Reactivation from latency displays HIV particle budding at plasma membrane, accompanying CD44 upregulation and recruitment

<p>Abstract</p> <p>Background</p> <p>It has been accepted that HIV buds from the cell surface in T lymphocytes, whereas in macrophages it buds into intracellular endosomes. Recent studies, on the other hand, suggest that HIV preferentially buds from the cell surface even in monocytic cells. However, most studies are based on observations in acutely infected cells and little is known about HIV budding concomitant with reactivation from latency. Such studies would provide a better understanding of a reservoir for HIV.</p> <p>Results</p> <p>We observed HIV budding in latently infected T lymphocytic and monocytic cell lines following TNF-α stimulation and examined the upregulation of host factors that may be involved in particle production. Electron microscopy analysis revealed that reactivation of latently infected J1.1 cells (latently infected Jurkat cells with HIV-1) and U1 cells (latently infected U937 cells with HIV-1) displayed HIV particle budding predominantly at the plasma membrane, a morphology that is similar to particle budding in acutely infected Jurkat and U937 cells. When mRNA expression levels were quantified by qRT-PCR, we found that particle production from reactivated J1.1 and U1 cells was accompanied by CD44 upregulation. This upregulation was similarly observed when Jurkat and U937 cells were acutely infected with HIV-1 but not when just stimulated with TNF-α, suggesting that CD44 upregulation was linked with HIV production but not with cell stimulation. The molecules in endocytic pathways such as CD63 and HRS were also upregulated when U1 cells were reactivated and U937 cells were acutely infected with HIV-1. Confocal microscopy revealed that these upregulated host molecules were recruited to and accumulated at the sites where mature particles were formed at the plasma membrane.</p> <p>Conclusion</p> <p>Our study indicates that HIV particles are budded at the plasma membrane upon reactivation from latency, a morphology that is similar to particle budding in acute infection. Our data also suggest that HIV expression may lead to the upregulation of certain host cell molecules that are recruited to sites of particle assembly, possibly coordinating particle production.</p

Identification and functional analysis of glutamine transporter inStreptococcus mutans

Background Streptococcus mutans, a biofilm-forming bacterium, possesses several transporters that function as import/export molecules. Among them, the PII protein family is composed of members that regulate glutamine synthesis in bacterial species. Objective In this study, we characterized the function of the glutamine transporter in S. mutans MT8148. Methods The SMU.732 gene, corresponding to glnP in S. mutans, is homologous to the glutamine transporter gene in Bacillus subtilis. We constructed a glnP-inactivated mutant strain (GEMR) and a complement strain (comp-GEMR) and evaluated their biological functions. Results Growth of GEMR was similar in the presence and absence of glutamine, whereas the growth rates of MT8148 and comp-GEMR were significantly lower in the presence of glutamine as compared to its absence. Furthermore, biofilms formed by MT8148 and comp-GEMR were significantly thicker than that formed by GEMR, while the GEMR strain showed a significantly lower survival rate in an acidic environment than the other strains. Addition of n-phenyl-2-naphthylamine, used to label of the membrane, led to increased fluorescence intensity of MT8148 and GEMR, albeit that was significantly lower in the latter. Conclusions These results suggest that glnP is associated with glutamine transport in S. mutans, especially the import of glutamine involved in biofilm formation

Identification of the P-TEFb complex-interacting domain of Brd4 as an inhibitor of HIV-1 replication by functional cDNA library screening in MT-4 cells

AbstractWe conducted a phenotypic cDNA screening using a T cell line-based assay to identify human genes that render cells resistant to human immunodeficiency virus type 1 (HIV-1). We isolated potential HIV-1 resistance genes, including the carboxy terminal domain (CTD) of bromodomain-containing protein 4 (Brd4). Expression of GFP-Brd4-CTD was tolerated in MT-4 and Jurkat cells in which HIV-1 replication was markedly inhibited. We provide direct experimental data demonstrating that Brd4-CTD serves as a specific inhibitor of HIV-1 replication in T cells. Our method is a powerful tool for the identification of host factors that regulate HIV-1 replication in T cells

Adaptability and selectivity of human peroxisome proliferator-activated receptor (PPAR) pan agonists revealed from crystal structures

The structures of the ligand-binding domains (LBDs) of human peroxisome proliferator-activated receptors (PPARα, PPARγ and PPARδ) in complexes with a pan agonist, an α/δ dual agonist and a PPARδ-specific agonist were determined. The results explain how each ligand is recognized by the PPAR LBDs at an atomic level

Substitution of the myristoylation signal of human immunodeficiency virus type 1 Pr55Gag with the phospholipase C-δ1 pleckstrin homology domain results in infectious pseudovirion production

