed from a replication-competent 1.3mer HBV genome, it formed particles with more heterogeneous structures. The presence of the precore protein, a protein related to the core protein, led to the formation of chimeric particles in the cytoplasm that consisted of both precore and core proteins. When the precore protein was expressed by itself, it could also form particulate structures in association with cellular RNAs in the nucleus. Our further analysis revealed that, in cells with replicating HBV, only the fast-migrating core particles on the gel contained the viral RNA and DNA, and the membrane-associated core particles contained more mature HBV DNA than the cytosolic core particles. In addition, the precore protein reduced the level of core particle-associated HBV DNA. Interestingly, the precore protein level in the cells could be increased by the degradative autophagy inhibitor bafilomycin A1 but not by the proteasome inhibitor MG132, suggesting that autophagy might regulate the biological activities of the precore protein. In conclusion, our results indicated that membranes and the precore protein could regulate HBV core particle assembly and DNA replication and suggested a role of autophagy in the regulation of HBV precore protein activities.

Importance: Hepatitis B virus (HBV) is an important human pathogen that chronically infects 254 million people in the world. This virus contains a core particle, which plays an important role in the transport and replication of the viral DNA genome. The major protein constituent of this particle is the viral core protein. In this report, we examined how the subcellular compartments and the related precore protein might affect the core particle structure and viral DNA replication. We found that the subcellular localizations could affect the core particle assembly, and membranes and the precore protein could regulate HBV DNA replication. We also found that the inhibition of autophagic degradation increased the precore protein level, suggesting a role of autophagy in the regulation of precore protein activities. These findings provided important information for further understanding the HBV life cycle, which will aid in the development of novel drugs for the treatment of HBV patients.

The evolution of the antibody response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is impacted by the nature and number of antigenic exposures. First-generation coronavirus disease 2019 (COVID-19) vaccines encoded an ancestral spike protein. Updated bivalent vaccines and breakthrough infections have shaped the intricate diversity of the polyclonal antibody response and specificity of individual antibody clones. We and others previously showed that bivalent vaccines containing the ancestral and Omicron (BA.5) spikes induce high levels of cross-reactive antibodies but undetectable BA.5-specific antibodies in serum. Here, we assessed sera collected before as well as 1 and 3 months following administration of an updated XBB.1.5 monovalent vaccine to individuals with diverse infection and vaccination histories. Vaccination increased neutralization against recent variants of concern, including HV.1, JN.1, and the vaccine-homologous XBB.1.5. Antibody binding and avidity against ancestral and XBB.1.5 antigens significantly increased after vaccination. However, antibody depletion experiments showed that most of the response was cross-reactive to the ancestral spike, and only low levels of XBB.1.5-specific antibodies to the spike or the receptor-binding domain were detected. Importantly, increased antibody levels were still detectable in circulation 3 months post-vaccination and cross-reacted with severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) as measured by pseudovirus neutralization and binding assays. Overall, our data suggest that the XBB.1.5 monovalent vaccine predominantly elicits a cross-reactive response imprinted by viral spike antigens encountered early during the pandemic.IMPORTANCEUpdated COVID-19 vaccine formulations and SARS-CoV-2 exposure history affect the antibody response to SARS-CoV-2. High titers of antibodies are induced in serum by XBB.1.5 monovalent vaccination. Antibody depletion experiments reveal that the majority of the antibody response is cross-reactive to the ancestral spike, despite vaccination increasing neutralization against recently circulating Omicron variants. Vaccine-induced SARS-CoV-2 antibodies cross-react with SARS-CoV-1 and remain in the bloodstream for at least 3 months after immunization.

