SERINC genes encode for homologous multipass transmembrane proteins with unknown cellular function, despite being highly conserved across eukaryotes. Among the five SERINC genes found in humans, SERINC5 was shown to act as a powerful inhibitor of retroviruses. It is efficiently incorporated into virions and blocks the penetration of the viral core into target cells, by impairing the fusion process with a yet unclear mechanism. SERINC5 was also found to promote human immunodeficiency virus 1 (HIV-1) virion neutralization by antibodies, indicating a pleiotropic activity, which remains mostly unexplored. Counteracting factors have emerged independently in at least three retrovirus lineages, underscoring their fundamental importance during retrovirus evolution. Nef and S2 of primate and equine lentiviruses, and glycoGag of gammaretroviruses, act similarly by targeting SERINC5 to endosomes and excluding it from virions. Here, we discuss the features that distinguish SERINC5 from other known restriction factors, delineating a yet unique class of antiviral inhibitors.

HIV-1 transactivating factor Tat is released by infected cells. Extracellular Tat homodimerizes and engages several receptors, including integrins, vascular endothelial growth factor receptor 2 (VEGFR2) and heparan sulfate proteoglycan (HSPG) syndecan-1 expressed on various cells. By means of experimental cell models recapitulating the processes of lymphocyte trans-endothelial migration, here, we demonstrate that upon association with syndecan-1 expressed on lymphocytes, Tat triggers simultaneously the in cis activation of lymphocytes themselves and the in trans activation of endothelial cells (ECs). This “two-way” activation eventually induces lymphocyte adhesion and spreading onto the substrate and vascular endothelial (VE)-cadherin reorganization at the EC junctions, with consequent endothelial permeabilization, leading to an increased extravasation of Tat-presenting lymphocytes. By means of a panel of biochemical activation assays and specific synthetic inhibitors, we demonstrate that during the above-mentioned processes, syndecan-1, integrins, FAK, src and ERK_1/2 engagement and activation are needed in the lymphocytes, while VEGFR2, integrin, src and ERK_1/2 are needed in the endothelium. In conclusion, the Tat/syndecan-1 complex plays a central role in orchestrating the setup of the various in cis and in trans multimeric complexes at the EC/lymphocyte interface. Thus, by means of computational molecular modelling, docking and dynamics, we also provide a characterization at an atomic level of the binding modes of the Tat/heparin interaction, with heparin herein used as a structural analogue of the heparan sulfate chains of syndecan-1.

The vaccination campaign against SARS-CoV-2 relies on the world-wide availability of effective vaccines, with a potential need of 20 billion vaccine doses to fully vaccinate the world population. To reach this goal, the manufacturing and logistic processes should be affordable to all countries, irrespective of economical and climatic conditions. Outer membrane vesicles (OMVs) are bacterial-derived vesicles that can be engineered to incorporate heterologous antigens. Given the inherent adjuvanticity, such modified OMVs can be used as vaccines to induce potent immune responses against the associated proteins. Here, we show that OMVs engineered to incorporate peptides derived from the receptor binding motif (RBM) of the spike protein from SARS-CoV-2 elicit an effective immune response in vaccinated mice, resulting in the production of neutralizing antibodies (nAbs) with a titre higher than 1:300. The immunity induced by the vaccine is sufficient to protect the animals from intranasal challenge with SARS-CoV-2, preventing both virus replication in the lungs and the pathology associated with virus infection. Furthermore, we show that OMVs can be effectively decorated with the RBM of the Omicron BA.1 variant and that such engineered OMVs induce nAbs against Omicron BA.1 and BA.5, as measured using the pseudovirus neutralization infectivity assay. Importantly, we show that the RBM_438–509 ancestral-OMVs elicited antibodies which efficiently neutralize in vitro both the homologous ancestral strain, the Omicron BA.1 and BA.5 variants with a neutralization titre ranging from 1:100 to 1:1500, suggesting its potential use as a vaccine targeting diverse SARS-CoV-2 variants. Altogether, given the convenience associated with the ease of engineering, production and distribution, our results demonstrate that OMV-based SARS-CoV-2 vaccines can be a crucial addition to the vaccines currently available.

