Envelope protein-specific B cell receptors direct lentiviral vector tropism in vivo.

While studying transgene expression after systemic administration of lentiviral vectors, we found that splenic B cells are robustly transduced, regardless of the types of pseudotyped envelope proteins. However, the administration of two different pseudotypes resulted in transduction of two distinct B cell populations, suggesting that each pseudotype uses unique and specific receptors for its attachment and entry into splenic B cells. Single-cell RNA sequencing analysis of the transduced cells demonstrated that different pseudotypes transduce distinct B cell subpopulations characterized by specific B cell receptor (BCR) genotypes. Functional analysis of the BCRs of the transduced cells demonstrated that BCRs specific to the pseudotyping envelope proteins mediate viral entry, enabling the vectors to selectively transduce the B cell populations that are capable of producing antibodies specific to their envelope proteins. Lentiviral vector entry via the BCR activated the transduced B cells and induced proliferation and differentiation into mature effectors, such as memory B and plasma cells. BCR-mediated viral entry into clonally specific B cell subpopulations raises new concepts for understanding the biodistribution of transgene expression after systemic administration of lentiviral vectors and offers new opportunities for BCR-targeted gene delivery by pseudotyped lentiviral vectors.

Development of a blocker of the universal phosphatidylserine- and phosphatidylethanolamine-dependent viral entry pathways.

Envelope phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtr) have been shown to mediate binding of enveloped viruses. However, commonly used PtdSer binding molecules such as Annexin V cannot block PtdSer-mediated viral infection. Lack of reagents that can conceal envelope PtdSer and PtdEtr and subsequently inhibit infection hinders elucidation of the roles of the envelope phospholipids in viral infection. Here, we developed sTIM1dMLDR801, a reagent capable of blocking PtdSer- and PtdEtr-dependent infection of enveloped viruses. Using sTIM1dMLDR801, we found that envelope PtdSer and/or PtdEtr can support ZIKV infection of not only human but also mosquito cells. In a mouse model for ZIKV infection, sTIM1dMLDR801 reduced ZIKV load in serum and the spleen, indicating envelope PtdSer and/or PtdEtr support in viral infection in vivo. sTIM1dMLDR801 will enable elucidation of the roles of envelope PtdSer and PtdEtr in infection of various virus species, thereby facilitating identification of their receptors and transmission mechanisms.

Roles of phosphatidylserine exposed on the viral envelope and cell membrane in HIV-1 replication.

Phosphatidylserine (PtdSer) is usually present only in the inner leaf of the lipid bilayers of the cell membrane, but is exposed on the outer leaf when cells are activated and/or die. Exposure of PtdSer has physiological functions. For example, the PtdSer exposed on dead cells can serve as "eat-me signals" for phagocytes to clear dead cells by phagocytosis, which prevents autoimmune reactions and inflammation. HIV-1 induces PtdSer exposure on infected and target cells and it also exposes PtdSer on its envelope. Recent studies showed that PtdSer exposed on the HIV-1 envelope and infected and target cells can facilitate or inhibit multiple steps of HIV-1 replication.At the virus binding and entry steps, interaction of the envelope PtdSer and the host's PtdSer-binding molecules can enhance HIV-1 infection of cells by facilitating virus attachment. At the virus budding step, HIV-1 can be trapped on the cell surface by one family of PtdSer-binding receptors, T-cell immunoglobulin mucin domain proteins (TIM)-1, 3, and 4 expressed on virus producer cells. Although this trapping can inhibit release of HIV-1, one of the HIV-1 accessory gene products, Negative Factor (Nef), can counteract virus trapping by TIM family receptors (TIMs) by inducing the internalization of these receptors. HIV-1 infection can induce exposure of PtdSer on infected cells by inducing cell death. A soluble PtdSer-binding protein in serum, protein S, bridges PtdSer exposed on HIV-1-infected cells and a receptor tyrosine kinase, Mer, expressed on macrophages and mediate phagocytic clearance of HIV-1 infected cells. HIV-1 can also induce exposure of PtdSer on target cells at the virus binding step. Binding of HIV-1 envelope proteins to its receptor (CD4) and co-receptors (CXCR4 or CCR5) elicit signals that induce PtdSer exposure on target cells by activating TMEM16F, a phospholipid scramblase. PtdSer exposed on target cells enhances HIV-1 infection by facilitating fusion between the viral envelope and target cell membrane. Because various other phospholipid channels mediating PtdSer exposure have recently been identified, it will be of interest to examine how HIV-1 actively interacts with these molecules to manipulate PtdSer exposure levels on cells and viral envelope to support its replication.

