Retroelements are expressed in diverse types of cancer and are related to tumorigenesis and to cancer progression. We characterized the expression of retroelements in cervical cancer and explored their interplay with HPV infection and their association with expression of neighboring genes. Forty biopsies of invasive cervical carcinoma (squamous cell carcinomas and adenocarcinomas) with genotyped HPV were selected and analyzed for human endogenous retrovirus (HERV) and long interspersed nuclear element 1 (L1) expression through RNA-seq data. We found 8060 retroelements expressed in the samples and a negative correlation of DNA methyltransferase 1 expression with the two most expressed L1 elements. A total of 103 retroelements were found differentially expressed between tumor histological types and between HPV types, including several HERV families (HERV-K, HERV-H, HERV-E, HERV-I and HERV-L). The comparison between HPV mono- and co-infections showed the highest proportion of differentially expressed L1 elements. The location of retroelements affected neighboring gene expression, such as shown for the interleukin-20 gene family. Three HERVs and seven L1 were located close to this gene family and two L1 showed a positive association with IL20RB expression. This study describes the expression of retroelements in cervical cancer and shows their association with HPV status and host gene expression.

We generated a replication-competent OC43 human seasonal coronavirus (CoV) expressing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike in place of the native spike (rOC43-CoV2 S). This virus is highly attenuated relative to OC43 and SARS-CoV-2 in cultured cells and animals and is classified as a biosafety level 2 (BSL-2) agent by the NIH biosafety committee. Neutralization of rOC43-CoV2 S and SARS-CoV-2 by S-specific monoclonal antibodies and human sera is highly correlated, unlike recombinant vesicular stomatitis virus-CoV2 S. Single-dose immunization with rOC43-CoV2 S generates high levels of neutralizing antibodies against SARS-CoV-2 and fully protects human ACE2 transgenic mice from SARS-CoV-2 lethal challenge, despite nondetectable replication in respiratory and nonrespiratory organs. rOC43-CoV2 S induces S-specific serum and airway mucosal immunoglobulin A and IgG responses in rhesus macaques. rOC43-CoV2 S has enormous value as a BSL-2 agent to measure S-specific antibodies in the context of a bona fide CoV and is a candidate live attenuated SARS-CoV-2 mucosal vaccine that preferentially replicates in the upper airway.

In coronavirus (CoV)-infected cells, several structural and accessory proteins are synthesized from subgenome RNAs (sgRNA) containing a common genomic 5ʹ-leader followed by a given open reading frame (ORF). We report that the abundance of these sgRNAs varies with distance from the 3′-end of the genome. Thus, there are more sgRNAs encoding nucleocapsid (N) than spike (S), presumably the results from discontinuous 5ʹ−3ʹ transcription template switch mediated by the viral replication and transcription complex (RTC). We optimized the circular polymerase extension reaction (CPER) methodology to generate infectious double-stranded circular cDNA (ds-circDNA) containing the mNeonGreen (NG) reporter in accessory ORFs of human CoVs OC43 and SARS-CoV-2. In each CoV, we found that levels of sgRNAs and NG expression increased with 3ʹ proximal genomic NG location. By reinfection of HCT-8 cells with the same MOI 0.01, however, we found that the slow-growing OC43 NG-ns2 virions exhibited equal infectivity and productivity as the fast-growing OC43 NG-ns12.9 virions. Introduction of point-mutations into the mapped TRS _B motif for synthesis of the OC43 ns12.9 and M sgRNAs led to disrupt TRS _B -TRS _L cross-interactions and block production of the corresponding sgRNAs and infectious virions. Together, our data indicate that, by using an optimized CPER approach for positional NG insertions, we demonstrated the progressional reduction of the RTC-mediated template switch (read-through individual TRS _B ) efficiency in production of the corresponding sgRNAs in an order from the 3ʹ viral genome end. IMPORTANCE: The mechanism for sgRNA synthesis in the coronavirus life cycle is poorly understood. The current model suggests discontinuous template-switch transcription mediated by viral replication and transcription complex (RTC) for synthesis of individual sgRNAs to translate corresponding structural and accessory proteins but lacks experimental data support. This report provides the first experimental evidence that, in both hCoV-OC43 and SARS-CoV-2, viral RTC synthesizes its sgRNAs by long-range base-pairing between a distal transcription regulatory body sequence (TRS _B ) upstream of each structural/accessory ORF and the transcription regulatory leader sequence (TRS _L ) from the viral genome 5ʹ-UTR, leading to the production of viral sgRNAs in abundance order from the viral genome 3ʹ -end, with more N sgRNAs but less S sgRNAs. Our data support a “first-come, first-serving” model in TRS _B -TRS _L cross-interaction and read-through TRS _B process to mediate discontinuous transcription switch in coronavirus sgRNA synthesis in a 5ʹ−3ʹ transcription direction from the 3ʹ viral genome during coronavirus infection.

Single-cell resolution spatial transcriptomics enables transcriptome-wide molecular profiling within intact tissue architecture, providing unprecedented opportunities to investigate cellular organization and disease-associated molecular states in the human brain. However, applying these technologies to post-mortem human brain tissue remains challenging due to RNA degradation, heterogeneity in tissue preservation, and a lack of standardized analytical workflows. These challenges are particularly pronounced for whole-transcriptome platforms, where successful implementation requires optimization of both experimental and computational procedures. Here, we present an end-to-end framework for single-cell-resolution, whole-transcriptome spatial transcriptomics of fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) post-mortem human brain tissue. The framework combines an optimized experimental workflow with a preservation-agnostic bioinformatics pipeline for data processing, integration, and annotation. Experimentally, we show that a condensed two-day Visium HD workflow provides improved library quality, lowered qPCR cycle thresholds, and more consistent fragment size distributions. Sequencing saturation analyses further identified cost-effective sequencing depths that maximize transcript recovery while minimizing redundant sequencing. Computationally, we established a scalable workflow incorporating DAPI-based nuclear segmentation, transcript assignment, quality control, reference-guided integration, clustering, and cell-type annotation. We implemented a reference-based highly variable gene selection strategy to enable robust cross-sample harmonization independent of tissue preservation method. Application of this framework to seven Alzheimer's disease frontal cortex specimens (five FF and two FFPE) generated a unified single-cell spatial transcriptomic atlas comprising more than 530,000 spatially resolved cells. The integrated dataset resolved major neuronal, glial, and vascular cell populations, recapitulated expected cortical architecture, and enabled direct comparison of FF- and FFPE-derived spatial transcriptomic profiles. Together, this work provides a practical experimental and computational framework for single-cell resolution, whole-transcriptome spatial transcriptomics in post-mortem human brain tissue and delivers a publicly available resource that expands the utility of archived and frozen specimens for studies of neurodegeneration and other neurological disorders.