Differences in endosomal Rab gene expression between positive and negative COVID-19 patients.

OBJECTIVE: SARS CoV-2, the etiologic agent of coronavirus disease-2019 (COVID-19) is well-known to use ACE2 to begin internalization. Some viruses enter the host cell through the endocytosis process and involve some endocytosis proteins, such as the Rab family. However, the relationship between SARS CoV-2 infection with endocytic mRNA RAB5, RAB7, and RAB11B is unknown. This study aims to compare the expression of RAB5, RAB7, and RAB11B between positive and negative COVID-19 patient groups. RESULTS: Both viral and human epithelial RNA Isolation and RT-PCR were performed from 249 samples. The genes expression was analysed using appropriate statistical tests. We found the Median (inter-quartile range/IQR) of RAB5, RAB7, and RAB11B expression among the COVID-19 patient group was 2.99 (1.88), 0.17 (0.47), 0.47 (1.49), and 1.60 (2.88), 1.05 (2.49), 1.10 (3.96) among control group respectively. We proceeded with Mann Whitney U Test and found that RAB5 expression was significantly increased (P < 0.001), and RAB7 and RAB11B expression was significantly decreased (P < 0.001 and P = 0.036) in the COVID-19 patient group compared to the control group. This first report showed significant differences in RAB5, RAB7, and RAB11B exist between COVID-19 positive and negative patients.

Structural mechanism for regulation of Rab7 by site-specific monoubiquitination.

Site-specific ubiquitination can regulate the functions of Rab proteins in membrane trafficking. Previously we showed that site-specific monoubiquitination on Rab5 downregulates its function. Rab7 acts in the downstream of Rab5. Although site-specific ubiquitination of Rab7 can affect its function, it remains elusive how the ubiquitination is involved in modulation of the function of Rab7 at molecular level. Here, we report molecular basis for the regulation of Rab7 by site-specific monoubiquitination. Rab7 was predominantly monoubiquitinated at multiple sites in the membrane fraction of cultured cells. Two major ubiquitination sites (K191 and K194), identified by mutational analysis with single K mutants, were responsible for membrane localization of monoubiquitinated Rab7. Using small-angle X-ray scattering, we derived structural models of site-specifically monoubiquitinated Rab7 in solution. Structural analysis combined with molecular dynamics simulation corroborated that the ubiquitin moieties on K191 and K194 are key determinants for exclusion of Rab7 from the endosomal membrane. Ubiquitination on the two major sites apparently mitigated colocalization of Rab7 with ORF3a of SARS-CoV-2, potentially deterring the egression of SARS-CoV-2. Our results establish that the regulatory effects of a Rab protein through site-specific monoubiquitination are commonly observed among Rab GTPases while the ubiquitination sites differ in each Rab protein.

Differential Expression of Rab5 and Rab7 Small GTPase Proteins in Placental Tissues From Pregnancies Affected by Maternal Coronavirus Disease 2019.

