GITR Intrinsically Sustains Early Type 1 and Late Follicular Helper CD4 T Cell Accumulation to Control a Chronic Viral Infection

CD4 T cells are critical for control of persistent infections; however, the key signals that regulate CD4 T help during chronic infection remain incompletely defined. While several studies have addressed the role of inhibitory receptors and soluble factors such as PD-1 and IL-10, significantly less work has addressed the role of T cell co-stimulatory molecules during chronic viral infection. Here we show that during a persistent infection with lymphocytic choriomeningitis virus (LCMV) clone 13, mice lacking the glucocorticoid-induced tumor necrosis factor receptor related protein (GITR) exhibit defective CD8 T cell accumulation, increased T cell exhaustion and impaired viral control. Differences in CD8 T cells and viral control between GITR^+/+ and GITR^-/- mice were lost when CD4 T cells were depleted. Moreover, mixed bone marrow chimeric mice, as well as transfer of LCMV epitope-specific CD4 or CD8 T cells, demonstrated that these effects of GITR are largely CD4 T cell-intrinsic. GITR is dispensable for initial CD4 T cell proliferation and differentiation, but supports the post-priming accumulation of IFNγ⁺IL-2⁺ Th1 cells, facilitating CD8 T cell expansion and early viral control. GITR-dependent phosphorylation of the p65 subunit of NF-κB as well as phosphorylation of the downstream mTORC1 target, S6 ribosomal protein, were detected at day three post-infection (p.i.), and defects in CD4 T cell accumulation in GITR-deficient T cells were apparent starting at day five p.i. Consistently, we pinpoint IL-2-dependent CD4 T cell help for CD8 T cells to between days four and eight p.i. GITR also increases the ratio of T follicular helper to T follicular regulatory cells and thereby enhances LCMV-specific IgG production. Together, these findings identify a CD4 T cell-intrinsic role for GITR in sustaining early CD8 and late humoral responses to collectively promote control of chronic LCMV clone 13 infection.     

The Bipartite Geminivirus Coat Protein Aids BR1 Function in Viral Movement by Affecting the Accumulation of Viral Single-Stranded DNA

The movement of bipartite geminiviruses such as squash leaf curl virus (SqLCV) requires the cooperative interaction of two essential virus-encoded movement proteins, BR1 and BL1. While the viral coat protein AR1 is not essential for systemic infection, genetic studies demonstrate that its presence masks the defective phenotype of certain BR1 missense mutants, thus suggesting that coat protein does interact with the viral movement pathway. To further examine the mechanism of this interaction, we have constructed alanine-scanning mutants of AR1 and studied them for the ability to mask the infectivity defects of appropriate BR1 mutants, for the ability to target to the nucleus and to bind viral single-stranded DNA (ssDNA) and multimerize, and for effects on the accumulation of replicated viral ssDNA. We identified a specific region of AR1 required for masking of appropriate BR1 mutants and showed that this same region of AR1 was also important for ssDNA binding and the accumulation of viral replicated ssDNA. This region of AR1 also overlapped that involved in multimerization of the coat protein. We also found that the accumulation in protoplasts of single-stranded forms of a recombinant plasmid that included the SqLCV replication origin but was too large to be encapsidated was dependent on the presence of AR1 but did not appear to require encapsidation. These findings extend our model for SqLCV movement, demonstrating that coat protein affects viral movement through its ability to induce the accumulation of replicated viral ssDNA genomes. They further suggested that encapsidation was not required for the AR1-dependent accumulation of viral ssDNA.     

A Lytic Viral Long Noncoding RNA Modulates the Function of a Latent Protein

Latent Kaposi''s sarcoma-associated herpesvirus (KSHV) episomes are coated with viral latency-associated nuclear antigen (LANA). In contrast, LANA rapidly disassociates from episomes during reactivation. Lytic KSHV expresses polyadenylated nuclear RNA (PAN RNA), a long noncoding RNA (lncRNA). We report that PAN RNA promotes LANA-episome disassociation through an interaction with LANA which facilitates LANA sequestration away from KSHV episomes during reactivation. These findings suggest that KSHV may have evolved an RNA aptamer to regulate latent protein function.     

Abundance and half-life of the distinct oat phytochrome A3 and A4 mRNAs

Gene-preferential oligonucleotide probes were used to determined the relative abundance and half-lives of distinct oat phytochrome A (PHYA) mRNAs. Oat PHYA mRNAs are highly conserved in the 5-untranslated region and the coding region, but the 3-untranslated region has an overall lower sequence conservation and was the source of gene-preferential probes. PHYA3 mRNA was estimated to be ca. 61% of the oat PHYA mRNA pool present in poly(A)⁺ RNA from dark-grown seedlings. The half-lives for PHYA3 and PHYA4 mRNAs were both estimated to be ca. 30 min, and a similar short half-life was estimated for the average PHYA mRNA. Sequence comparisons of PHYA mRNAs from four grass species identified conserved sequences within the 5- and 3-untranslated regions that might be important for PHYA mRNA degradation.     

