We describe the first example of combining bacterial artificial chromosome (BAC) recombination-mediated mutagenesis with reverse genetics for a negative strand RNA virus. A BAC-based respiratory syncytial virus (RSV) rescue system was established. An important advantage of this system is that RSV antigenomic cDNA was stabilized in the BAC vector. The RSV genotype chosen was A2-line19F, a chimeric strain previously shown to recapitulate in mice key features of RSV pathogenesis. We recovered two RSV reporter viruses, one expressing the red fluorescent protein monomeric Katushka 2 (A2-K-line19F) and one expressing Renilla luciferase (A2-RL-line19F). As proof of principle, we efficiently generated a RSV gene deletion mutant (A2-line19FΔNS1/NS2) and a point mutant (A2-K-line19F-I557V) by recombination-mediated BAC mutagenesis. Together with sequence-optimized helper expression plasmids, BAC-RSV is a stable, versatile, and efficient reverse genetics platform for generation of a recombinant Pneumovirus.
Blockade of the programmed death 1 (PD-1) pathway has emerged as a novel therapy for cancer. Therefore, development of biomarkers for response prediction, such as PD-ligand 1 (PD-L1) expression by immunohistochemistry, may help to stratify patients. Solid tumors with CD8 T-cell rich tumor microenvironment have been implicated to be associated with increased PD-L1 expression. We hypothesized that gastric cancers associated with Epstein-Barr virus infection (EBV+) or microsatellite instability (MSI), both of which are known to harbor such tumor microenvironment, are associated with increased PD-L1 expression. Forty-four resected gastric cancers including 7 EBV+, 16 MSI, and 21 microsatellite stable cancers without EBV (EBV-/MSS) were studied for PD-L1 expression and T-cell subpopulations by immunohistochemistry. Positive PD-L1 expression (PD-L1+), defined as membranous staining in either tumor cells or tumor immune infiltrates, was seen in 32 (72%) gastric cancers. EBV+ or MSI cancers showed significantly higher rates of PD-L1+ compared with EBV-/MSS cancers (7/7, 100%; 14/16, 87%; 11/21, 52%; P=0.013). PD-L1+/EBV+ and PD-L1+/MSI cancers had significantly more CD8 T cells at tumor invasive front than PD-L1+/EBV-/MSS cancers (P<0.001). PD-L1+ was not associated with the depth of invasion or nodal metastasis (P=0.534, 0.288). Multivariate analysis showed PD-L1+ was not an independent predictor of disease-free survival while MSI was (P=0.548, 0.043). In summary, EBV+ or MSI gastric cancers are more likely to express PD-L1 and have increased CD8 T cells at tumor invasive front than EBV-/MSS cancers. Our results suggest EBV infection and MSI should be investigated for predicting response to PD-1 blockade.
Respiratory syncytial virus (RSV) is a single-stranded RNA virus that assembles into viral filaments at the cell surface. Virus assembly often depends on the ability of a virus to use host proteins to accomplish viral tasks. Since the fusion protein cytoplasmic tail (FCT) is critical for viral filamentous assembly, we hypothesized that host proteins important for viral assembly may be recruited by the FCT. Using a yeast two-hybrid screen, we found that filamin A interacted with FCT, and mammalian cell experiments showed it localized to viral filaments but did not affect viral replication. Furthermore, we found that a number of actin-associated proteins also were excluded from viral filaments. Actin or tubulin cytoskeletal rearrangement was not necessary for F trafficking to the cell surface or for viral assembly into filaments, but was necessary for optimal viral replication and may be important for anchoring viral filaments. These findings suggest that RSV assembly into filaments occurs independently of actin polymerization and that viral proteins are the principal drivers for the mechanical tasks involved with formation of complex, structured RSV filaments at the host cell plasma membrane.
The microbiome is increasingly recognized for its role in multiple aspects of cancer development and treatment, specifically in response to checkpoint inhibitors. While checkpoint inhibitors have revolutionized cancer treatment by producing durable anti-tumor responses, only a minority of patients respond to the available immunotherapy drugs and accurate, sensitive and specific microbiome predictors of response to treatment remain elusive. Additionally, the specific mechanisms linking the microbiome and host immunological responses remain unclear. In this review, we examine the evidence for the gut microbiome's association with anti-tumor responses to checkpoint inhibitors in the treatment of melanoma, non-small cell lung cancer, and renal cell carcinoma. Furthermore, we discuss the current evidence available from murine models seeking to explain the immunological mechanisms that may drive this process. While this work is promising in defining the impact of gut microbiota in cancer treatment, many unanswered questions indicate the need for additional human and experimental studies.
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