PURPOSE: The majority of pregnancies affected by maternal coronavirus disease 2019 (COVID-19) do not result in fetal transmission. However, several studies have identified parenchymal changes in their placental tissues, suggesting a placental response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the maternal-fetal interface. Although many COVID-19 placental studies have focused on the expression of the canonical SARS-CoV-2 entry proteins angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2, further characterization of subcellular molecules involved in viral trafficking have not yet been investigated in these tissues. Of interest are Rab proteins, a family of small GTPase proteins that direct intracellular transport between different endocytic organelles. Rab5 and Rab7 in particular have previously been implicated in HIV and cytomegalovirus invasion of placental trophoblast cells in vitro; the localization of these molecules has not been fully characterized within the human maternal-fetal interface, however, or within placental tissues from SARS-CoV-2-infected pregnancies. METHODS: Using fluorescent immunohistochemistry, Rab5 and Rab7 placental localization and comparative fluorescence intensity were explored in a cohort of placental tissues from pregnancies affected by maternal COVID-19 disease (COVID, n = 15) compared with contemporary control subjects (Control, n = 10). Fluorescence intensity was quantified by using corrected total cell fluorescence values. FINDINGS: Within placental villi, Rab5 was consistently localized in syncytiotrophoblast and cytotrophoblast cells. Rab5 had significantly higher mean (SEM) fluorescence intensity in the COVID cohort (Control, 1.96 [0.16]; COVID, 2.62 [0.09]; P = 0.0014). In contrast, although Rab7 was also localized within placental villous syncytiotrophoblast and cytotrophoblast cells, mean (SEM) Rab7 fluorescence intensity was significantly downregulated in COVID vs Control placentas (Control, 35.9 [4.1]; COVID, 20.1 [0.52]; P = 0.0001). IMPLICATIONS: This differential expression of Rab5 and Rab7 suggests that placental endocytic pathways may be altered at the maternal-fetal interface in pregnancies affected by maternal SARS-CoV-2 infection. As key molecules governing intracellular vesicle transport, including viral trafficking, Rab GTPase proteins may be of interest for ongoing studies examining placental responses to COVID-19 in pregnancy.

Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells.

To better understand host-virus genetic dependencies and find potential therapeutic targets for COVID-19, we performed a genome-scale CRISPR loss-of-function screen to identify host factors required for SARS-CoV-2 viral infection of human alveolar epithelial cells. Top-ranked genes cluster into distinct pathways, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. We validate these gene targets using several orthogonal methods such as CRISPR knockout, RNA interference knockdown, and small-molecule inhibitors. Using single-cell RNA-sequencing, we identify shared transcriptional changes in cholesterol biosynthesis upon loss of top-ranked genes. In addition, given the key role of the ACE2 receptor in the early stages of viral entry, we show that loss of RAB7A reduces viral entry by sequestering the ACE2 receptor inside cells. Overall, this work provides a genome-scale, quantitative resource of the impact of the loss of each host gene on fitness/response to viral infection.

Dysregulation of endocytic machinery and ACE2 in small airways of smokers and COPD patients can augment their susceptibility to SARS-CoV-2 (COVID-19) infections.

Lungs of smokers and chronic obstructive pulmonary disease (COPD) are severely compromised and are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) attack. The dangerous combination of enhanced SARS-CoV-2 attachment receptor protein ACE2 along with an increase in endocytic vacuoles will enable viral attachment, entry, and replication. The objective of the study was to identify the presence of SARS-CoV-2 host attachment receptor angiotensin-converting enzyme-2 (ACE2) along with endocytic vacuoles, early endosome antigen-1 (EEA1), late endosome marker RAB7, cathepsin-L, and lysosomal associated membrane protein-1 (LAMP-1) as lysosome markers in the airways of smokers and COPD patients. The study design was cross-sectional and involved lung resections from 39 patients in total, which included 19 patients with Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage I or GOLD stage II COPD, of which 9 were current smokers with COPD (COPD-CS) and 10 were ex-smokers with COPD (COPD-ES), 10 were normal lung function smokers, and 10 were never-smoking normal controls. Immunostaining for ACE2, EEA1, RAB7, and cathepsin-L was done. A comparative description for ACE2, EEA1, RAB7, and cathepsin-L expression pattern is provided for the patient groups. Furthermore, staining intensity for LAMP-1 lysosomes was measured as the ratio of the LAMP-1-stained areas per total area of epithelium or subepithelium, using Image ProPlus v7.0 software. LAMP-1 expression showed a positive correlation to patient smoking history while in COPD LAMP-1 negatively correlated to lung function. The active presence of ACE2 protein along with endocytic vacuoles such as early/late endosomes and lysosomes in the small airways of smokers and COPD patients provides evidence that these patient groups could be more susceptible to COVID-19.

ß-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway.

ß-Coronaviruses are a family of positive-strand enveloped RNA viruses that includes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we demonstrate that ß-coronaviruses utilize lysosomal trafficking for egress rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of ß-coronaviruses results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation pathways. ß-Coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.