Journal of Virology最新文献

Even in antiretroviral therapy (ART)-suppressed human immunodeficiency virus (HIV)-infected individuals, there are heterogeneous populations of HIV-expressing cells exhibiting variable degrees of progression through blocks to HIV transcriptional initiation, elongation, completion, and splicing. These HIV-transcribing cells likely contribute to HIV-associated immune activation and inflammation as well as the viral rebound that occurs after stopping ART. However, it is unclear whether the blocks to HIV transcription are present before ART and how the timing and duration of ART may affect the clearance of cells expressing HIV transcripts that differ in their processivity and/or presence of mutations. To investigate these questions, we quantified different types of HIV transcripts and the corresponding HIV DNA regions/proviruses in longitudinal blood samples obtained before ART initiation (T1) and after 6 months (T2) and 1 year (T3) of ART in 16 individuals who initiated ART during acute HIV infection. Before ART, the pattern of HIV transcripts suggested blocks to elongation and splicing, and only ~10% of intact proviruses were transcribing intact HIV RNA. During the first 6 months of ART, we detected progressively greater reductions in initiated, 5'-elongated, mid-transcribed, completed, and multiply spliced HIV transcripts. Completed HIV RNA decayed faster than initiated or 5'-elongated HIV RNA, and intact HIV RNA tended to decay faster than defective HIV RNA. HIV DNA and RNA levels at T1-T3 correlated inversely with baseline CD4+ T-cell counts. Our findings suggest the existence of immune responses that act selectively to reduce HIV transcriptional completion and/or preferentially kill cells making completed or intact HIV RNA.IMPORTANCEEven in virologically suppressed HIV-infected individuals, expression of viral products from both intact and defective proviruses may contribute to HIV-associated immune activation and inflammation, which are thought to underlie the organ damage that persists despite suppressive ART. We investigated how the timing of ART initiation and the duration of ART affect the heterogeneous populations of HIV-transcribing cells, including a detailed characterization of the different HIV transcripts produced before ART and the rate at which they decay after ART initiation during acute HIV infection. Even during untreated infection, most cells (~90%) have blocks at some stage of transcription. Furthermore, different HIV transcripts decline at different rates on ART, with the fastest decay of cells making completed and intact HIV RNA. Our results suggest that intrinsic or extrinsic immune responses act selectively to either reduce particular stages of HIV transcription or cause selective killing of cells making particular HIV transcripts.

Usutu virus (USUV) is an arbovirus and has emerged as a potential cause of encephalitis in humans and other vertebrates. The increasing detection of USUV in mosquitoes and birds across Africa and Central Europe, along with the lack of specific treatments or vaccines for many encephalitic orthoflaviviruses, underscores the need for focused research. In this study, we developed a USUV infection model in immunocompetent C57BL/6 mice (8-12 weeks old) to characterize disease development and associated inflammatory mechanisms. Mice were intracranially infected with 10⁴ PFU of USUV, leading to neurological symptoms such as hunched posture, paralysis, conjunctivitis, and eventual death by day 6 post-infection. Meningeal cell infiltration and microglia activation were most prevalent in mouse brains; however, neuronal loss was not observed at the peak of the disease, which coincided with increased viral load and leukocyte infiltration. The immune response in the brain was marked by the systematic recruitment and activation of macrophages, neutrophils, and T lymphocytes. A noticeable shift was seen in CD4+ T cells toward T helper 1 (Th1) polarization, which corroborates a massive increase in the expression of Th1-associated cytokines and chemokines at the peak of infection, indicative of an augmented proinflammatory state. Additionally, a rise in regulatory T cells was observed, peaking on day 6 post-infection. These findings highlight the dynamic nature of the host response to USUV infection, enhance our understanding of the disease pathogenesis, and address the scarcity of immunocompetent experimental models for the investigation of neglected emerging flaviviruses.IMPORTANCEMosquito-borne viruses, including USUV, are maintained in nature through complex cycles involving arthropod vectors and vertebrate hosts. A comprehensive understanding of USUV biology and host-pathogen interactions is crucial for developing effective treatments, which necessitates reliable experimental models (G. J. Sips, J. Wilschut, and J. M. Smit, Rev Med Virol 22:69-87, 2012, https://doi.org/10.1002/rmv.712; T. C. Pierson and M. S. Diamond, Nat Microbiol 5:796-812, 2020, https://doi.org/10.1038/s41564-020-0714-0). The establishment of a USUV infection model in immunocompetent adult mice brings new perspectives on the inflammatory component of viral encephalitis, which is difficult to study in mice lacking antiviral interferon responses. Moreover, USUV is an emerging viral disease lacking therapeutic and preventive measures. The interplay of USUV pathogenesis and the host's immune response indicates that lymphocytes and monocytes participate in USUV infection in this model and could be explored in search of treatments targeting immunopathogenic processes triggered by infection.

