Murine norovirus mutants adapted to replicate in human cells reveal a post-entry restriction.

RNA viruses lack proofreading in their RNA polymerases and therefore exist as genetically diverse populations. By exposing these diverse viral populations to selective pressures, viruses with mutations that confer fitness advantages can be enriched. To examine factors important for viral tropism and host restriction, we passaged murine norovirus (MNV) in a human cell line, HeLa cells, to select mutant viruses with increased fitness in non-murine cells. A major determinant of host range is expression of the MNV receptor CD300lf on mouse cells, but additional host factors may limit MNV replication in human cells. We found that viruses passaged six times in HeLa cells had enhanced replication compared with the parental virus. The passaged viruses had several mutations throughout the viral genome, which were primarily located in the viral non-structural coding regions. Although viral attachment was not altered for the passaged viruses, their replication was higher than the parental virus when the entry was bypassed, suggesting that the mutant viruses overcame a post-entry block in human cells. Three mutations in the viral NS1 protein were sufficient for enhanced post-entry replication in human cells. We found that the human cell-adapted MNV variants had reduced fitness in murine BV2 cells and infected mice, with reduced viral titers. These results suggest a fitness tradeoff, where increased fitness in a non-native host cell reduces fitness in a natural host environment. Overall, this work suggests that MNV tropism is determined by the presence of not only the viral receptor but also post-entry factors. IMPORTANCE: Viruses infect specific species and cell types, which is dictated by the expression of host factors required for viral entry as well as downstream replication steps. Murine norovirus (MNV) infects mouse cells, but not human cells. However, human cells expressing the murine CD300lf receptor support MNV replication, suggesting that receptor expression is a major determinant of MNV tropism. To determine whether other factors influence MNV tropism, we selected for variants with enhanced replication in human cells. We identified mutations that enhance MNV replication in human cells and demonstrated that these mutations enhance infection at a post-entry replication step. Therefore, MNV infection of human cells is restricted at both entry and post-entry stages. These results shed new light on factors that influence viral tropism and host range.

Murine norovirus mutants adapted to replicate in human cells reveal a post-entry restriction.

RNA viruses lack proofreading in their RNA polymerases and therefore exist as genetically diverse populations. By exposing these diverse viral populations to selective pressures, viruses with mutations that confer fitness advantages can be enriched. To examine factors important for viral tropism and host restriction, we passaged murine norovirus (MNV) in a human cell line, HeLa cells, to select for mutant viruses with increased fitness in non-murine cells. A major determinant of host range is expression of the MNV receptor CD300lf on mouse cells, but additional host factors may limit MNV replication in human cells. We found that viruses passaged six times in HeLa cells had enhanced replication compared with the parental virus. The passaged viruses had several mutations throughout the viral genome, which were primarily located in the viral non-structural coding regions. While viral attachment was not altered for the passaged viruses, their replication was higher than the parental virus when entry was bypassed, suggesting the mutant viruses overcame a post-entry block in human cells. Three mutations in the viral NS1 protein were sufficient for enhanced post-entry replication in human cells. We found that the human cell-adapted MNV variants had reduced fitness in mouse BV2 cells. Although the mutant viruses had increased fitness in HeLa cells, they did not have increased fitness in mice. Overall, this work suggests that MNV tropism is not only determined by the presence of the viral receptor but also post-entry factors.

Mice with humanized immune system as novel models to study HIV-associated pulmonary hypertension.

People living with HIV and who receive antiretroviral therapy have a significantly improved lifespan, compared to the early days without therapy. Unfortunately, persisting viral replication in the lungs sustains chronic inflammation, which may cause pulmonary vascular dysfunction and ultimate life-threatening Pulmonary Hypertension (PH). The mechanisms involved in the progression of HIV and PH remain unclear. The study of HIV-PH is limited due to the lack of tractable animal models that recapitulate infection and pathobiological aspects of PH. On one hand, mice with humanized immune systems (hu-mice) are highly relevant to HIV research but their suitability for HIV-PH research deserves investigation. On another hand, the Hypoxia-Sugen is a well-established model for experimental PH that combines hypoxia with the VEGF antagonist SU5416. To test the suitability of hu-mice, we combined HIV with either SU5416 or hypoxia. Using right heart catheterization, we found that combining HIV+SU5416 exacerbated PH. HIV infection increases human pro-inflammatory cytokines in the lungs, compared to uninfected mice. Histopathological examinations showed pulmonary vascular inflammation with arterial muscularization in HIV-PH. We also found an increase in endothelial-monocyte activating polypeptide II (EMAP II) when combining HIV+SU5416. Therefore, combinations of HIV with SU5416 or hypoxia recapitulate PH in hu-mice, creating well-suited models for infectious mechanistic pulmonary vascular research in small animals.

Targeting of protease activator receptor-2 (PAR-2) antagonist FSLLRY-NH2 as an asthma adjuvant therapy.

Asthma is a chronic inflammatory and multifactorial respiratory tract disease. It affects over 18 million adults and 6 million children in the USA with Puerto Ricans showing the highest prevalence (12%-19%). This airways illness can be triggered by an environmental stimulus such as grass pollen, fungi spores, cockroaches allergens, dust mites metabolic compounds, and importantly, by environmental proteases such as trypsin and tryptase. Because of the pivotal role of proteases in the onset of asthma pathophysiology, we focused this study on the serine Protease Activated Receptor-2 (PAR-2), a G-protein-coupled receptor widely expressed in cells across the respiratory tract. Herein, we measured the activation of PAR-2 on primary pulmonary bronchial/tracheal epithelial cells, human small airway epithelial cells, lung bronchial smooth muscle cells (with and without asthma). We tested human-derived eosinophils from 61 Puerto Rican participants (33 asthmatic and 28 non-asthmatic). As surrogate of PAR-2 activation or inhibition we used intracellular calcium mobilization assay. We hypothesized that following exposure of the PAR-2 agonist (AC264613), the studied human primary cell types will increase the mobilization of intracellular calcium levels. In contrast, we expected a decrease of the intracellular calcium levels upon exposure to a PAR-2 antagonist (FSLLRY-NH2). The Puerto Rican-derived eosinophils were analyzed for the proinflammatory markers MAPK/PI3K using flow cytometry (n = 8). As expected, the PAR-2 agonist significantly increased the activation of PAR-2 on the bronchial/tracheal epithelial cells, bronchial smooth muscle cells and human small airway epithelial cells (P = .01). The PAR-2 antagonist significantly decreased the intracellular calcium levels of these lung primary down to undetectable levels (P = .01). Remarkably, the asthmatic-derived eosinophils showed a striking 300% increase of intracellular calcium mobilization suggesting a severe response to the PAR-2 agonist stimuli in asthmatics. In contrast, there were no significant changes between groups after adding the PAR-2 antagonist. Our outcomes revealed that PAR-2 antagonist effectively inhibited the studied primary cells, expecting to decrease the immune response of eosinophils. Most importantly, our results reveal a promising role for the PAR-2 antagonist in targeting bronchial/tracheal epithelial cells, human small airway epithelial cells and bronchial smooth muscle cells with the potential to oblige an asthma adjuvant therapy.