Development of a Rapid Assay for Determining the Relative Abundance of Bacteria

A sandwich hybridization assay for high-throughput, rapid, simple, and inexpensive quantification of specific microbial populations was evaluated. The assay is based on the hybridization of a target rRNA with differentially labeled capture and detector probes. Betaproteobacterial ammonia-oxidizing bacteria (AOB) were selected as the target group for the study, since they represent a phylogenetically coherent group of organisms that perform a well-defined geochemical function in natural and engineered environments. Reagent concentrations, probe combinations, and washing, blocking, and hybridization conditions were optimized to improve signal and reduce background. The detection limits for the optimized RNA assay were equivalent to approximately 10³ to 10⁴ and 10⁴ to 10⁵ bacterial cells, respectively, for E. coli rRNA and RNA extracted from activated sludge, by using probes targeting the majority of bacteria. Furthermore, the RNA assay had good specificity, permitted discrimination of rRNA sequences that differed by a 2-bp mismatch in the probe target region, and could distinguish the sizes of AOB populations in nitrifying and nonnitrifying wastewater treatment plants.     

Circadian Control of the Accumulation of mRNAs for Light- and Heat-Inducible Chloroplast Proteins in Pea (Pisum sativum L.)

The levels of the mRNAs for light-inducible, nuclear-coded chloroplast proteins vary rhythmically in pea (Pisum sativum L.) plants either grown in a dark-light cycle or under constant light conditions. This has been observed for the early light-inducible protein, the light-harvesting chlorophyll a/b protein, and the small subunit of the ribulose-1,5-bisphosphate carboxylase. The mRNA levels are high in the morning, exhibit a minimum in the first half of the night, and increase again during the second half of the night. The amplitude of fluctuation is between 5- and 10-fold. A similar change in the mRNA abundance was found for four nuclear encoded heat-shock proteins of 18, 24, 26, and 30 kilodaltons. The ability of plants to transcribe heat-shock genes upon heat-shock for 2 hours varies through the day. The maxima for induction are found in the second half of the night and the morning. The minima are reached during the afternoon. The degree of fluctuation is between 3- and 5-fold. The levels of mRNAs for cytosolic as well as for plastid heat-shock proteins oscillate in parallel.     

Relative Contributions of Ectopic Liver and Abdominal Fat Accumulation to Arterial Stiffness

Objective: To compare the arterial stiffness between the abdominal obese population without nonalcoholic fatty liver disease (NAFLD) (referred to as abdominal fat accumulation), the normal-weight population with NAFLD (ectopic liver fat accumulation), and the population with both abdominal obesity and NAFLD (accumulation of both abdominal and ectopic liver fat).Methods: A total of 111,552 Chinese adults who underwent the health checkups from January 2012 to December 2012 were screened. Clinical and biochemical parameters were measured in each subject. NAFLD was diagnosed by ultrasonography. Arterial stiffness was evaluated by cardio-ankle vascular index (CAVI).Results: Normal-weight subjects with NAFLD had significantly higher CAVI than subjects with abdominal obesity with or without NAFLD (8.12 ± 1.16 vs. 7.93 ± 1.38, 7.96 ± 1.20; P<.01). When the presence of abdominal obesity, NAFLD, and both diseases (abdominal obesity and NAFLD) were included in regression analyses individually, CAVI was independently associated with abdominal obesity or NAFLD or both after adjusting for confounders. When the presence of abdominal obesity and NAFLD and both diseases were included simultaneously in regression analyses, the association between NAFLD and CAVI and the association between both diseases and CAVI remained significant, whereas the association between abdominal obesity and CAVI was no longer significant. The presence of NAFLD conferred a greater odds ratios of having an elevated CAVI than did the presence of abdominal obesity or even the presence of both diseases.Conclusion: Ectopic liver fat accumulation is associated with greater risk of arterial stiffness compared with abdominal fat accumulation or accumulation of both abdominal and ectopic liver fat.Abbreviations: ALT = alanine aminotransferase AST = aspartate aminotransferase BMI = body mass index CAVI = cardioankle vascular index CI = confidence interval Cr = creatinine FFA = free fatty acid FPG = fasting plasma glucose ?-GGT = ?-glutamyl transferase HDL-C = high-density-lipoprotein cholesterol LDL-C = lowdensity-lipoprotein cholesterol NAFLD = nonalcoholic fatty liver disease OR = odds ratio TC = total cholesterol TG = triglyceride UA = uric acid WC = waist circumference