Class B scavenger receptors (SRBs) have been well-studied in bacteria-induced immune responses in invertebrates. However, the status of SRB-defending viruses remains unclear. In this study, we identified a scavenger receptor in Procambarus clarkii (crayfish), which is homologous to mammalian SRBs, and designated it as PcSRB. The expression of PcSRB was upregulated after the WSSV challenge. The survival rate of crayfish was decreased, but the WSSV copy number increased after PcSRB knockdown during virus invasion. In addition, PcSRB bound to WSSV. Furthermore, we detected how PcSRB interacted with WSSV, and we found that PcSRB could bind to cholesta-3,5-diene, (CD3,5), a novel WSSV lipid ligand, rather than dibutyl phthalate (DBP). Besides, PcSRB could bind to VP19, VP26, and VP28, rather than VP24. Mutant-binding experiments demonstrated that the hydrophobic domain (130-180 aa) of PcSRB is important for recognizing WSSV. Furthermore, PcSRB might promote lysosomal eliminating function to degrade WSSV. Altogether, we identified a new mechanism for scavenger receptor recognition and resistance to WSSV.IMPORTANCEPcSRB could bind to WSSV directly. PcSRB could interact with WSSV via binding to lipid molecule CD3,5 and viral envelope proteins. PcSRB could influence lysosomal activation.

RAF1 is a key player in growth factor receptor signaling, which has been linked to multiple viral infections, including human cytomegalovirus (HCMV) infection. Although HCMV remains latent in most individuals, it can cause acute infection in immunocompromised populations, such as transplant recipients, neonates, and cancer patients. Current treatments are suboptimal, highlighting the need for novel therapies. Multiple points in the growth factor signaling pathway are important for HCMV infection, but the relationship between HCMV and RAF1, a component of the mitogen-activated protein kinase (MAPK) cascade, is not well understood. The AMP-activated protein kinase (AMPK) is a known regulator of RAF1, and AMPK activity is induced by HCMV infection, which is important for productive HCMV replication. Our data indicate that HCMV infection induces AMPK-specific changes in RAF1 protein phosphorylation, including increasing phosphorylation at RAF1-Ser621, a known AMPK phospho-site, which results in increased binding to the 14-3-3 scaffolding protein, an important aspect of RAF1 protein activation. Inhibition of RAF1, either pharmacologically or via shRNA or CRISPR-mediated targeting, inhibits viral replication and spread in both fibroblasts and epithelial cells. Collectively, our data indicate that HCMV infection and AMPK activation modulate RAF1 activity, which is important for viral replication.

Importance: Human cytomegalovirus (HCMV) infection is a widespread infection impacting approximately 60-90% of the global population. Although latent in healthy individuals, acute infection in immunocompromised populations, such as neonates, transplant recipients, and cancer patients, can result in retinal and gastrointestinal problems, hearing loss, and even death. Current antivirals are suboptimal due to the development of viral resistance or toxicity in patients, highlighting the need for novel treatments. Our research suggests a new potential target, RAF1, which is a regulator of cellular growth and proliferation. We find that RAF1 is phosphorylated by AMP-activated protein kinase, and that inhibition of RAF1 negatively impacts viral infection. Furthermore, drugs currently used to treat certain cancers also inhibit RAF1 and may have an additional anti-HCMV therapeutic effect in HCMV-susceptible cancer patients.

