Increased circulating microparticles contribute to severe infection and adverse outcomes of COVID-19 in patients with diabetes.

Patients with diabetes infected with COVID-19 have greater mortality than those without comorbidities, but the underlying mechanisms remain unknown. This study aims to identify the mechanistic interactions between diabetes and severe COVID-19. Microparticles (MPs), the cell membrane-derived vesicles released on cell activation, are largely increased in patients with diabetes. To date, many mechanisms have been postulated for increased severity of COVID-19 in patients with underlying conditions, but the contributions of excessive MPs in patients with diabetes have been overlooked. This study characterizes plasma MPs from normal human subjects and patients with type 2 diabetes in terms of amount, cell origins, surface adhesive properties, ACE2 expression, spike protein binding capacity, and their roles in SARS-CoV-2 infection. Results showed that over 90% of plasma MPs express ACE2 that binds the spike protein of SARS-CoV-2. MPs in patients with diabetes increase 13-fold in quantity and 11-fold in adhesiveness when compared with normal subjects. Perfusion of human plasma with pseudo-typed SARS-CoV-2 virus or spike protein-bound MPs into human endothelial cell-formed microvessels-on-a chip demonstrated that MPs from patients with diabetes, not normal subjects, interact with endothelium and carry SARS-CoV-2 into cells through endocytosis, providing additional virus entry pathways and enhanced infection. Results also showed a large percentage of platelet-derived tissue factor-bearing MPs in diabetic plasma, which could contribute to thrombotic complications with SARS-CoV-2 infection. This study reveals a dual role of diabetic MPs in promoting SARS-CoV-2 entry and propagating vascular inflammation. These findings provide novel mechanistic insight into the high prevalence of COVID-19 in patients with diabetes and their propensity to develop severe vascular complications.NEW & NOTEWORTHY This study provides the first evidence that over 90% of human plasma microparticles express ACE2 that binds SARS-CoV-2 S protein with high affinity. Thus, the highly elevated adhesive circulating microparticles identified in patients with diabetes not only have greater SARS-CoV-2 binding capacity but also enable additional viral entry through virus-bound microparticle-endothelium interactions and enhanced infection. These findings reveal a novel mechanistic insight into the adverse outcomes of COVID-19 in patients with diabetes.

The Immune-Specific E3 Ubiquitin Ligase MARCH1 Is Upregulated during Human Cytomegalovirus Infection to Regulate Iron Levels.

Human cytomegalovirus (HCMV) modulates numerous cellular pathways to facilitate infection. Iron is essential to many cellular processes and is often incorporated into proteins and enzymes involved in oxidative phosphorylation and DNA synthesis and repair, among others. Despite its prominent role in the cell, little is known about the regulation of iron metabolism during HCMV infection. Herein, we observe modulation of the transferrin receptor (TfR) during infection and a corresponding change in the cellular labile iron pool. TfR and the iron pool are increased early during infection and then return to mock levels at the late stages of infection. We identified the cellular ubiquitin ligase MARCH1 as an important regulator of TfR. MARCH1 plays a proviral role during infection, as its knockdown leads to a decrease in infectious titers. Knockdown of MARCH1 also leads to an increase in ROS, lipid peroxidation, and mitochondrial dysfunction. Inhibiting an early increase in TfR expression during infection also decreases virus production. These findings indicate the importance of tightly regulating iron metabolism during HCMV infection to facilitate efficient virus production. IMPORTANCE Iron is essential for cells, playing important roles in energy generation, DNA replication, and gene expression. During infection, HCMV alters many cellular processes to aid its replication. We found that iron levels are tightly regulated during infection and that dysregulation of iron levels alters the ability to produce infectious virions. We also found that HCMV inactivates many of the cellular safeguards put in place to deal with excess iron. Thus, infected cells become more susceptible to variations in iron levels, which could be exploited as a therapeutic strategy for dealing with HCMV infections.

Monoclonal Antibodies to S and N SARS-CoV-2 Proteins as Probes to Assess Structural and Antigenic Properties of Coronaviruses.

Antibodies targeting the spike (S) and nucleocapsid (N) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential tools. In addition to important roles in the treatment and diagnosis of infection, the availability of high-quality specific antibodies for the S and N proteins is essential to facilitate basic research of virus replication and in the characterization of mutations responsible for variants of concern. We have developed panels of mouse and rabbit monoclonal antibodies (mAbs) to the SARS-CoV-2 spike receptor-binding domain (S-RBD) and N protein for functional and antigenic analyses. The mAbs to the S-RBD were tested for neutralization of native SARS-CoV-2, with several exhibiting neutralizing activity. The panels of mAbs to the N protein were assessed for cross-reactivity with the SARS-CoV and Middle East respiratory syndrome (MERS)-CoV N proteins and could be subdivided into sets that showed unique specificity for SARS-CoV-2 N protein, cross-reactivity between SARS-CoV-2 and SARS-CoV N proteins only, or cross-reactivity to all three coronavirus N proteins tested. Partial mapping of N-reactive mAbs were conducted using truncated fragments of the SARS-CoV-2 N protein and revealed near complete coverage of the N protein. Collectively, these sets of mouse and rabbit monoclonal antibodies can be used to examine structure/function studies for N proteins and to define the surface location of virus neutralizing epitopes on the RBD of the S protein.

