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Journal of Virology Aug 2023The APOBEC3 family of DNA cytosine deaminases comprises an important arm of the innate antiviral defense system. The gamma-herpesviruses Epstein-Barr virus and Kaposi's...
The APOBEC3 family of DNA cytosine deaminases comprises an important arm of the innate antiviral defense system. The gamma-herpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus and the alpha-herpesviruses herpes simplex virus (HSV)-1 and HSV-2 have evolved an efficient mechanism to avoid APOBEC3 restriction by directly binding to APOBEC3B and facilitating its exclusion from the nuclear compartment. The only viral protein required for APOBEC3B relocalization is the large subunit of the ribonucleotide reductase (RNR). Here, we ask whether this APOBEC3B relocalization mechanism is conserved with the beta-herpesvirus human cytomegalovirus (HCMV). Although HCMV infection causes APOBEC3B relocalization from the nucleus to the cytoplasm in multiple cell types, the viral RNR (UL45) is not required. APOBEC3B relocalization occurs rapidly following infection suggesting the involvement of an immediate early or early (IE/E) viral protein. In support of this possibility, genetic (IE1 mutant) and pharmacologic (cycloheximide) strategies that prevent the expression of IE/E viral proteins also block APOBEC3B relocalization. In comparison, the treatment of infected cells with phosphonoacetic acid, which interferes with viral late protein expression, still permits A3B relocalization. These results combine to indicate that the beta-herpesvirus HCMV uses an RNR-independent, yet phenotypically similar, molecular mechanism to antagonize APOBEC3B. IMPORTANCE Human cytomegalovirus (HCMV) infections can range from asymptomatic to severe, particularly in neonates and immunocompromised patients. HCMV has evolved strategies to overcome host-encoded antiviral defenses to achieve lytic viral DNA replication and dissemination and, under some conditions, latency and long-term persistence. Here, we show that HCMV infection causes the antiviral factor, APOBEC3B, to relocalize from the nuclear compartment to the cytoplasm. This overall strategy resembles that used by related herpesviruses. However, the HCMV relocalization mechanism utilizes a different viral factor(s) and available evidence suggests the involvement of at least one protein expressed at the early stages of infection. This knowledge is important because a greater understanding of this mechanism could lead to novel antiviral strategies that enable APOBEC3B to naturally restrict HCMV infection.
Topics: Humans; Infant, Newborn; Cytidine Deaminase; Cytomegalovirus; DNA Replication; DNA, Viral; Epstein-Barr Virus Infections; Herpesviridae Infections; Herpesvirus 1, Human; Herpesvirus 4, Human; Immediate-Early Proteins; Minor Histocompatibility Antigens; Ribonucleotide Reductases; Viral Proteins; Virus Replication
PubMed: 37565748
DOI: 10.1128/jvi.00781-23 -
The Journal of International Medical... Jan 2024This study aimed to examine the mechanism of hyperphosphatemia-induced vascular calcification (HPVC).
OBJECTIVE
This study aimed to examine the mechanism of hyperphosphatemia-induced vascular calcification (HPVC).
METHODS
Primary human aortic smooth muscle cells and rat aortic rings were cultured in Dulbecco's modified Eagle's medium supplemented with 0.9 mM or 2.5 mM phosphorus concentrations. Type III sodium-dependent phosphate cotransporter-1 (Pit-1) small interfering RNA and phosphonoformic acid (PFA), a Pit-1 inhibitor, were used to investigate the effects and mechanisms of Pit-1 on HPVC. Calcium content shown by Alizarin red staining, expression levels of Pit-1, and characteristic molecules for phenotypic transition of vascular smooth muscle cells were examined.
RESULTS
Hyperphosphatemia induced the upregulation of Pit-1 expression, facilitated phenotypic transition of vascular smooth muscle cells, and led to HPVC in cellular and organ models. Treatment with Pit-1 small interfering RNA or PFA significantly inhibited Pit-1 expression, suppressed phenotypic transition, and attenuated HPVC.
CONCLUSIONS
Our findings suggest that Pit-1 plays a pivotal role in the development of HPVC. The use of PFA as a Pit-1 inhibitor has the potential for therapeutic intervention in patients with HPVC. However, further rigorous clinical investigations are required to ensure the safety and efficacy of PFA before it can be considered for widespread implementation in clinical practice.
Topics: Animals; Humans; Rats; Aorta; Foscarnet; Hyperphosphatemia; RNA, Small Interfering; Transcription Factors; Vascular Calcification; Sodium-Phosphate Cotransporter Proteins, Type III
PubMed: 38180904
DOI: 10.1177/03000605231222156 -
Journal of Clinical Virology : the... Oct 2023Antiviral resistance in human herpes simplex viruses (HSV) remains a significant clinical challenge in immunocompromised populations. Although molecular tests have...