Dengue virus (DENV) is a mosquito-borne flavivirus which coexists as four genetically and immunologically distinct serotypes (DENV-1 to -4). In secondary heterologous DENV infection, pre-existing immunity is believed to contribute to severe disease through antibody-dependent enhancement (ADE). Although the elevated pathology observed in ADE conditions has been described, the cell-intrinsic mechanisms governing this process remain unclear. Using single-cell RNA sequencing (scRNAseq), we investigated the transcriptomic profiles of human monocyte-derived macrophages infected by DENV-2 in ADE compared to conventional infection conditions. Unsupervised analysis of scRNAseq data enabled the identification of two distinct cell populations in a heterogeneous cell culture, likely representing infected and bystander/uninfected cells. Differential gene expression and ingenuity pathway analyses revealed a number of significantly upregulated and downregulated genes and gene networks between cells infected by ADE compared to conventional infection. Specifically, these pathways indicated mechanisms such as suppressed interferon signaling and inflammatory chemokine transcription in cells infected via ADE. Further analysis revealed that transcriptomic changes were independent of viral RNA within infected cells, suggesting that the observed changes are reflective of cell-intrinsic responses and not simply a function of per-cell viral burden. The interpreted "bystander" cell population also demonstrated distinct profiles in ADE conditions, indicating an immunologically activated phenotype enriched for the expression of gene networks involved with protein translation, cytokine production, and antigen presentation. Together, these findings support the concept that DENV infection via ADE induces a qualitatively different transcriptomic response in infected cells, contributing to our understanding of ADE as a mechanistic driver of disease and pathogenesis.IMPORTANCEDengue virus (DENV) is a mosquito-borne human pathogen with a significant and growing global health burden. Although correlates of severe dengue disease are poorly understood, pre-existing immunity to DENV has been associated with severe disease risk and known to contribute to an alternative route of viral entry termed antibody-dependent enhancement (ADE). Using single-cell RNA sequencing, we identified distinct transcriptomic processes involved in antibody-mediated DENV entry compared to conventional receptor-mediated entry. These data provide meaningful insight into the discrete processes contributing to DENV pathogenesis in ADE conditions.

Type III interferons (IFNs) primarily act on epithelial cells and protect against virus infection of the mucosa, whereas type I IFNs act more systemically. To date, it has been unknown which epithelial subtypes in the upper airways, the primary site for initial infection for most respiratory viruses, primarily rely on type III IFN or type I IFNs for antiviral protection. To address this question, we performed a single-cell transcriptomics analysis of the epithelial IFN-mediated response focusing on the upper airways of mice. This work identified nine distinct cell types derived from the olfactory epithelium and thirteen distinct cell types from the respiratory epithelium. Interestingly, type I IFNs induced a stronger antiviral transcriptional response than type III IFN in respiratory epithelial cells, whereas in olfactory epithelial cells, including sustentacular (SUS) and Bowman's gland cells (BGC), type III IFN was more dominant compared to type I IFN. SUS and BGC, which provide structural support and maintain the integrity of olfactory sensory neurons, were highly susceptible to infection with a mouse-adapted variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 MA20) but were protected against infection if the animals were prophylactically treated with type III IFN. These findings demonstrate a high degree of cell type heterogeneity in terms of interferon-mediated antiviral responses and reveal a potent role for type III IFNs in protecting the olfactory epithelium.IMPORTANCESARS-CoV-2 infects SUS and BGC in the olfactory epithelium, causing an impairment of structural support and integrity of olfactory sensory neurons that can result in severe olfactory dysfunctions. We observed an unexpected compartmentalization of the IFN-mediated transcriptional response within the airway epithelium, and we found that olfactory epithelial cells preferentially respond to type III IFN, which resulted in robust antiviral protection of SUS and BGC. Given the proximity of the olfactory epithelium to the central nervous system, we hypothesize that evolution favored a type III IFN-biased antiviral immune response in this tissue to limit inflammatory responses in the brain. Cell type-specific antiviral responses in the upper airways, triggered by the different types of IFNs, should be investigated in more detail and carefully taken into consideration during the development of IFN-based antivirals for clinical use.

In a subset of SARS-CoV-2-infected individuals treated with the antiviral nirmatrelvir-ritonavir, the virus rebounds following treatment. The mechanisms driving this rebound are not well understood. We used a mathematical model to describe the longitudinal viral load dynamics of 51 individuals treated with nirmatrelvir-ritonavir, 20 of whom rebounded. Target cell preservation, either by a robust innate immune response or initiation of N-R near the time of symptom onset, coupled with incomplete viral clearance, appears to be the main factor leading to viral rebound. Moreover, the occurrence of viral rebound is likely influenced by the time of treatment initiation relative to the progression of the infection, with earlier treatments leading to a higher chance of rebound. A comparison with an untreated cohort suggests that early treatments with nirmatrelvir-ritonavir may be associated with a delay in the onset of an adaptive immune response. Nevertheless, our model demonstrates that extending the course of nirmatrelvir-ritonavir treatment to a 10-day regimen may greatly diminish the chance of rebound in people with mild-to-moderate COVID-19 and who are at high risk of progression to severe disease. Altogether, our results suggest that in some individuals, a standard 5-day course of nirmatrelvir-ritonavir starting around the time of symptom onset may not completely eliminate the virus. Thus, after treatment ends, the virus can rebound if an effective adaptive immune response has not fully developed. These findings on the role of target cell preservation and incomplete viral clearance also offer a possible explanation for viral rebounds following other antiviral treatments for SARS-CoV-2.