Versatile targeting system for lentiviral vectors involving biotinylated targeting molecules.

Conjugating certain types of lentiviral vectors with targeting ligands can redirect the vectors to specifically transduce desired cell types. However, extensive genetic and/or biochemical manipulations are required for conjugation, which hinders applications for targeting lentiviral vectors for broader research fields. We developed envelope proteins fused with biotin-binding molecules to conjugate the pseudotyped vectors with biotinylated targeting molecules by simply mixing them. The envelope proteins fused with the monomeric, but not tetrameric, biotin-binding molecules can pseudotype lentiviral vectors and be conjugated with biotinylated targeting ligands. The conjugation is stable enough to redirect lentiviral transduction in the presence of serum, indicating their potential in in vivo . When a signaling molecule is conjugated with the vector, the conjugation facilitates transduction and signaling in a receptor-specific manner. This simple method of ligand conjugation and ease of obtaining various types of biotinylated ligands will make targeted lentiviral transduction easily applicable to broad fields of research.

Protein S and Gas6 induce efferocytosis of HIV-1-infected cells.

Efferocytosis, the phagocytic clearance of apoptotic cells, can provide host protection against certain types of viruses by mediating phagocytic clearance of infected cells undergoing apoptosis. It is known that HIV-1 induces apoptosis and HIV-1-infected cells are efferocytosed by macrophages, although its molecular mechanisms are unknown. To elucidate the roles that efferocytosis of HIV-1-infected cells play in clearance of infected cells, we sought to identify molecules that mediate these processes. We found that protein S, present in human serum, and its homologue, Gas6, can mediate phagocytosis of HIV-1-infected cells by bridging receptor tyrosine kinase Mer, expressed on macrophages, to phosphatidylserine exposed on infected cells. Efferocytosis of live infected cells was less efficient than dead infected cells; however, a significant fraction of live infected cells were phagocytosed over 12h. Our results suggest that efferocytosis not only removes dead cells, but may also contribute to macrophage removal of live virus producing cells.

Zucchini-dependent piRNA processing is triggered by recruitment to the cytoplasmic processing machinery.

The piRNA pathway represses transposable elements in the gonads and thereby plays a vital role in protecting the integrity of germline genomes of animals. Mature piRNAs are processed from longer transcripts, piRNA precursors (pre-piRNAs). In Drosophila, processing of pre-piRNAs is initiated by piRNA-guided Slicer cleavage or the endonuclease Zucchini (Zuc). As Zuc does not have any sequence or structure preferences in vitro, it is not known how piRNA precursors are selected and channeled into the Zuc-dependent processing pathway. We show that a heterologous RNA that lacks complementary piRNAs is processed into piRNAs upon recruitment of several piRNA pathway factors. This processing requires Zuc and the helicase Armitage (Armi). Aubergine (Aub), Argonaute 3 (Ago3), and components of the nuclear RDC complex, which are required for normal piRNA biogenesis in germ cells, are dispensable. Our approach allows discrimination of proteins involved in the transcription and export of piRNA precursors from components required for the cytoplasmic processing steps. piRNA processing correlates with localization of the substrate RNA to nuage, a distinct membraneless cytoplasmic compartment, which surrounds the nucleus of germ cells, suggesting that sequestration of RNA to this subcellular compartment is both necessary and sufficient for selecting piRNA biogenesis substrates.