BACKGROUND
Antiviral resistance in human herpes simplex viruses (HSV) remains a significant clinical challenge in immunocompromised populations. Although molecular tests have largely replaced viral culture for HSV diagnosis and molecular antiviral resistance testing is available for many viruses, HSV resistance testing continues to rely on phenotypic, viral culture-based methods, requiring weeks for results. Consequently, treatment of suspected HSV resistance remains largely empiric.
METHODS
We used HSV whole genome sequencing and a database of previously characterized HSV acyclovir and foscarnet resistance mutations to evaluate the performance of genotypic antiviral resistance testing among 19 control strains compared to in-house plaque reduction assay (PRA) and 25 clinical isolates sent for reference lab PRA antiviral resistance testing.
RESULTS
Among control strains, 23/29 (79.3%) results were concordant, 5 (17.2%) were indeterminate, and 1 (3.4%) was discordant. Indeterminate results were caused by variants of uncertain significance (VUS), including mutations without published phenotypes and mutations with contradictory results. Among clinical isolates, 14/40 (35%) results were concordant, 17 (42.5%) were indeterminate, and 9 (22.5%) were discordant. All discordant results were in reportedly phenotypically-susceptible HSV-1 strains yet possessed resistance mutations. Three contained resistant subpopulations. 6/8 (75%) discordant phenotypes were concordant with resistant genotypes upon repeat PRA.
CONCLUSIONS
These data support the combination of genotypic and phenotypic testing to diagnose HSV resistance more accurately and likely more rapidly than phenotypic testing alone. Genotypic context of resistance mutations and the ability of viral strains to form plaques in culture may affect phenotypic resistance results, highlighting the limitations of PRA alone as a gold standard method.
Topics: Humans; Antiviral Agents; Herpesvirus 2, Human; Acyclovir; Foscarnet; Herpesvirus 1, Human; Genotype; Drug Resistance, Viral; Herpes Simplex
PubMed: 37586184
DOI: 10.1016/j.jcv.2023.105554 -
Yakugaku Zasshi : Journal of the... 2024Quantitative NMR (qNMR), particularly H-qNMR, is useful for determining the absolute purity of organic molecules. However, identifying the target signal(s) for...
Quantitative NMR (qNMR), particularly H-qNMR, is useful for determining the absolute purity of organic molecules. However, identifying the target signal(s) for quantification is difficult, because of the overlap and complexity of organic molecules. Therefore, we focused on the P nucleus, owing to the simplicity of its signals, and investigated the P-qNMR absolute determination method by using organophosphorus drugs, water-soluble cyclophosphamide hydrate (CP), and water-insoluble sofosbuvir (SOF). The optimized and reproducible P-qNMR conditions, such as qNMR sample preparation [i.e., selecting suitable deuterated solvents and a reference standard (RS) for P-qNMR], hygroscopicity and solution stability of the analyte and RS, and qNMR measurements-such as acquisition time, relaxation delay time, and spectral width-were examined. The CP purities determined using P-qNMR agreed well with those for the established H-qNMR method in DO. In contrast, the SOF purity determined using P-qNMR was 1.6% higher than that for H-qNMR in the protic solvent CDOD. Therefore, using a protic solvent, such as CDOD, was not suitable for P-qNMR; the deuterium exchange with the RS for P-qNMR (i.e., phosphonoacetic acid) resulted in a small integrated intensity. Consequently, the aprotic solvent DMSO-d was employed to determine the SOF purity. The data revealed that the SOF purities determined using P-qNMR agreed well with the established H-qNMR values, indicating that the absolute quantification of SOF using both P-qNMR and H-qNMR is possible in DMSO-d. Thus, we established an optimized and reproducible P-qNMR method in validation study across multiple laboratories.
Topics: Organophosphorus Compounds; Dimethyl Sulfoxide; Water; Solvents; Pharmaceutical Preparations
PubMed: 38556308
DOI: 10.1248/yakushi.23-00151-3 -
Biochemistry Dec 2023CRISPR gene editing and control systems continue to emerge and inspire novel research and clinical applications. Advances in CRISPR performance such as optimizing the...
