In the early phase of HIV infection, as studied in vitro, high levels of unintegrated (both linear and circular) and integrated (provirus) forms of viral DNA are seen, and cells produce high levels of virus. In time, the level of unintegrated DNA declines, followed by a progressive decline in virus expression. Extensive studies of the proviral landscape in people living with HIV (PLWH) on antiretroviral therapy (ART) show that only about 2% of proviruses are intact; the remainder are characterized as defective and contain numerous deletions of proviral DNA segments and hypermutations. In the current study, we investigated the decline of viral expression in infected T cells in search of mechanisms involved in proviral inactivation.
We derived clonal lines from Jurkat cells infected with HIV MN and monitored them for viral expression over time in culture. In a subset of clones that displayed a decline in expression, we found provirus containing large deletions and the integration of a retrotranscribed molecule of tRNAGly adjacent to the 3'-end of the proviral DNA. We provide evidence linking the proviral deletions to the insertion of a reverse transcribed tRNAGly molecule and propose a mechanism for its self-primed reverse transcription.
Large deletions of proviral DNA have been reported in PLWH on ART and attributed to errors that occurred in the synthesis of the minus strand during the reverse transcription of the viral genome. Our results support an additional mechanism for proviral deletions, mediated by tRNAGly, in the inactivation of the provirus.
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Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) is a coinhibitory receptor that plays an essential role in maintaining immune system homeostasis by suppressing T-cell activation. We report a sporadic case of CTLA4 haploinsufficiency in a patient with Epstein-Barr virus-positive diffuse large B-cell lymphoma and subsequent benign lymphadenopathy. A missense mutation in exon 2 of the CTLA4 gene (c.251T>C, p.V84A) was found in the patient's peripheral blood and buccal cell DNA, but not in her parents' DNA. CTLA4 expression decreased in the peripheral regulatory T cells upon stimulation, whereas CTLA4 and PD-1-positive T cell subsets increased, possibly to compensate for the defective CTLA4 function. This case suggests that some adult lymphoma patients with no remarkable medical history have primary immune disorder. As immune-targeted therapies are now widely used for the treatment of malignancies, it is increasingly important to recognize the underlying primary immune disorders to properly manage the disease and avoid unexpected complications of immunotherapies.

Nucleic acid content can be quantified by flow cytometry through the use of intercalating compounds; however, measuring the presence of specific sequences has hitherto been difficult to achieve by this methodology. The primary obstacle to detecting discrete nucleic acid sequences by flow cytometry is their low quantity and the presence of high background signals, rendering the detection of hybridized fluorescent probes challenging. Amplification of nucleic acid sequences by molecular techniques such as in situ PCR have been applied to single-cell suspensions, but these approaches have not been easily adapted to conventional flow cytometry. An alternative strategy implements a Branched DNA technique, comprising target-specific probes and sequentially hybridized amplification reagents, resulting in a theoretical 8,000- to 16,000-fold increase in fluorescence signal amplification. The Branched DNA technique allows for the quantification of native and unmanipulated mRNA content with increased signal detection and reduced background. This procedure utilizes gentle fixation steps with low hybridization temperatures, leaving the assayed cells intact to permit their concomitant immunophenotyping. This technology has the potential to advance scientific discovery by correlating potentially small quantities of mRNA with many biological measurements at the single-cell level.
Copyright © 2016 John Wiley & Sons, Inc.

Hepatitis C virus (HCV) E2 protein binds to CD81, which is a component of the B cell co-stimulatory complex. The E2-CD81 interaction leads to B cell proliferation, protein tyrosine phosphorylation and to the hypermutation of immunoglobulin genes. Epidemiological studies have reported a high prevalence of B cell non-Hodgkin lymphoma (NHL) in HCV-positive patients, suggesting a potential association between HCV and Epstein-Barr virus (EBV) in the genesis of B lymphocyte proliferative disorders. In the present study, in order to investigate the association between EBV and HCV in B cells, we created an in vitro EBV-induced B cell transformation model. CD81 was gradually overexpressed during transformation by EBV. B cells isolated from HCV-positive patients grew more rapidly and clumped together earlier than B cells isolated from healthy donors following EBV infection. Pre-stimulation of CD81 expressed by resting B cells with anti-CD81 monoclonal antibody (mAb) or HCV E2 accelerated the generation of lymphoblastoid cell lines (LCLs) by EBV infection. These cells proliferated prominently through the early expression of interleukin-10 and intracellular latent membrane protein (LMP)-l. By contrast, the overexpression of CD81 on EBV-transformed B cells by anti-CD81 mAb or HCV E2 protein induced apoptosis through reactive oxygen species (ROS)-mediated mitochondrial dysfunction. These results suggest that the engagement of CD81 expressed by B cells has differential effects on B cell fate (proliferation or apoptosis) according to EBV infection and the expression level of CD81.