Cellular APOBEC3A deaminase drives mutations in the SARS-CoV-2 genome.

The number of genetic variations in the SARS-CoV-2 genome has been increasing primarily due to continuous viral mutations. Here, we report that the human APOBEC3A (A3A) cytidine deaminase plays a critical role in the induction of C-to-U substitutions in the SARS-CoV-2 genome. Bioinformatic analysis of the chronological genetic changes in a sequence database indicated that the largest UC-to-UU mutation signature, consistent with APOBEC-recognized nucleotide motifs, was predominant in single-stranded RNA regions of the viral genome. In SARS-CoV-2-infected cells, exogenous expression of A3A but not expression of other APOBEC proteins induced UC-to-UU mutations in viral RNA (vRNA). Additionally, the mutated C bases were often located at the tips in bulge or loop regions in the vRNA secondary structure. Interestingly, A3A mRNA expression was drastically increased by interferons (IFNs) and tumour necrosis factor-α (TNF-α) in epithelial cells derived from the respiratory system, a site of efficient SARS-CoV-2 replication. Moreover, the UC-to-UU mutation rate was increased in SARS-CoV-2 produced from lung epithelial cells treated with IFN-ß and TNF-α, but not from CRISPR/Cas9-based A3A knockout cells. Collectively, these findings demonstrate that A3A is a primary host factor that drives mutations in the SARS-CoV-2 RNA genome via RNA editing.

Nanopore Sequencing for Characterization of HIV-1 Recombinant Forms.

High genetic diversity, including the emergence of recombinant forms (RFs), is one of the most prominent features of human immunodeficiency virus type 1 (HIV-1). Conventional detection of HIV-1 RFs requires pretreatments, i.e., cloning or single-genome amplification, to distinguish them from dual- or multiple-infection variants. However, these processes are time-consuming and labor-intensive. Here, we constructed a new nanopore sequencing-based platform that enables us to obtain distinctive genetic information for intersubtype RFs and dual-infection HIV-1 variants by using amplicons of HIV-1 near-full-length genomes or two overlapping half-length genome fragments. Repeated benchmark tests of HIV-1 proviral DNA revealed consensus sequence inference with a reduced error rate, allowing us to obtain sufficiently accurate sequence data. In addition, we applied the platform for sequence analyses of 9 clinical samples with suspected HIV-1 RF infection or dual infection according to Sanger sequencing-based genotyping tests for HIV-1 drug resistance. For each RF infection case, replicated analyses involving our nanopore sequencing-based platform consistently produced long consecutive analogous consensus sequences with mosaic genomic structures consisting of two different subtypes. In contrast, we detected multiple heterologous sequences in each dual-infection case. These results demonstrate that our new nanopore sequencing platform is applicable to identify the full-length HIV-1 genome structure of intersubtype RFs as well as dual-infection heterologous HIV-1. Since the genetic diversity of HIV-1 continues to gradually increase, this system will help accelerate full-length genome analysis and molecular epidemiological surveillance for HIV-1. IMPORTANCE HIV-1 is characterized by large genetic differences, including HIV-1 recombinant forms (RFs). Conventional genetic analyses require time-consuming pretreatments, i.e., cloning or single-genome amplification, to distinguish RFs from dual- or multiple-infection cases. In this study, we developed a new analytical system for HIV-1 sequence data obtained by nanopore sequencing. The error rate of this method was reduced to ~0.06%. We applied this system for sequence analyses of 9 clinical samples with suspected HIV-1 RF infection or dual infection, which were extracted from 373 cases of HIV patients based on our retrospective analysis of HIV-1 drug resistance genotyping test results. We found that our new nanopore sequencing platform is applicable to identify the full-length HIV-1 genome structure of intersubtype RFs as well as dual-infection heterologous HIV-1. Our protocol will be useful for epidemiological surveillance to examine HIV-1 transmission as well as for genotypic tests of HIV-1 drug resistance in clinical settings.

Retrospective Analysis of the Efficacy of Early Antiretroviral Therapy in HIV-1-Infected Patients Coinfected with Pneumocystis jirovecii.