Candida albicans stably colonizes humans but is a major fungal pathogen that occupies a wide range of divergent niches within the host. Rapid and effective adaptation to dynamic and contrasting host niches is associated with its pathogenicity. Recent studies have focused on genome evolution implicated in adaptive processes. Here, we demonstrate that modulation of TOR signaling is a mechanism underlying adaptive evolution in C. albicans. Clinical isolates of C. albicans exhibited enhanced commensal fitness in competition with the lab reference strain SC5314, which could be attributed to the diminished GlcNAc-responsive hypha-associated transcription in the gut. In vitro passaging of clinical isolates confers a reduction in TOR signaling, which is detrimental to fitness attributes in evolved strains, including stress response, antifungal drug tolerance, as well as in vivo commensal fitness and invasive infection. This phenomenon is observed independent of strain background and passaging environment. Importantly, inhibition of TOR signaling by rapamycin suppresses the fitness advantage observed in clinical isolates relative to their in vitro passaged derivatives. Thus, C. albicans undergoes rapid evolution via modulating TOR signaling that enables this fungus to adapt to diverse host niches.

Importance: Pathogens must be proficient at adapting to their surroundings to survive in the face of a changing microenvironment in the host and cause disease. This is particularly important for commensal-pathogenic organisms such as C. albicans as this fungus colonizes and infects mammalian hosts. Previous studies have focused on genome evolution such as aneuploidies, accumulation of point mutations, or loss of heterozygosity. Here, we demonstrate that C. albicans undergoes rapid adaptive evolution via modulating the TOR pathway. Alterations in TOR activity underlie some evolved traits with important consequences for both host adaptation and pathogenicity in C. albicans. Such mechanisms of adaptive evolution may be exploited by other organisms.

Herpesviruses are enveloped viruses with large double-stranded DNA genomes that are highly prevalent in the human population and elicit numerous types of clinical manifestations, from mild to severe. These viruses are classified into three subfamilies: alpha-, beta-, and gammaherpesvirinae, all capable of establishing life-long persistent infections in the host. As strict intracellular parasites, these viruses have evolved molecular determinants to support and modulate viral and host gene transcription processes during infection and the translation of messenger RNAs (mRNAs) to synthesize proteins that participate in cellular pathways promoting their replication cycles and virion formation. Notably, some of these proteins have functional RNA-binding domains consisting of arginine-glycine-glycine (RGG) amino acid (aa) sequences that, when methylated, regulate their nucleic acid-binding capacities and can influence the export of mRNAs lacking introns from the nucleus into the cytoplasm. Additional domains and motifs in these proteins mediate their interactions with regulatory proteins related to RNA splicing, either promoting or repressing mRNA processing. Notably, all human herpesviruses (HHVs) encode in their genomes proteins that share homology with infected cell protein 27 (ICP27) of herpes simplex virus type 1 (HSV-1), which can significantly impact the biogenesis of mRNAs and their processing during infection. Here, we review and discuss the roles of ICP27 and the corresponding homologs encoded in different human herpesviruses, focusing on their similarities and differences in structure and function. A more profound knowledge of the role of key viral factors required for effective herpesvirus replication could aid in the design and identification of novel antivirals to treat the diseases produced by these viruses.

Antisense oligomers (ASOs) hold promise as antibiotics for the selective targeting of bacterial pathogens and as tools for the modulation of gene expression in microbes that are not amenable to genetic engineering. However, their efficient delivery across the complex bacterial envelope remains a major challenge. There are few methods to assess the efficiency of carrier-mediated ASO uptake by bacteria. Here, we have developed a "switch-on" reporter assay to measure ASO uptake efficiency in a semi-quantitative manner. The assay uses a synthetic RNA toehold switch fused to the mRNA of a fluorescent reporter protein, which is activated in vivo by a peptide nucleic acid (PNA)-based ASO upon delivery into the bacterial cytosol. We have used this assay to screen different cell-penetrating peptides (CPPs) as ASO carriers in Escherichia coli and Salmonella enterica and observed up to 60-fold activation, depending on the CPP and bacterial strain used. Our assay shows high dynamic range and sensitivity, which should enable high-throughput screens for bacterial ASO carriers. We also show that the reporter can be used to study routes of PNA uptake, as demonstrated by reduced reporter activity in the absence of the inner membrane protein SbmA. In summary, we present a tool for the discovery of species-specific and efficient ASO carriers that will also be useful for a broader investigation of cellular uptake mechanisms of antibacterial ASOs.IMPORTANCEThe rise of antimicrobial resistance presents a major global health challenge. If not addressed, the death toll from resistant infections is expected to rise dramatically in the coming years. As a result, it is essential to explore alternative antimicrobial therapies. One promising approach is to target bacterial mRNAs using antisense oligomers (ASOs) to silence genes involved in essential functions, virulence, or resistance. However, delivering ASOs across bacterial membranes remains a major challenge and effective methods to monitor their uptake are limited. In this study, we develop a reporter assay to facilitate the high-throughput discovery of bacterial ASO carriers. This research paves the way for developing novel precision antisense-based antibacterial therapies.

