Physiological magnesium concentrations increase fidelity of diverse reverse transcriptases from HIV-1, HIV-2, and foamy virus, but not MuLV or AMV.

Reverse transcriptases (RTs) are typically assayed using optimized Mg2+ concentrations (~5-10 mM) several-fold higher than physiological cellular free Mg2+ (~0.5 mM). Recent analyses demonstrated that HIV-1, but not Moloney murine leukaemia (MuLV) or avain myeloblastosis (AMV) virus RTs has higher fidelity in low Mg2+. In the current report, lacZα-based α-complementation assays were used to measure the fidelity of several RTs including HIV-1 (subtype B and A/E), several drug-resistant HIV-1 derivatives, HIV-2, and prototype foamy virus (PFV), all which showed higher fidelity using physiological Mg2+, while MuLV and AMV RTs demonstrated equivalent fidelity in low and high Mg2+. In 0.5 mM Mg2+, all RTs demonstrated approximately equal fidelity, except for PFV which showed higher fidelity. A Next Generation Sequencing (NGS) approach that used barcoding to determine mutation profiles was used to examine the types of mutations made by HIV-1 RT (type B) in low (0.5 mM) and high (6 mM) Mg2+ on a lacZα template. Unlike α-complementation assays which are dependent on LacZα activity, the NGS assay scores mutations at all positions and of every type. Consistent with α-complementation assays, a ~four-fold increase in mutations was observed in high Mg2+. These findings help explain why HIV-1 RT displays lower fidelity in vitro (with high Mg2+ concentrations) than other RTs (e.g. MuLV and AMV), yet cellular fidelity for these viruses is comparable. Establishing in vitro conditions that accurately represent RT's activity in cells is pivotal to determining the contribution of RT and other factors to the mutation profile observed with HIV-1.

The transition of M-CSF-derived human macrophages to a growth-promoting phenotype.

Stimulated macrophages are potent producers of inflammatory mediators. This activity is highly regulated, in part, by resolving molecules to prevent tissue damage. In this study, we demonstrate that inflammation induced by Toll-like receptor stimulation is followed by the upregulation of receptors for adenosine (Ado) and prostaglandin E2 (PGE2), which help terminate macrophage activation and initiate tissue remodeling and angiogenesis. Macrophages can be hematopoietically derived from monocytes in response to 2 growth factors: macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). We examine how exposure to either of these differentiation factors shapes the macrophage response to resolving molecules. We analyzed the transcriptomes of human monocyte-derived macrophages stimulated in the presence of Ado or PGE2 and demonstrated that, in macrophages differentiated in M-CSF, Ado and PGE2 induce a shared transcriptional program involving the downregulation of inflammatory mediators and the upregulation of growth factors. In contrast, macrophages generated in GM-CSF fail to convert to a growth-promoting phenotype, which we attribute to the suppression of receptors for Ado and PGE2 and lower production of these endogenous regulators. These observations indicate that M-CSF macrophages are better prepared to transition to a program of tissue repair, whereas GM-CSF macrophages undergo more profound activation. We implicate the differential sensitivity to pro-resolving mediators as a contributor to these divergent phenotypes. This research highlights a number of molecular targets that can be exploited to regulate the strength and duration of macrophage activation.

Host and parasite responses in human diffuse cutaneous leishmaniasis caused by L. amazonensis.

Diffuse cutaneous leishmaniasis (DCL) is a rare form of leishmaniasis where parasites grow uncontrolled in diffuse lesions across the skin. Meta-transcriptomic analysis of biopsies from DCL patients infected with Leishmania amazonensis demonstrated an infiltration of atypical B cells producing a surprising preponderance of the IgG4 isotype. DCL lesions contained minimal CD8+ T cell transcripts and no evidence of persistent TH2 responses. Whereas localized disease exhibited activated (so-called M1) macrophage presence, transcripts in DCL suggested a regulatory macrophage (R-Mϕ) phenotype with higher levels of ABCB5, DCSTAMP, SPP1, SLAMF9, PPARG, MMPs, and TM4SF19. The high levels of parasite transcripts in DCL and the remarkable uniformity among patients afforded a unique opportunity to study parasite gene expression in this disease. Patterns of parasite gene expression in DCL more closely resembled in vitro parasite growth in resting macrophages, in the absence of T cells. In contrast, parasite gene expression in LCL revealed 336 parasite genes that were differently upregulated, relative to DCL and in vitro macrophage growth, and these transcripts may represent transcripts that are produced by the parasite in response to host immune pressure.

