Sergei Nekhai

Sickle cell disease (SCD) is an inherited hemoglobinopathy characterized by chronic hemolytic anemia. Individuals with SCD have a reduced risk of HIV-1 infection, and their peripheral blood mononuclear cells (PBMCs) express antiviral factors and are resistant to HIV-1 infection ex vivo. To identify factors associated with HIV-1 resistance in SCD, we recruited patients with both SCD and HIV-1 infection (SCD+HIV+) and compared them with SCD patients without HIV-1 infection (SCD+HIV-), HIV-1–positive individuals without SCD (SCD-HIV+), and healthy controls (SCD-HIV-). We evaluated clinical and hematologic parameters, ex vivo susceptibility to HIV-1 infection, and the expression of antiviral and iron-regulatory genes in these groups of participants. Compared with SCD+HIV- patients, SCD+HIV+ patients showed less hemolysis and reduced inflammation. SCD+HIV+ PBMCs were more susceptible to HIV-1 infection ex vivo and contained higher levels of intracellular labile iron than SCD+HIV- PBMCs. RNA-seq analysis further revealed reduced expression of iron-regulatory and antiviral genes in SCD+HIV+ PBMCs relative to SCD+HIV− PBMCs. Variance partition analysis identified hemoglobin levels, as well as lymphocyte and monocyte counts, as key contributors to variability in gene expression. Among the antiviral factors examined, MX2, APOBEC3A, SERINC5, SLF11 and CH25H emerged as top candidate mediators of protection against HIV-1 in SCD. Overall, these findings suggest that HIV-1 infection in SCD may occur in individuals with milder hemolysis and less severe disease manifestations.

Protein phosphorylation is a crucial regulatory mechanism in cellular homeostasis. The human cytomegalovirus (HCMV) incorporates protein phosphatase 1 (PP1) into its tegument, yet the biological relevance and mechanisms of this incorporation remain unclear. Our study offers the first characterization of the PP1 interactome during HCMV infection and its alterations. Using co-immunoprecipitation, mass spectrometry, and quantitative proteomics, we identified 159 high-confidence interacting proteins (HCIPs) in the PP1 interactome, consisting of 126 human and 33 viral proteins. We observed significant temporal changes in the PP1 interactome following HCMV infection, including the altered interactions of PP1 regulatory subunits. Further analysis highlighted the central roles of these PP1 interacting proteins in intracellular trafficking, with particular emphasis on the trafficking protein particle complex and Rab GTPases, which are crucial for the virus’s manipulation of host cellular processes in virion assembly and egress. Additionally, our study on the noncatalytic PP1 inhibitor 1E7-03 revealed a decrease in PP1’s interaction with key HCMV proteins, supporting its potential as an antiviral agent. Our findings suggest that PP1 docking motifs are critical in viral–host interactions and offer new insights for antiviral strategies.

Genome-wide association studies (GWAS) have become well-powered to detect loci associated with telomere length. However, no prior work has validated genes nominated by GWAS to examine their role in telomere length regulation. We conducted a multi-ancestry meta-analysis of 211,369 individuals and identified five novel association signals. Enrichment analyses of chromatin state and cell-type heritability suggested that blood/immune cells are the most relevant cell type to examine telomere length association signals. We validated specific GWAS associations by overexpressing KBTBD6 or POP5 and demonstrated that both lengthened telomeres. CRISPR/Cas9 deletion of the predicted causal regions in K562 blood cells reduced expression of these genes, demonstrating that these loci are related to transcriptional regulation of KBTBD6 and POP5. Our results demonstrate the utility of telomere length GWAS in the identification of telomere length regulation mechanisms and validate KBTBD6 and POP5 as genes affecting telomere length regulation.