CRISPR gene editing and control systems continue to emerge and inspire novel research and clinical applications. Advances in CRISPR performance such as optimizing the duration of activity in cells, tissues, and organisms, as well as limiting off-target activities, have been extremely important for expanding the utility of CRISPR-based systems. By investigating the effects of various chemical modifications in guide RNAs (gRNAs) at defined positions and combinations, we find that 2'--methyl-3'-phosphonoacetate (MP) modifications can be substantially more effective than 2'--methyl-3'-phosphorothioate (MS) modifications at the 3' ends of single-guide RNAs (sgRNAs) to promote high editing yields, in some instances showing an order of magnitude higher editing yield in human cells. MP-modified 3' ends are especially effective at promoting the activity of guide RNAs cotransfected with Cas messenger RNA (mRNA), as the gRNA must persist in cells until the Cas protein is expressed. We demonstrate such an MP enhancement for sgRNAs cotransfected with a BE4 mRNA for cytidine base editing and also demonstrate that MP at the 3' ends of prime editing guide RNAs (pegRNAs) cotransfected with PE2 mRNA can promote maximal prime editing yields. In the presence of serum, sgRNAs with MP-modified 3' ends showed marked improvements in editing efficiency over sgRNAs with MS-modified 3' ends codelivered with Cas9 mRNA and showed more modest improvements at enhancing the activity of transfected ribonucleoprotein (RNP) complexes. Our results suggest that MP should be considered as a performance-enhancing modification for the 3' ends of synthetic gRNAs, especially in situations where the guide RNAs may be susceptible to exonuclease-mediated degradation.
Topics: Humans; CRISPR-Cas Systems; RNA, Guide, CRISPR-Cas Systems; Phosphonoacetic Acid; Gene Editing; RNA, Messenger
PubMed: 35436085
DOI: 10.1021/acs.biochem.1c00768 -
Chemical & Pharmaceutical Bulletin Jan 2024The spectrum of P-NMR is fundamentally simpler than that of H-NMR; consequently identifying the target signal(s) for quantitation is simpler using quantitative P-NMR...
The spectrum of P-NMR is fundamentally simpler than that of H-NMR; consequently identifying the target signal(s) for quantitation is simpler using quantitative P-NMR (P-qNMR) than using quantitative H-NMR (H-qNMR), which has been already established as an absolute determination method. We have previously reported a P-qNMR method for the absolute determination of cyclophosphamide hydrate and sofosbuvir as water-soluble and water-insoluble organophosphorus compounds, respectively. This study introduces the purity determination of brigatinib (BR), an organophosphorus compound with limited water solubility, using P-qNMR at multiple laboratories. Phosphonoacetic acid (PAA) and 1,4-BTMSB-d were selected as the reference standards (RSs) for P-qNMR and H-qNMR, respectively. The qNMR solvents were chosen based on the solubilities of BR and the RSs for qNMR. CDOH was selected as the solvent for P-qNMR measurements to prevent the influence of deuterium exchange caused by the presence of exchangeable intramolecular protons of BR and PAA on the quantitative values, while CDOD was the solvent of choice for the H-qNMR measurements to prevent the influence of water signals and the exchangeable intramolecular protons of BR and PAA. The mean purity of BR determined by P-qNMR was 97.94 ± 0.69%, which was in agreement with that determined by H-qNMR (97.26 ± 0.71%), thus indicating the feasibility of purity determination of BR by P-qNMR. Therefore, the findings of this study may provide an effective method that is simpler than conventional H-qNMR for the determination of organophosphorus compounds.
Topics: Protons; Organophosphorus Compounds; Reference Standards; Water; Solvents
PubMed: 37899177
DOI: 10.1248/cpb.c23-00635 -
Journal of Infection in Developing... Jan 2024Human herpesvirus 6B (HHV-6B) encephalitis is common in immunosuppressed patients and presents a diagnostic challenge for physicians. Metagenomic next-generation...
Metagenomic Next-Generation Sequencing (mNGS) of cerebrospinal fluid for diagnosis of human herpesvirus 6B encephalitis following transplantation for severe aplastic anemia.
INTRODUCTION
Human herpesvirus 6B (HHV-6B) encephalitis is common in immunosuppressed patients and presents a diagnostic challenge for physicians. Metagenomic next-generation sequencing (mNGS) may facilitate early diagnosis of HHV-6B encephalitis. Herein, we described a case of HHV-6B encephalitis following transplantation for severe aplastic anemia (SAA) diagnosed by mNGS.
CASE SUMMARY
A 31-year-old male underwent myeloablative haploid hematopoietic stem cell transplantation for the treatment of SAA. On day + 21 after transplantation, the patient developed symptoms such as sudden epilepsy, drowsiness, memory dislocation, and memory loss. HHV-6B encephalitis was confirmed based on cranial MRI and mNGS of cerebrospinal fluid. Following antiviral therapy with sodium foscarnet, the symptoms improved and HHV-6B was negative by mNGS. There were no serious sequelae. Currently, the patient is in good health and is still under follow-up.
CONCLUSIONS
A case of HHV-6B encephalitis after SAA transplantation was diagnosed by mNGS of cerebrospinal fluid in time and was effectively treated with sodium foscarnet.
Topics: Male; Humans; Adult; Foscarnet; Herpesvirus 6, Human; Anemia, Aplastic; Encephalitis, Viral; Roseolovirus Infections; Hematopoietic Stem Cell Transplantation; Encephalitis; High-Throughput Nucleotide Sequencing; Sodium
PubMed: 38377081
DOI: 10.3855/jidc.18152