Differential Content of Proteins, mRNAs, and miRNAs Suggests that MDSC and Their Exosomes May Mediate Distinct Immune Suppressive Functions.

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells that accumulate in the circulation and the tumor microenvironment of most cancer patients. There, MDSC suppress both adaptive and innate immunity, hindering immunotherapies. The inflammatory milieu often present in cancers facilitates MDSC suppressive activity, causing aggressive tumor progression and metastasis. MDSC from tumor-bearing mice release exosomes, which carry biologically active proteins and mediate some of the immunosuppressive functions characteristic of MDSC. Studies on other cell types have shown that exosomes may also carry RNAs which can be transferred to local and distant cells, yet the mRNA and microRNA cargo of MDSC-derived exosomes has not been studied to date. Here, the cargo of MDSC and their exosomes was interrogated with the goal of identifying and characterizing molecules that may facilitate MDSC suppressive potency. Because inflammation is an established driving force for MDSC suppressive activity, we used the well-established 4T1 mouse mammary carcinoma system, which includes "conventional" as well as "inflammatory" MDSC. We provide evidence that MDSC-derived exosomes carry proteins, mRNAs, and microRNAs with different quantitative profiles than those of their parental cells. Several of these molecules have known or predicted functions consistent with MDSC suppressive activity, suggesting a potential mechanistic redundancy.

The Transcriptional Regulator CpsY Is Important for Innate Immune Evasion in Streptococcus pyogenes.

As an exclusively human pathogen, Streptococcus pyogenes (the group A streptococcus [GAS]) has specifically adapted to evade host innate immunity and survive in multiple tissue niches, including blood. GAS can overcome the metabolic constraints of the blood environment and expresses various immunomodulatory factors necessary for survival and immune cell resistance. Here we present our investigation of one such factor, the predicted LysR family transcriptional regulator CpsY. The encoding gene, cpsY, was initially identified as being required for GAS survival in a transposon-site hybridization (TraSH) screen in whole human blood. CpsY is homologous with transcriptional regulators of Streptococcus mutans (MetR), Streptococcus iniae (CpsY), and Streptococcus agalactiae (MtaR) that regulate methionine transport, amino acid metabolism, resistance to neutrophil-mediated killing, and survival in vivo Our investigation indicated that CpsY is involved in GAS resistance to innate immune cells of its human host. However, GAS CpsY does not manifest the in vitro phenotypes of its homologs in other streptococcal species. GAS CpsY appears to regulate a small set of genes that is markedly different from the regulons of its homologs. The differential expression of these genes depends on the growth medium, and CpsY modestly influences their expression. The GAS CpsY regulon includes known virulence factors (mntE, speB, spd, nga [spn], prtS [SpyCEP], and sse) and cell surface-associated factors of GAS (emm1, mur1.2, sibA [cdhA], and M5005_Spy0500). Intriguingly, the loss of CpsY in GAS does not result in virulence defects in murine models of infection, suggesting that CpsY function in immune evasion is specific to the human host.

Cell cycle control (and more) by programmed -1 ribosomal frameshifting: implications for disease and therapeutics.

Abstract Like most basic molecular mechanisms, programmed -1 ribosomal frameshifting (-1 PRF) was first identified in viruses. Early observations that global dysregulation of -1 PRF had deleterious effects on yeast cell growth suggested that -1 PRF may be used to control cellular gene expression, and the cell cycle in particular. Collection of sufficient numbers of viral -1 PRF signals coupled with advances in computer sciences enabled 2 complementary computational approaches to identify -1 PRF signals in free living organisms. The unexpected observation that almost all -1 PRF events on eukaryotic mRNAs direct ribosomes to premature termination codons engendered the hypothesis that -1 PRF signals post-transcriptionally regulate gene expression by functioning as mRNA destabilizing elements. Emerging research suggests that some human diseases are associated with global defects in -1 PRF. The recent discovery of -1 PRF signal-specific trans-acting regulators may provide insight into novel therapeutic strategies aimed at treating diseases caused by changes in gene expression patterns.