The SARS-CoV-2 coronavirus encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin-like ISG15 protein modifications as well as, with lower activity, Lys48-linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin-binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, noncovalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self-processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.

During unresolved infections, some viruses escape immunological control and establish a persistant reservoir in certain cell types, such as human immunodeficiency virus (HIV), which persists in follicular helper T cells (TFH cells), and Epstein-Barr virus (EBV), which persists in B cells. Here we identified a specialized group of cytotoxic T cells (TC cells) that expressed the chemokine receptor CXCR5, selectively entered B cell follicles and eradicated infected TFH cells and B cells. The differentiation of these cells, which we have called 'follicular cytotoxic T cells' (TFC cells), required the transcription factors Bcl6, E2A and TCF-1 but was inhibited by the transcriptional regulators Blimp1, Id2 and Id3. Blimp1 and E2A directly regulated Cxcr5 expression and, together with Bcl6 and TCF-1, formed a transcriptional circuit that guided TFC cell development. The identification of TFC cells has far-reaching implications for the development of strategies to control infections that target B cells and TFH cells and to treat B cell-derived malignancies.

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Neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19. Here, we identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2. Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody-Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.

We have shown that cellular inhibitor of apoptosis proteins (cIAPs) impair clearance of hepatitis B virus (HBV) infection by preventing TNF-mediated killing/death of infected cells. A key question, with profound therapeutic implications, is whether this finding can be translated to the development of drugs that promote elimination of infected cells. Drug inhibitors of cIAPs were developed as cancer therapeutics to promote TNF-mediated tumor killing. These drugs are also known as Smac mimetics, because they mimic the action of the endogenous protein Smac/Diablo that antagonizes cIAP function. Here, we show using an immunocompetent mouse model of chronic HBV infection that birinapant and other Smac mimetics are able to rapidly reduce serum HBV DNA and serum HBV surface antigen, and they promote the elimination of hepatocytes containing HBV core antigen. The efficacy of Smac mimetics in treating HBV infection is dependent on their chemistry, host CD4 + T cells, and TNF. Birinapant enhances the ability of entecavir, an antiviral nucleoside analog, to reduce viral DNA production in HBV-infected animals. These results indicate that birinapant and other Smac mimetics may have efficacy in treating HBV infection and perhaps, other intracellular infections.hepatitis B virus | cellular inhibitor of apoptosis proteins | TNF |

SummaryThe cytosolic innate immune molecule, NOD1, recognizes peptidoglycan (PG) delivered to epithelial cells via the Helicobacter pylori cag pathogenicity island (cagPAI), and has been implicated in host defence against cagPAI + H. pylori bacteria. To further clarify the role of NOD1 in host defence, we investigated NOD1-dependent regulation of human b-defensins (DEFBs) in two epithelial cell lines. Our findings identify that NOD1 activation, via either cagPAI + bacteria or internalized PG, was required for DEFB4 and DEFB103 expression in HEK293 cells. To investigate cell type-specific induction of DEFB4 and DEFB103, we generated stable NOD1 'knockdown' (KD) and control AGS cells. Reporter gene assay and RT-PCR analyses revealed that only DEFB4 was induced in an NOD1-/cagPAIdependent fashion in AGS cells. Moreover, culture supernatants from AGS control, but not AGS NOD1 KD cells, stimulated with cagPAI + H. pylori, significantly reduced H. pylori bacterial numbers. siRNA studies confirmed that human b-defensin 2 (hBD-2), but not hBD-3, contributes to the antimicrobial activity of AGS cell supernatants against H. pylori. This study demonstrates, for the first time, the involvement of NOD1 and hBD-2 in direct killing of H. pylori bacteria by epithelial cells and confirms the importance of NOD1 in host defence mechanisms against cagPAI + H. pylori infection.

The ehx plasmids of these strains are highly related, which suggests acquisition of the large plasmid was central to the strains’ emergence.

Hepatitis B virus (HBV) infection can result in a spectrum of outcomes from immune-mediated control to disease progression, cirrhosis, and liver cancer. The host molecular pathways that influence and contribute to these outcomes need to be defined. Using an immunocompetent mouse model of chronic HBV infection, we identified some of the host cellular and molecular factors that impact on infection outcomes. Here, we show that cellular inhibitor of apoptosis proteins (cIAPs) attenuate TNF signaling during hepatitis B infection, and they restrict the death of infected hepatocytes, thus allowing viral persistence. Animals with a liver-specific cIAP1 and total cIAP2 deficiency efficiently control HBV infection compared with WT mice. This phenotype was partly recapitulated in mice that were deficient in cIAP2 alone. These results indicate that antagonizing the function of cIAPs may promote the clearance of HBV infection.hepatitis B virus | cellular inhibitor of apoptosis proteins | cIAP1 | cIAP2 | TNF I t is estimated that 2 billion people currently living in the world have been infected with hepatitis B virus (HBV), and among these, 360 million people are chronic carriers (1). HBV causes 780,000 deaths each year and is responsible for 50% and 33% of deaths attributable to liver cancer and cirrhosis, respectively (2). The host factors and molecular pathways that impact on HBV disease and clinical outcomes are not well-understood (3). What is becoming clear is that immunosuppressive agents and particularly, biological agents, including anti-TNF therapy, can cause major flares in HBV-related disease, leading to morbidity and mortality (4, 5). Animal models and particularly, immunocompetent mouse models of persistent HBV infection have been used to dissect host-pathogen interactions that influence infection outcomes (6-8). These animal models can be used to define host cell signaling and cell death pathways that contribute to the persistence or control of HBV infection.We induced HBV infection in two mouse models to examine the relevance of host factors in controlling infection. In a model that mimics partial control of infection, we were able to determine the importance of host cell signaling pathways through the use of gene-targeted mice. By identifying the relevant host cell signaling molecules that impact on HBV clinical outcomes, it may be possible to develop therapeutics that target host cell pathways and alter the course of HBV-related disease. ResultsChronic HBV Infection Can Be Mimicked in a Mouse Model. We used a previously described method to induce HBV persistence in immunocompetent mice (6). A plasmid containing a 1.2 over length sequence of HBV genotype A was hydrodynamically injected into mice, but in contrast to the previously published protocol, we did not anesthetize animals. Using this modified technique, we did not observe any injection-associated mortality, and C57BL/6 mice showed persistently high serum HBV DNA levels over 8-12 wk (Fig. 1A). Eventually, HBV DNA levels fell in all animals along with t...