Herpes simplex virus-1 (HSV-1) is a neurotropic virus that can infect the brain, and an uncontrolled infection can lead to a range of diseases, including chronic nerve pain, encephalitis, and neurobehavioral abnormalities. These outcomes are often severe and have lasting consequences, highlighting the need to identify host factors that contribute to disease severity. In this study, we report that intranasal HSV-1 infection in murine model, which promotes viral dissemination into the brain, implicates the host protein heparanase (HPSE) as a key mediator of neuroinflammation. Specifically, we observed that the HPSE activity during HSV-1 infection in naïve animals promotes the upregulation of proinflammatory cytokines, enhances microglial activity in the brain, and contributes to cognitive impairment, anxiety, and motor coordination deficits. Such effects are significantly less detectable in heparanase deficient (Hpse-/-) mice. Additionally, we found that moderate activation of toll-like receptors (TLRs), particularly in Hpse+/+ mice, may contribute to the activation of the inflammasome pathway. This, in turn, leads to the activation of caspase-1 (Casp1) and caspase-3 (Casp3), which may play a role in nerve function loss. Our findings position HPSE as a potential therapeutic target for mitigating virus-induced neuroinflammation and neurobehavioral defects.

Importance: Herpes simplex virus-1 (HSV-1) infection in the brain can lead to severe and often permanent neurological consequences. Host factors influence disease outcomes in response to infection, and understanding these factors is crucial for developing effective therapies. This study identifies the host protein HPSE as a key mediator of neuroinflammation in response to HSV-1 infection. We demonstrate that the HPSE activity drives proinflammatory cytokine expression and microglial activation and promotes a signaling cascade involving toll-like receptors and caspase activation, potentially intensifying neuroinflammatory responses. These findings implicate HPSE as an important player in HSV-1 pathogenesis in the central nervous system and suggest that targeting HPSE could provide a novel therapeutic strategy to mitigate virus-induced neuroinflammation and neurobehavioral disturbance.

Human immunodeficiency virus type 1 (HIV-1) capsid, which is the target of the antiviral lenacapavir, protects the viral genome and binds multiple host proteins to influence intracellular trafficking, nuclear import, and integration. Previously, we showed that capsid binding to cleavage and polyadenylation specificity factor 6 (CPSF6) in the cytoplasm is competitively inhibited by cyclophilin A (CypA) binding and regulates capsid trafficking, nuclear import, and infection. Here, we determined that a capsid mutant with increased CypA binding affinity had significantly reduced nuclear entry and mislocalized integration. However, disruption of CypA binding to the mutant capsid restored nuclear entry, integration, and infection in a CPSF6-dependent manner. Furthermore, relocalization of CypA expression from the cell cytoplasm to the nucleus failed to restore mutant HIV-1 infection. Our results clarify that sequential binding of CypA and CPSF6 to HIV-1 capsid is required for optimal nuclear entry and integration targeting, providing insights for the development of antiretroviral therapies, such as lenacapavir.

Importance: Human immunodeficiency virus (HIV) encodes a protein that forms a conical shell, called a capsid, that surrounds its genome. The capsid has been shown to protect the viral genome from innate immune sensors in the cell, to help transport the genome toward and into the nucleus, to keep the components of reverse transcription together for conversion of the RNA genome into DNA, and to target viral DNA integration into specific regions of the host genome. In this study, we show that HIV hijacks two host proteins to bind to capsid sequentially in order to choreograph the precise order and timing of these virus replication steps. Disruption of binding of these proteins to capsid or their location in the cell leads to defective HIV nuclear import, integration, and infection. Mutations that exist in the capsid protein of HIV in infected individuals may reduce the efficacy of antiretroviral drugs that target capsid.