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Although the SARS-CoV-2 Omicron variant (BA.1) spread rapidly across the world and effectively evaded immune responses, its viral fitness in cell and animal models was reduced. The precise nature of this attenuation remains unknown as generating replication-competent viral genomes is challenging because of the length of the viral genome (~30 kb). Here, we present a plasmid-based viral genome assembly and rescue strategy (pGLUE) that constructs complete infectious viruses or noninfectious subgenomic replicons in a single ligation reaction with >80% efficiency. Fully sequenced replicons and infectious viral stocks can be generated in 1 and 3 weeks, respectively. By testing a series of naturally occurring viruses as well as Delta-Omicron chimeric replicons, we show that Omicron nonstructural protein 6 harbors critical attenuating mutations, which dampen viral RNA replication and reduce lipid droplet consumption. Thus, pGLUE overcomes remaining barriers to broadly study SARS-CoV-2 replication and reveals deficits in nonstructural protein function underlying Omicron attenuation.

Inhibitors of bromodomain and extraterminal domain (BET) proteins are possible anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prophylactics as they downregulate angiotensin-converting enzyme 2 (ACE2). Here we show that BET proteins should not be inactivated therapeutically because they are critical antiviral factors at the post-entry level. Depletion of BRD3 or BRD4 in cells overexpressing ACE2 exacerbates SARS-CoV-2 infection; the same is observed when cells with endogenous ACE2 expression are treated with BET inhibitors during infection and not before. Viral replication and mortality are also enhanced in BET inhibitor-treated mice overexpressing ACE2. BET inactivation suppresses interferon production induced by SARS-CoV-2, a process phenocopied by the envelope (E) protein previously identified as a possible "histone mimetic." E protein, in an acetylated form, directly binds the second bromodomain of BRD4. Our data support a model where SARS-CoV-2 E protein evolved to antagonize interferon responses via BET protein inhibition; this neutralization should not be further enhanced with BET inhibitor treatment.

Pregnancy confers unique immune responses to infection and vaccination across gestation. To date, there are limited data comparing vaccine- and infection-induced neutralizing Abs (nAbs) against COVID-19 variants in mothers during pregnancy. We analyzed paired maternal and cord plasma samples from 60 pregnant individuals. Thirty women vaccinated with mRNA vaccines (from December 2020 through August 2021) were matched with 30 naturally infected women (from March 2020 through January 2021) by gestational age of exposure. Neutralization activity against the 5 SARS-CoV-2 spike sequences was measured by a SARS-CoV-2-pseudotyped spike virion assay. Effective nAbs against SARS-CoV-2 were present in maternal and cord plasma after both infection and vaccination. Compared with WT spike protein, these nAbs were less effective against the Delta and Mu spike variants. Vaccination during the third trimester induced higher cord-nAb levels at delivery than did infection during the third trimester. In contrast, vaccine-induced nAb levels were lower at the time of delivery compared with infection during the first trimester. The transfer ratio (cord nAb level divided by maternal nAb level) was greatest in mothers vaccinated in the second trimester. SARS-CoV-2 vaccination or infection in pregnancy elicits effective nAbs with differing neutralization kinetics that are influenced by gestational time of exposure.

SARS-CoV-2 non-structural protein Nsp14 is a highly conserved enzyme necessary for viral replication. Nsp14 forms a stable complex with non-structural protein Nsp10 and exhibits exoribonuclease and N7-methyltransferase activities. Protein-interactome studies identified human sirtuin 5 (SIRT5) as a putative binding partner of Nsp14. SIRT5 is an NAD-dependent protein deacylase critical for cellular metabolism that removes succinyl and malonyl groups from lysine residues. Here we investigated the nature of this interaction and the role of SIRT5 during SARS-CoV-2 infection. We showed that SIRT5 interacts with Nsp14, but not with Nsp10, suggesting that SIRT5 and Nsp10 are parts of separate complexes. We found that SIRT5 catalytic domain is necessary for the interaction with Nsp14, but that Nsp14 does not appear to be directly deacylated by SIRT5. Furthermore, knock-out of SIRT5 or treatment with specific SIRT5 inhibitors reduced SARS-CoV-2 viral levels in cell-culture experiments. SIRT5 knock-out cells expressed higher basal levels of innate immunity markers and mounted a stronger antiviral response, independently of the Mitochondrial Antiviral Signaling Protein MAVS. Our results indicate that SIRT5 is a proviral factor necessary for efficient viral replication, which opens novel avenues for therapeutic interventions.