In humans, mutations in sterile α motif and histidine-aspartate domain–containing protein 1 (SAMHD1) lead to the development of a type I interferonopathy known as Aicardi–Goutières syndrome (AGS). AGS can present with a variety of severe phenotypes in patients, and a hallmark of this disease is chronic activation of type I interferon (IFN) signaling. However, the mechanism through which type I IFN signaling is activated in the absence of functional SAMHD1 is not known. Here, we investigated the molecular pathways that lead to type I IFN signaling activation in the absence of SAMHD1. Our investigations revealed that chronic activation of type I IFN signaling in SAMHD1-knockout (KO) monocytes is cyclic GMP–AMP synthase (cGAS)-dependent. Analysis of other nucleic acid sensors showed that type I IFN signaling in SAMHD1-KO cells is not dependent on melanoma differentiation-associated protein 5 (MDA5) or retinoic acid–inducible gene I (RIG-I). In agreement with our observation that type I IFN signaling is dependent on cGAS, two inhibitors of the cGAS–stimulator of IFN genes pathway, G140 and H151, effectively prevented type I IFN activation in SAMHD1-KO monocytes. We also found that type I IFN signaling in SAMHD1-KO monocytes is dependent on type I IFN receptor expression. Further exploration revealed mitochondrial malfunction in SAMHD1-KO monocytes that is likely to leak mitochondrial components into the cytoplasm. Overall, our work suggests that genetic knock out of SAMHD1 leads to mitochondrial disfunction, resulting in the presence of mitochondrial DNA in the cytoplasm, which triggers cGAS and the type I IFN response.

The host transmembrane protein SERINC5 is incorporated into retrovirus particles and inhibits HIV-1 infectivity. The lentiviral Nef protein counteracts SERINC5 by downregulating it from the cell surface and preventing its incorporation into virions. The ability of Nef to antagonize the host factor varies in magnitude between different HIV-1 isolates. After having identified a subtype H nef allele unable to promote HIV-1 infectivity in the presence of SERINC5, we investigated the molecular determinants responsible for the defective counteraction of the host factor. Chimeric molecules with a subtype C Nef highly active against SERINC5 were constructed to locate Nef residues crucial for the activity against SERINC5. An Asn at the base of the C-terminal loop of the defective nef allele was found in place of a highly conserved acidic residue (D/E 150). The conversion of Asn to Asp restored the ability of the defective Nef to downregulate SERINC5 and promote HIV-1 infectivity. The substitution was also found to be crucial for the ability of Nef to downregulate CD4, but not for Nef activities that do not rely on the internalization of receptors from the cell surface, suggesting a general implication in promoting clathrin-mediated endocytosis. Accordingly, bimolecular fluorescence complementation revealed that the conserved acidic residue contributes to the recruitment of AP2 by Nef. Altogether, our results confirm that Nef downregulates SERINC5 and CD4 by engaging a similar machinery and indicates that, in addition to the di-leucine motif, other residues in the C-terminal flexible loop are important for the ability of the protein to sustain clathrin-mediated endocytosis.

The vaccination campaign against SARS-CoV-2 relies on the world-wide availability of effective vaccines, with a potential need of 20 billion vaccine doses to fully vaccinate the world population. To reach this goal, the manufacturing and logistic processes should be affordable to all countries, irrespectively of economical and climatic conditions. Outer membrane vesicles (OMV) are bacterial-derived vesicles that can be engineered to incorporate heterologous antigens. Given the inherent adjuvanticity, such modified OMV can be used as vaccine to induce potent immune responses against the associated protein. Here we show that OMVs engineered to incorporate peptides derived from the receptor binding motif (RBM) of the spike protein from SARS-CoV-2 elicit an effective immune response in immunized mice, resulting in the production of neutralizing antibodies (nAbs). The immunity induced by the vaccine is sufficient to protect the animals from intranasal challenge with SARS-CoV-2, preventing both virus replication in the lungs and the pathology associated with virus infection. Furthermore, we show that OMVs can be effectively decorated with the RBM of the Omicron BA.1 variant and that such engineered OMVs induced nAbs against Omicron BA.1 and BA.5, as judged by pseudovirus infectivity assay. Importantly, we show that the RBM 438-509 ancestral-OMVs elicited antibodies which efficiently neutralized in vitro both the homologous ancestral strain, the Omicron BA.1 and BA.5 variants, suggesting its potential use as a pan Coronavirus vaccine. Altogether, given the convenience associated with ease of engineering, production and distribution, our results demonstrate that OMV-based SARS-CoV-2 vaccines can be a crucial addition to the vaccines currently available.