The early initiation of antiretroviral therapy (ART) in HIV-infected patients shortly after the initiation of treatment for Pneumocystis pneumonia (PCP) has not been fully validated in a clinical setting. We retrospectively extracted all patients diagnosed with HIV-related PCP (HIV-PCP), including those with severe cases, who were treated with first-line ART in our hospital. The HIV-PCP patients were divided into two groups: an early ART group (patients who commenced ART within 21 days after the start of PCP treatment) and a deferred ART group (patients who started ART after 22 days). We compared the incidence of AIDS progression or death, the virological suppression rate, and changes in the CD4+ cell count at 24 weeks after the initiation of ART between the two groups. In addition, we analyzed the incidences of immune reconstitution inflammatory syndrome and grade 3 or 4 laboratory and clinical adverse events within 24 weeks as safety outcomes. Ninety-one HIV-PCP patients (36 in the early ART group and 55 in the deferred group) were included in this study. We found no significant difference in the incidence of AIDS progression or death between the two groups. Virological outcomes tended to be better in the early ART group but were not significantly different. Increases in the CD4+ cell counts at 24 weeks were comparable in both groups, suggesting that the safety was not significantly different. Analysis of the propensity-score matched cohort was performed to adjust for selection bias, and no significant difference was found in any outcome. Our results suggest that early ART introduction can be considered for untreated HIV-positive patients with PCP on the basis of efficacy and safety.

Efficacy of endobronchial ultrasound-guided transbronchial biopsy without guide sheath for small peripheral pulmonary lesions (≤15 mm): A retrospective cohort study.

INTRODUCTION: Endobronchial ultrasonography-guided transbronchial biopsy (EBUS-TBB) with guide sheath (GS) is an effective procedure for diagnosing small peripheral pulmonary lesions (PPLs) (≤20 mm in the largest diameter). However, samples obtained using EBUS-TBB with GS are small, and the diagnostic yield of small PPLs biopsied using EBUS-TBB with GS is unsatisfactory. OBJECTIVES: The aim of this study was to evaluate the diagnostic yield of small PPLs using EBUS-TBB without GS compared to that with GS. MATERIALS AND METHODS: Between 1 April 2013 and 31 March 2015, 276 consecutive lesions were biopsied using EBUS-TBB with GS or without GS. We retrospectively compared EBUS-TBB with and without GS in terms of the diagnostic yield and complications related to small PPLs (≤20 mm). RESULTS: Of the 276 lesions who underwent EBUS-TBB with or without GS, we identified 80 lesions with small PPLs (≤20 mm). Sixty-two lesions were successfully diagnosed by EBUS-TBB (77.5%, diagnostic yield). The diagnostic yield of PPLs using EBUS-TBB without GS was not significantly higher than that using EBUS-TBB with GS (34/41 = 82.9% and 28/39 = 71.7%, respectively; p = 0.233). However, according to size (≤15 mm or > 15 mm), location (upper, middle/lingular, or lower area), and structure (solid nodule or ground-glass opacity), the diagnostic yield of small PPLs (≤15 mm) using EBUS-TBB without GS was significantly higher than with GS (21/26 = 80.7% vs. 8/16 = 50.0%, p = 0.036). There were no complications among the two groups. CONCLUSIONS: EBUS-TBB without GS is an effective and safe procedure for diagnosing small PPLs (≤15 mm) compared to that with GS.

Regulation of DNA polymerase POLD4 influences genomic instability in lung cancer.

Genomic instability is an important factor in cancer susceptibility, but a mechanistic understanding of how it arises remains unclear. We examined hypothesized contributions of the replicative DNA polymerase δ (pol δ) subunit POLD4 to the generation of genomic instability in lung cancer. In examinations of 158 lung cancers and 5 mixtures of 10 normal lungs, cell cycle- and checkpoint-related genes generally showed mRNA expression increases in cancer, whereas POLD4 showed reduced mRNA in small cell lung cancer (SCLC). A fraction of non-small cell lung cancer patients also showed low expression comparable with that in SCLC, which was associated with poor prognosis. The lung cancer cell line ACC-LC-48 was found to have low POLD4 expression, with higher histone H3K9 methylation and lower acetylation in the POLD4 promoter, as compared with the A549 cell line with high POLD4 expression. In the absence of POLD4, pol δ exhibited impaired in vitro DNA synthesis activity. Augmenting POLD4 expression in cells where it was attenuated altered the sensitivity to the chemical carcinogen 4-nitroquinoline-1-oxide. Conversely, siRNA-mediated reduction of POLD4 in cells with abundant expression resulted in a cell cycle delay, checkpoint activation, and an elevated frequency of chromosomal gap/break formation. Overexpression of an engineered POLD4 carrying silent mutations at the siRNA target site rescued these phenotypes, firmly establishing the role of POLD4 in these effects. Furthermore, POLD4 overexpression reduced intrinsically high induction of γ-H2AX, a well-accepted marker of double-stranded DNA breaks. Together, our findings suggest that reduced expression of POLD4 plays a role in genomic instability in lung cancer.