The matrix domain (MA) of human immunodeficiency virus type 1 Pr55Gag is covalently modified with a myristoyl group that mediates efficient viral production. However, the role of myristoylation, particularly in the viral entry process, remains uninvestigated. This study replaced the myristoylation signal of MA with a well-studied phosphatidylinositol 4,5-biphosphate-binding plasma membrane (PM) targeting motif, the phospholipase C-δ1 pleckstrin homology (PH) domain. PH–Gag–Pol PM targeting and viral production efficiencies were improved compared with Gag–Pol, consistent with the estimated increases in Gag–PM affinity. Both virions were recovered in similar sucrose density-gradient fractions and had similar mature virion morphologies. Importantly, PH–Gag–Pol and Gag–Pol pseudovirions had almost identical infectivity, suggesting a dispensable role for myristoylation in the virus life cycle. PH–Gag–Pol might be useful in separating the myristoylation-dependent processes from the myristoylation-independent processes. This the first report demonstrating infectious pseudovirion production without myristoylated Pr55Gag

血清ペプシノゲン検査に基づく胃がん発生率と有効性に関する15年間のコホート研究

Objectives : The incidence of and mortality from gastric cancer in Japan have remained high and prophylaxis is important. However, the number of the individuals undergoing gastric mass radiography has decreased in recent years because the examination has a big burden at the time of the consultation. Many studies have reported the ease and effectiveness of the pepsinogen test and a higher incidence of gastric cancer in positive groups. However, the longest survey period was 10 years. Therefore, we conducted a 15-year cohort study to examine the validity of the testing period and the incidence of gastric cancer in serum pepsinogen positive and negative groups at a private company utilizing pepsinogen test. Methods : Subjects were 4383 employees who received a pepsinogen test. Subjects were followed for 15 years. For the purpose of examining the three periods over five-, 10-, and 15-year periods, we analyzed the validity of testing during each period, carried out a log-rank test, and analyzed hazard ratio in the Cox proportional hazard model. Results : The number of individuals who developed gastric cancer during the survey was nine in the five-year negative group, 18 in the five-year positive group, 16 in the 10-year negative group, 27 in the 10-year positive group, 31 in the 15-year negative group, and 29 in the 15-year positive group. The sensitivity of testing was 0.667 over the first five years, 0.628 over 10 years, and 0.483 over 15 years, and the specificity was 0.744 over the first five years, 0.745 over 10 years, and 0.745 over 15 years. The five-year incidence of gastric cancer was 57 per 100,000 person years in the negative group and 350 per 100,000 person years in the positive group. The ten-year incidences were 53 per 100,000 person years in the negative group and 279 per 100,000 person years in the positive group. The 15-year incidence was 75 per 100,000 person years in the negative group and 231 per 100,000 person years in the positive group. The hazard ratio of the positive group toward the negative group was 4.98 over the first five years, 4.71 over 10 years, and 2.76 over 15 years (p＜0.001). Conclusions : This study clarified that the first five years after the testing showed the highest hazard ratio and validity, therefore, the validity of testing was approximately 10 years. 【目的】我が国の胃がんの死亡率及び罹患率は現在も上位であり、その予防対策は重要で ある。けれども、近年X線の胃集団検診の受診者は、受診時の負担が大きいことから減少 している。一方職域の胃がん予防対策は、法的義務が無いため、企業により様々であるが 地域同様減少している。多くの先行研究では、血清ペプシノゲン検査法が簡便で有益とし ており、陽性群に発症率が高いとしているが、調査期間は最長10年であり、15年間の調査 はなかった。そこで、一企業において15年間の“陽性群”と“陰性群”での発症率の違い と検査の有効性の期間を検討することを目的にコホート調査を行った。 【方法】ペプシノゲン検査を受診した4,383名を15年間追跡した。５年間、10年間、15年間の ３期間に区切って各期間の検査の有効性を算出し、発症率をLog-rankで検定し、発症危険 度をCox比例ハザードで分析した。 【結果】追跡期間中に陰性群と陽性群のそれぞれの胃がん発症数は、５年間で９人と18人、 10年間で16人と27人、15年間で31人と29人であった。検査の感度は５年間で0.667、10年間 で0.628、15年間で0.483であり、特異度は、５年間で0.744、10年間で0.745，15年間で0.745 であった。発症率では、５年間の陰性群と陽性群で57、350per 100,000 person yearsであり、 10年では53、279 per 100,000 person years であり、15年では 75、231 per 100,000 person yearsであった。陰性群に対する陽性群の発生危険度は、５年間4.98、10年間4.71、15年間 2.76であった (p＜0.001)。 【結論】胃がんの発症率及び発症危険度や検査の有効性が最も高かったのは、2000年まで の５年間であり、検査の有効性は、約10年であることが明らかになった。Thesis of Takami Okuno / 奥野 敬生 博士論文 金沢大学医薬保健学総合研究科（保健学専攻