Efforts to determine why new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants demonstrate improved fitness have been limited to analyzing mutations in the spike (S) protein with the use of S-pseudotyped particles. In this study, we show that SARS-CoV-2 virus-like particles (SC2-VLPs) can package and deliver exogenous transcripts, enabling analysis of mutations within all structural proteins and at multiple steps in the viral life cycle. In SC2-VLPs, four nucleocapsid (N) mutations found universally in more-transmissible variants independently increased messenger RNA delivery and expression ~10-fold, and in a reverse genetics model, the serine-202→arginine (S202R) and arginine-203→methionine (R203M) mutations each produced >50 times as much virus. SC2-VLPs provide a platform for rapid testing of viral variants outside of a biosafety level 3 setting and demonstrate N mutations and particle assembly to be mechanisms that could explain the increased spread of variants, including B.1.617.2 (Delta, which contains the R203M mutation).

We identified an emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant by viral whole-genome sequencing of 2,172 nasal/nasopharyngeal swab samples from 44 counties in California, a state in the western United States. Named B.1.427/B.1.429 to denote its two lineages, the variant emerged in May 2020 and increased from 0% to >50% of sequenced cases from September 2020 to January 2021, showing 18.6%-24% increased transmissibility relative to wild-type circulating strains. The variant carries three mutations in the spike protein, including an L452R substitution. We found 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation common to variants B.1.1.7, B.1.351, and P.1. Antibody neutralization assays revealed 4.0- to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California exhibiting decreased antibody neutralization warrants further investigation.

The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.

Lipid droplets (LDs) are organelles involved in lipid storage, maintenance of energy homeostasis, protein sequestration, signaling events and inter-organelle interactions. Recently, LDs have been shown to favor the replication of members from different viral families, such as the Flaviviridae and Coronaviridae. In this work, we show that LDs are essential organelles for members of the Arenaviridae family. A virus-driven reduction of LD number was observed in cultures infected with Junı́n mammarenavirus (JUNV), caused in part by action of the viral nucleoprotein. Notably, we identified a new pool of nucleoprotein and viral RNA that localizes in the vicinity of LDs, suggesting that LDs play a role during the viral replication cycle. Regarding the mechanism behind LD exhaustion, we found evidence that lipophagy is involved in LD degradation with the resulting fatty acids being substrates of fatty acid β-oxidation, which fuels viral multiplication. This work highlights the importance of LDs during the replication cycle of JUNV, contributing to the knowledge of the metabolic changes these mammarenaviruses cause in their hosts. [ABSTRACT FROM AUTHOR]

Lipid droplets (LDs) are organelles involved in lipid storage, maintenance of energy homeostasis, protein sequestration, signaling events and inter-organelle interactions. Recently, LDs have been shown to favor the replication of members from different viral families, such as the Flaviviridae and Coronaviridae. In this work, we show that LDs are essential organelles for members of the Arenaviridae family. A virus-driven reduction of LD number was observed in cultures infected with Junín mammarenavirus (JUNV), caused in part by action of the viral nucleoprotein. Notably, we identified a new pool of nucleoprotein and viral RNA that localizes in the vicinity of LDs, suggesting that LDs play a role during the viral replication cycle. Regarding the mechanism behind LD exhaustion, we found evidence that lipophagy is involved in LD degradation with the resulting fatty acids being substrates of fatty acid β-oxidation, which fuels viral multiplication. This work highlights the importance of LDs during the replication cycle of JUNV, contributing to the knowledge of the metabolic changes these mammarenaviruses cause in their hosts., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Mycobacteria, like many other prokaryotic organisms, do not appear to have membrane-bound organelles to organize the subcellular space. Nevertheless, mycobacteria and related bacteria grow their cell envelope in a spatially controlled manner, restricting cell elongation to the polar regions of the rod-shaped cell. This spatial organization demands that de novo synthesized cell envelope components must be supplied to the polar ends of the cell. Because many cell envelope components are either lipids or built as lipid-anchored precursors, the plasma membrane is the major site of the biosynthesis. Thus, there are logistical questions of where in the plasma membrane these lipids and lipid precursors are made and how they are subsequently delivered to the growing poles of the cell. Our discovery of an intracellular membrane domain (IMD) fills in this gap. Currently available data suggest that the IMD is a membrane domain within the plasma membrane of mycobacteria, which mediates key biosynthetic reactions for cell envelope and other lipid biosynthetic reactions. Consistent with its role in polar growth, the IMD is enriched in the polar regions of actively growing cells and becomes less polarized when the cells experience non-growing conditions. We discuss how such membrane compartmentalization may be generated and maintained in a mycobacterial cell and why it has not evolved into a bona fide organelle. In a broader perspective, we suggest that segregation of biosynthetic pathways into different domains of a planar membrane could be more widespread than we currently think.