Host-microbe interactions that facilitate entry into mammalian cells are essential for obligate intracellular bacterial survival and pathogenesis. Anaplasma phagocytophilum is an obligate intracellular bacterium that invades neutrophils to cause granulocytic anaplasmosis. The invasin-receptor pairs and signaling events that induce Anaplasma uptake are inadequately defined. A. phagocytophilum invasion protein A orchestrates entry via residues 9-21 (AipA9-21) engaging an unknown receptor. Yeast two-hybrid screening suggested that AipA binds within C-terminal amino acids 851-967 of CD13 (aminopeptidase N), a multifunctional protein that, when crosslinked, initiates Src kinase and Syk signaling that culminates in endocytosis. Co-immunoprecipitation validated the interaction and confirmed that it requires the AipA N-terminus. CD13 ectopic expression on non-phagocytic cells increased susceptibility to A. phagocytophilum infection. Antibody blocking and enzymatic inhibition experiments found that the microbe exploits CD13 but not its ectopeptidase activity to infect myeloid cells. A. phagocytophilum induces Src and Syk phosphorylation during invasion. Inhibitor treatment established that Src is key for A. phagocytophilum infection, while Syk is dispensable and oriented the pathogen-invoked signaling pathway by showing that Src is activated before Syk. Disrupting the AipA-CD13 interaction with AipA9-21 or CD13781-967 antibody inhibited Src and Syk phosphorylation and also infection. CD13 crosslinking antibody that induces Src and Syk signaling restored infectivity of anti-AipA9-21-treated A. phagocytophilum. The bacterium poorly infected CD13 knockout mice, providing the first demonstration that CD13 is important for microbial infection in vivo. Overall, A. phagocytophilum AipA9-21 binds CD13 to induce Src signaling that mediates uptake into host cells, and CD13 is critical for infection in vivo.
Diverse microbes engage CD13 to infect host cells. Yet invasin-CD13 interactions, the signaling they invoke for pathogen entry, and the relevance of CD13 to infection in vivo are underexplored. Dissecting these concepts would advance fundamental understanding of a convergently evolved infection strategy and could have translational benefits. Anaplasma phagocytophilum infects neutrophils to cause granulocytic anaplasmosis, an emerging disease for which there is no vaccine and few therapeutic options. We found that A. phagocytophilum uses its surface protein and recently identified protective immunogen, AipA, to bind CD13 to elicit Src kinase signaling, which is critical for infection. We elucidated the AipA CD13 binding domain, which CD13 region AipA engages, and established that CD13 is key for A. phagocytophilum infection in vivo. Disrupting the AipA-CD13 interaction could be utilized to prevent granulocytic anaplasmosis and offers a model that could be applied to protect against multiple infectious diseases.

P-selectin glycoprotein ligand-1 (PSGL-1/CD162) has been studied extensively for its role in mediating leukocyte rolling through interactions with its cognate receptor, P-selectin. Recently, PSGL-1 was identified as a novel HIV-1 host restriction factor, particularly when expressed at high levels in the HIV envelope. Importantly, while the potent antiviral activity of PSGL-1 has been clearly demonstrated in various complementary model systems, the breadth of PSGL-1 incorporation across genetically diverse viral isolates and clinical isolates has yet to be described. Additionally, the biological activity of virion-incorporated PSGL-1 has also yet to be shown.
Herein we assessed the levels of PSGL-1 on viruses produced through transfection with various amounts of PSGL-1 plasmid DNA (0-250 ng), compared to levels of PSGL-1 on viruses produced through infection of T cell lines and primary PBMC. We found that very low levels of PSGL-1 plasmid DNA (< 2.5 ng/well) were necessary to generate virus models that could closely mirror the phenotype of viruses produced via infection of T cells and PBMC. Unique to this study, we show that PSGL-1 is incorporated in a broad range of HIV-1 and SIV isolates and that virions with incorporated PSGL-1 are detectable in plasma from viremic HIV-1-infected individuals, corroborating the relevance of PSGL-1 in natural infection. Additionally, we show that PSGL-1 on viruses can bind its cognate selectin receptors, P-, E-, and L-selectins. Finally, we show viruses with endogenous levels of PSGL-1 can be captured by P-selectin and transferred to HIV-permissive bystander cells, highlighting a novel role for PSGL-1 in HIV-1 infection. Notably, viruses which contained high levels of PSGL-1 were noninfectious in our hands, in line with previous findings reporting the potent antiviral activity of PSGL-1.
Our results indicate that levels of PSGL-1 incorporation into virions can vary widely among model systems tested, and that careful tailoring of plasmid levels is required to recapitulate physiological systems when using pseudovirus models. Taken together, our data suggest that PSGL-1 may play diverse roles in the physiology of HIV-1 infection, particularly due to the functionally active state of PSGL-1 on virion surfaces and the breadth of PSGL-1 incorporation among a wide range of viral isolates.
© 2022. The Author(s).