Importance: Nirmatrelvir-ritonavir is an effective treatment for SARS-CoV-2. In a subset of individuals treated with nirmatrelvir-ritonavir, the initial reduction in viral load is followed by viral rebound once treatment is stopped. We show that the timing of treatment initiation with nirmatrelvir-ritonavir may influence the risk of viral rebound. Nirmatrelvir-ritonavir stops viral growth and preserves target cells but may not lead to full clearance of the virus. Thus, once treatment ends, if an effective adaptive immune response has not adequately developed, the remaining virus can lead to rebound. Our results provide insights into the mechanisms of rebound and can help develop better treatment strategies to minimize this possibility.

SARS-CoV-2 poses an ongoing threat to human health as variants continue to emerge. Several effective vaccines are available, but a diminishing number of Americans receive the updated vaccines (only 22% received the 2023 update). Public hesitancy towards vaccines and common occurrence of "breakthrough" infections (i.e., infections of vaccinated individuals) highlight the need for alternative methods to reduce viral transmission. SARS-CoV-2 enters cells by fusing its envelope with the target cell membrane in a process mediated by the viral spike protein, S. The S protein operates via a Class I fusion mechanism in which fusion between the viral envelope and host cell membrane is mediated by structural rearrangements of the S trimer. We previously reported lipopeptides derived from the C-terminal heptad repeat (HRC) domain of SARS-CoV-2 S that potently inhibit fusion by SARS-CoV-2, both in vitro and in vivo. These lipopeptides bear an attached cholesterol unit to anchor them in the membrane. Here, to improve prospects for experimental development and future clinical utility, we employed structure-guided design to incorporate charged residues at specific sites in the peptide to enhance aqueous solubility. This effort resulted in two new, potent lipopeptide inhibitors.

Importance: Despite the existence of vaccines for SARS-CoV-2, the constant evolution of new variants and the occurrence of breakthrough infections highlight the need for new and effective antiviral approaches. We have shown that lipopeptides designed to bind a conserved region on the SARS-CoV-2 spike protein can effectively block viral entry into cells and thereby block infection. To support the feasibility of using this approach in humans, we re-designed these lipopeptides to be more soluble, using information about the structure of the spike protein interacting with the peptides to modify the peptide chain. The new peptides are effective against both SARS-CoV-2 and MERS. The lipopeptides described here could serve as treatment for people who are unvaccinated or who experience breakthrough infections, and the approach to increasing solubility can be applied in a broad spectrum approach to treating infections with emerging viruses.

HIV persists in people living with HIV (PLHIV) on antiretroviral therapy (ART) in long-lived and proliferating latently infected CD4+ T cells that selectively express pro-survival proteins, including the zinc finger proteins, Ikaros and Aiolos. In this study, we investigated whether pomalidomide, an immunomodulatory agent that induces degradation of Ikaros and Aiolos, could increase the death of HIV-infected cells and/or reverse HIV latency. Using an in vitro model of CD4+ T cells infected with a green fluorescent protein (GFP) reporter virus, pomalidomide increased the expression of the pro-survival protein B cell lymphoma (Bcl)-2 and did not increase apoptosis of GFP+ HIV productively infected CD4+ T cells. Pomalidomide also increased the expression of CD155 and UL16-binding protein (ULBP) stress proteins on GFP+ HIV productively infected CD4+ T cells, but this did not translate to enhanced clearance following co-culture with a natural killer (NK) cell line. Using CD4+ T cells from PLHIV on ART, pomalidomide ex vivo activated memory CD4+ T cells resulting in elevated HLA-DR expression and induced CD4+ T cell proliferation but only in the presence of T cell receptor stimulation with anti-CD3 and anti-CD28. There was no effect on cell-associated HIV RNA or the frequency of intact HIV DNA. In conclusion, despite an increase in stress protein expression, promoting Ikaros and Aiolos degradation in CD4+ T cells using pomalidomide did not directly induce apoptosis of HIV-infected cells or induce HIV latency reversal.IMPORTANCEPeople living with HIV (PLHIV) require lifelong antiretroviral therapy (ART) due to the persistence of latently infected cells. The zinc finger proteins, Ikaros and Aiolos, have recently been implicated in promoting the persistence of latently infected cells. In this study, we investigated the effects of pomalidomide, an immunomodulatory imide drug that induces the degradation of Ikaros and Aiolos, on HIV latency reversal and death of infected cells. Using CD4+ T cells from people living with HIV on suppressive antiretroviral therapy, as well as an in vitro model of productive HIV infection, we found that pomalidomide induced T cell activation and expression of stress proteins but no evidence of latency reversal or selective death of infected cells.