UL88 Mediates the Incorporation of a Subset of Proteins into the Virion Tegument.

Little is known about the human cytomegalovirus (HCMV) tegument protein UL88. Large-scale genomic studies have reported disparate results for UL88-null viruses, reporting both no phenotype and a >1-log decrease in virus titers. UL88 has also been reported to interact with UL69 and UL48, but the functional relevance of this interaction is unknown. Here, we report that UL88, which is conserved among different viral strains, is dispensable for production of infectious HCMV virions in multiple HCMV strains and cell types. However, the specific infectivity of HCMV virions suffers in the absence of UL88, as more genomes are required per PFU. This may be a result of altered virion tegument protein composition, as Western blot analysis shows a significant reduction in the tegument levels of pp71, UL47, and UL48 in viruses lacking UL88. While an interaction between UL88 and UL48 has previously been reported, we show that UL88 can interact with UL47; however, UL88 does not appear to be part of a stable complex consisting of UL47 and UL48. These findings identify an important role for UL88 in incorporating the viral proteins UL47 and UL48 into the virion tegument layer.IMPORTANCE A better understanding of the role and functions of tegument proteins in HCMV, many of which remain uncharacterized, will contribute to our understanding of the biology of HCMV. The virus has a large genome, greater than 230 kb, and functional annotation of these genes is important for identifying novel targets for improving therapeutic intervention. This study identifies a role for a viral tegument protein with unknown function, UL88, in maintaining the proper tegument composition of HCMV virions. Virions produced in the absence of UL88 exhibit decreased fitness and require more genomes per infectious unit.

Dengue Virus Capsid Interacts with DDX3X-A Potential Mechanism for Suppression of Antiviral Functions in Dengue Infection.

Dengue virus is a pathogen of global concern and has a huge impact on public health system in low- and middle-income countries. The capsid protein of dengue virus is least conserved among related flavivirus and there is very limited information on the role of cytosolic proteins that interact with dengue virus capsid. We identified DEAD (Asp-Glu-Ala-Asp) Box Helicase 3, an X-Linked (DDX3X), cytosolic ATP-dependent RNA helicase as a dengue virus capsid-interacting protein. We show that the N-terminal region of capsid is important for interaction with DDX3X, while the N-terminal domain of DDX3X seems to be involved in interaction with dengue capsid. DDX3X was down-regulated in dengue virus infected cells at later stages of infection. Our results show that DDX3X is an antiviral protein as suppression of DDX3X expression by siRNA led to an increase in viral titers and overexpression of DDX3X led to inhibition of viral replication. Knock-down of DDX3X did not affect induction of type I interferon response upon infection suggesting that the effect of DDX3X knock-down is independent of the interferon-dependent pathways that DDX3X modulates under normal conditions. Thus, our study identifies DDX3X as a dengue virus capsid interacting protein and indicates a potential link between the antiviral functions of DDX3X and dengue capsid at later stages of dengue infection.

N-Desmethylclozapine, Fluoxetine, and Salmeterol Inhibit Postentry Stages of the Dengue Virus Life Cycle.

Around 10,000 people die each year due to severe dengue disease, and two-thirds of the world population lives in a region where dengue disease is endemic. There has been remarkable progress in dengue virus vaccine development; however, there are no licensed antivirals for dengue disease, and none appear to be in clinical trials. We took the approach of repositioning approved drugs for anti-dengue virus activity by screening a library of pharmacologically active compounds. We identified N-desmethylclozapine, fluoxetine hydrochloride, and salmeterol xinafoate as dengue virus inhibitors based on reductions in the numbers of infected cells and viral titers. Dengue virus RNA levels were diminished in inhibitor-treated cells, and this effect was specific to dengue virus, as other flaviviruses, such as Japanese encephalitis virus and West Nile virus, or other RNA viruses, such as respiratory syncytial virus and rotavirus, were not affected by these inhibitors. All three inhibitors specifically inhibited dengue virus replication with 50% inhibitory concentrations (IC50s) in the high-nanomolar range. Estimation of negative-strand RNA intermediates and time-of-addition experiments indicated that inhibition was occurring at a postentry stage, most probably at the initiation of viral RNA replication. Finally, we show that inhibition is most likely due to the modulation of the endolysosomal pathway and induction of autophagy.

Identification and characterization of the role of c-terminal Src kinase in dengue virus replication.

We screened a siRNA library targeting human tyrosine kinases in Huh-7 cells and identified c-terminal Src kinase (Csk) as one of the kinases involved in dengue virus replication. Knock-down of Csk expression by siRNAs or inhibition of Csk by an inhibitor reduced dengue virus RNA levels but did not affect viral entry. Csk partially colocalized with viral replication compartments. Dengue infection was drastically reduced in cells lacking the three ubiquitous src family kinases, Src, Fyn and Yes. Csk knock-down in these cells failed to block dengue virus replication suggesting that the effect of Csk is via regulation of Src family kinases. Csk was found to be hyper-phosphorylated during dengue infection and inhibition of protein kinase A led to a block in Csk phosphorylation and dengue virus replication. Overexpression studies suggest an important role for the kinase and SH3 domains in this process. Our results identified a novel role for Csk as a host tyrosine kinase involved in dengue virus replication and provide further insights into the role of host factors in dengue replication.