h4>ABSTRACT/h4> While P-selectin glycoprotein ligand-1 (PSGL-1/CD162) has been studied extensively for its role in mediating leukocyte rolling through interactions with its receptor, P-selectin, recently, it was identified as a novel HIV-1 host restriction factor. One key mechanism of HIV-1 restriction is the ability of PSGL-1 to be physically incorporated into the external viral envelope, which effectively reduces infectivity by blocking virus attachment through the steric hindrance caused by its large ectodomain. Importantly, a large portion of the literature demonstrating the antiviral activity of PSGL-1 has utilized viruses produced in transfected cells which express high levels of PSGL-1. However, herein we show that virion-incorporated PSGL-1 is far less abundant on the surface of viruses produced via infection of physiologically relevant models (T cell lines and primary cells) compared to transfection (overexpression) models. Unique to this study, we show that PSGL-1 is incorporated in a broad range of HIV-1 and SIV isolates, supporting the physiological relevance of this incorporation. We also report that high levels of virion-incorporated PSGL-1 are detectable in plasma from viremic HIV-1 infected individuals, further corroborating the clinical relevance of PSGL-1 in natural infection. Additionally, we show that PSGL-1 on viruses is functionally active and can bind its cognate receptor, P-selectin, and that virions captured via P-selectin can subsequently be transferred to HIV-permissive bystander cells in a model of trans-infection. Taken together, our data suggest that PSGL-1 may have diverse roles in the physiology of HIV-1 infection, not restricted to the current antiviral paradigm. h4>IMPORTANCE/h4> PSGL-1 is an HIV-1 host restriction factor which reduces viral infectivity by physically incorporating into the envelope of virions. While the antiviral effects of PSGL-1 in viruses produced by transfection models is profound, HIV-1 continues to remain infectious when produced through natural infection, even when PSGL-1 is incorporated. To study this discordance, we compared the differences in infectivity and PSGL-1 abundance in viruses produced by transfection or infection. Viruses produced via transfection contained unnaturally high levels of incorporated PSGL-1 compared to viruses from primary cells, and were much less infectious. We also found PSGL-1 to be present on a broad range of HIV-1 isolates, including those found in plasma from HIV-infected patients. Remarkably, we show that virion-incorporated PSGL-1 facilitates virus capture and transfer to HIV-permissive host cells via binding to P-selectin. These findings suggest that PSGL-1 may also work to enhance infection in vivo .

Mucins and mucin-like molecules are highly glycosylated, high-molecular-weight cell surface proteins that possess a semi-rigid and highly extended extracellular domain. P-selectin glycoprotein ligand-1 (PSGL-1), a mucin-like glycoprotein, has recently been found to restrict HIV-1 infectivity through virion incorporation that sterically hinders virus particle attachment to target cells. Here, we report the identification of a family of antiviral cellular proteins, named the Surface-Hinged, Rigidly-Extended Killer (SHREK) family of virion inactivators (PSGL-1, CD43, TIM-1, CD34, PODXL1, PODXL2, CD164, MUC1, MUC4, and TMEM123) that share similar structural characteristics with PSGL-1. We demonstrate that SHREK proteins block HIV-1 infectivity by inhibiting virus particle attachment to target cells. In addition, we demonstrate that SHREK proteins are broad-spectrum host antiviral factors that block the infection of diverse viruses such as influenza A. Furthermore, we demonstrate that a subset of SHREKs also blocks the infectivity of a hybrid alphavirus-SARS-CoV-2 (Ha-CoV-2) pseudovirus. These results suggest that SHREK proteins may be a part of host innate immunity against enveloped viruses.

The HIV-1 glycoprotein spike (gp120) is typically the first viral antigen that cells encounter before initiating immune responses, and is often the sole target in vaccine designs. Thus, characterizing the presence of cellular antigens on the surfaces of HIV particles may help identify new antiviral targets or impact targeting of gp120. Despite the importance of characterizing proteins on the virion surface, current techniques available for this purpose do not support high-throughput analysis of viruses, and typically only offer a semi-quantitative assessment of virus-associated proteins. Traditional bulk techniques often assess averages of viral preparations, which may mask subtle but important differences in viral subsets. On the other hand, microscopy techniques, which provide detail on individual virions, are difficult to use in a high-throughput manner and have low levels of sensitivity for antigen detection. Flow cytometry is a technique that traditionally has been used for rapid, high-sensitivity characterization of single cells, with limited use in detecting viruses, since the small size of viral particles hinders their detection. Herein, we report the detection and surface antigen characterization of HIV-1 pseudovirus particles by light scattering and fluorescence with flow cytometry, termed flow virometry for its specific application to viruses. We quantified three cellular proteins (integrin α4β7, CD14, and CD162/PSGL-1) in the viral envelope by directly staining virion-containing cell supernatants without the requirement of additional processing steps to distinguish virus particles or specific virus purification techniques. We also show that two antigens can be simultaneously detected on the surface of individual HIV virions, probing for the tetraspanin marker, CD81, in addition to α4β7, CD14, and CD162/PSGL-1. This study demonstrates new advances in calibrated flow virometry as a tool to provide sensitive, high-throughput characterization of the viral envelope in a more efficient, quantitative manner than previously reported techniques.