HIV-1 has eluded vaccine therapy for the past 40 years. The virus mutates rapidly and is protected by a shifting glycan shield of mannose sugars, which has hindered the broad neutralization of the virus by antibodies (Abs). Studies have shown that mannose residues are self-adhesive, but it is not known if these adhesions drive HIV-1 to aggregate in solution, further complicating Ab neutralization. The behavior of HIV-1 in culture media was monitored using Dynamic Light Scattering and complementary atomic force microscopy (AFM) imaging in the presence of anti-gp120 Abs, lectins, mannosidase, and mucin. After accounting for the serum contribution from the culture media, HIV-1 was found to be diffusing in solution in 400–700 nm clusters. These clusters could be sheared into single virus particles by filtration, but the dispersed particles clustered back within a short time frame. Sample preparation prior to AFM and transmission electron microscopy (TEM) imaging appears to disperse clusters, but the clusters become visible in AFM when they are stabilized by Abs in solution. The clustered form of the virus appears to restrict access of Abs, lectins, and glycosidases to surfaces within the cluster. Mannosidase treatment following virus dispersion by filtration prevented clustering, suggesting that the mannose glycan shield is involved in cluster formation. Dispersed HIV-1 particles that were bound by Abs did not re-cluster back. Free mucin molecules (porcine gastric mucin) effectively dispersed HIV-1 clusters, even those stabilized by Abs. HIV-1-loaded mucin dried on the AFM surface with a fern-like fractal pattern, similar to that seen clinically in cervical mucin during the more penetrable ovulation stage. IMPORTANCE: The phenomenon of reversible clustering is expected to further nuance HIV immune stealth because virus surfaces can escape interaction with antibodies (Abs) by hiding temporarily within clusters. It is well known that mucin reduces HIV virulence, and the current perspective is that mucin aggregates HIV-1 to reduce infections. Our findings, however, suggest that mucin is dispersing HIV clusters. The study proposes a new paradigm for how HIV-1 may broadly evade Ab recognition with reversible clustering and why mucin effectively neutralizes HIV-1.

In patients with sickle cell disease (SCD), chronic hemolysis and frequent blood transfusions cause iron overload and accumulation in the kidneys. The iron deposition is found in the renal cortex and correlates with the severity of hemolysis. In this study, we observed a significant accumulation of iron in the renal cortex of a mouse model of SCD, and assessed the expression of the proteins involved in maintaining renal iron homeostasis. Despite the intracellular iron accumulation, the levels of the transferrin receptor in the kidneys were increased, but the levels of the iron exporter ferroportin were not altered in SCD mice. Ferroportin is regulated by hepcidin, which binds to it and promotes its degradation. We found reduced serum hepcidin levels but increased renal hepcidin production in SCD mice. Furthermore, we observed significant macrophage infiltration and increased expression of intercellular adhesion molecule 1 in the endothelial cells of the kidneys in SCD mice. These observations correlated with elevated levels of proinflammatory cytokines IL-1β and IL-6, which can potentially stimulate hepcidin expression. Taken together, our results demonstrate that in individuals with SCD, a renal inflammation state induces renal hepcidin production that blocks the upregulation of ferroportin levels, resulting in dysregulation of iron homeostasis in the kidney and iron deposition in the renal cortex.

The Ebola virus (EBOV) transcriptional regulation involves host protein phosphatases PP1 and PP2A, which dephosphorylate the transcriptional cofactor of EBOV polymerase VP30. The 1E7-03 compound, which targets PP1, induces VP30 phosphorylation and inhibits EBOV infection. This study aimed to investigate the role of PP1 in EBOV replication. When EBOV-infected cells were continuously treated with 1E7-03, the NP E619K mutation was selected. This mutation moderately reduced EBOV minigenome transcription, which was restored by the treatment with 1E7-03. Formation of EBOV capsids, when NP was co-expressed with VP24 and VP35, was impaired with NPE 619K. Treatment with 1E7-03 restored capsid formation by NP E619K mutation, but inhibited capsids formed by WT NP. The dimerization of NP E619K, tested in a split NanoBiT assay, was significantly decreased (~ 15-fold) compared to WT NP. NP E619K bound more efficiently to PP1 (~ 3-fold) but not B56 subunit of PP2A or VP30. Cross-linking and co-immunoprecipitation experiments showed fewer monomers and dimers for NP E619K which were increased with 1E7-03 treatment. NP E619K showed increased co-localization with PP1α compared to WT NP. Mutations of potential PP1 binding sites and NP deletions disrupted its interaction with PP1. Collectively, our findings suggest that PP1 binding to the NP regulates NP dimerization and capsid formation, and that NP E619K mutation, which has the enhanced PP1 binding, disrupts these processes. Our results point to a new role for PP1 in EBOV replication in which NP binding to PP1 may facilitate viral transcription by delaying capsid formation and EBOV replication.

Yagahira Castro Sesquen, Santosh L Saraf, Victor R Gordeuk, Sergei Nekhai, Marina Jerebtsova; Use of Multiple Urinary Biomarkers for the Early Detection of Chro