Retroviruses integrate into a shared, non-palindromic DNA motif.

Many DNA-binding factors, such as transcription factors, form oligomeric complexes with structural symmetry that bind to palindromic DNA sequences1. Palindromic consensus nucleotide sequences are also found at the genomic integration sites of retroviruses2-6 and other transposable elements7-9, and it has been suggested that this palindromic consensus arises as a consequence of the structural symmetry in the integrase complex2,3. However, we show here that the palindromic consensus sequence is not present in individual integration sites of human T-cell lymphotropic virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1), but arises in the population average as a consequence of the existence of a non-palindromic nucleotide motif that occurs in approximately equal proportions on the plus strand and the minus strand of the host genome. We develop a generally applicable algorithm to sort the individual integration site sequences into plus-strand and minus-strand subpopulations, and use this to identify the integration site nucleotide motifs of five retroviruses of different genera: HTLV-1, HIV-1, murine leukaemia virus (MLV), avian sarcoma leucosis virus (ASLV) and prototype foamy virus (PFV). The results reveal a non-palindromic motif that is shared between these retroviruses

A frequent PLCγ1 mutation in adult T-cell leukemia/lymphoma determines functional properties of the malignant cells

BACKGROUND: Development of adult T-cell leukemia/lymphoma (ATL) involves human T-cell leukemia virus type 1 (HTLV-1) infection and accumulation of somatic mutations. The most frequently mutated gene in ATL (36 % of cases) is phospholipase C gamma1 (PLCG1). PLCG1 is also frequently mutated in other T-cell lymphomas. However, the functional consequences of the PLCG1 mutations in cancer cells have not been characterized. METHODS: We compared the activity of the wild-type PLCγ1 with that of a mutant carrying a hot-spot mutation of PLCγ1 (S345F) observed in ATL, both in cells and in cell-free assays. To analyse the impact of the mutation on cellular properties, we quantified cellular proliferation, aggregation, chemotaxis and apoptosis by live cell-imaging in an S345F+ ATL-derived cell line (KK1) and a KK1 cell line in which we reverted the mutation to the wild-type sequence using CRISPR/Cas9 and homology-directed repair. FINDINGS: The PLCγ1 S345F mutation results in an increase of basal PLC activity in vitro and in different cell types. This higher basal activity is further enhanced by upstream signalling. Reversion of the S345F mutation in the KK1 cell line resulted in reduction of the PLC activity, lower rates of proliferation and aggregation, and a marked reduction in chemotaxis towards CCL22. The PLCγ1-pathway inhibitors ibrutinib and ritonavir reduced both the PLC activity and the tested functions of KK1 cells. INTERPRETATION: Consistent with observations from clinical studies, our data provide direct evidence that activated variants of the PLCγ1 enzyme contribute to the properties of the malignant T-cell clone in ATL. FUNDING: MRC (UK) Project Grant (P028160)

Free serum haemoglobin is associated with brain atrophy in secondary progressive multiple sclerosis.

Background A major cause of disability in secondary progressive multiple sclerosis (SPMS) is progressive brain atrophy, whose pathogenesis is not fully understood. The objective of this study was to identify protein biomarkers of brain atrophy in SPMS. Methods We used surface-enhanced laser desorption-ionization time-of-flight mass spectrometry to carry out an unbiased search for serum proteins whose concentration correlated with the rate of brain atrophy, measured by serial MRI scans over a 2-year period in a well-characterized cohort of 140 patients with SPMS. Protein species were identified by liquid chromatography-electrospray ionization tandem mass spectrometry. Results There was a significant (p<0.004) correlation between the rate of brain atrophy and a rise in the concentration of proteins at 15.1 kDa and 15.9 kDa in the serum. Tandem mass spectrometry identified these proteins as alpha-haemoglobin and beta-haemoglobin, respectively.  The abnormal concentration of free serum haemoglobin was confirmed by ELISA (p<0.001). The serum lactate dehydrogenase activity was also highly significantly raised (p<10-12) in patients with secondary progressive multiple sclerosis. Conclusions An underlying low-grade chronic intravascular haemolysis is a potential source of the iron whose deposition along blood vessels in multiple sclerosis plaques contributes to the neurodegeneration and consequent brain atrophy seen in progressive disease. Chelators of free serum iron will be ineffective in preventing this neurodegeneration, because the iron (Fe2+) is chelated by haemoglobin

Balancing the robustness and predictive performance of biomarkers.

Recent studies have highlighted the importance of assessing the robustness of putative biomarkers identified from experimental data. This has given rise to the concept of stable biomarkers, which are ones that are consistently identified regardless of small perturbations to the data. Since stability is not by itself a useful objective, we present a number of strategies that combine assessments of stability and predictive performance in order to identify biomarkers that are both robust and diagnostically useful. Moreover, by wrapping these strategies around logistic regression classifiers regularized by the elastic net penalty, we are able to assess the effects of correlations between biomarkers upon their perceived stability. We use a synthetic example to illustrate the properties of our proposed strategies. In this example, we find that: (i) assessments of stability can help to reduce the number of false-positive biomarkers, although potentially at the cost of missing some true positives; (ii) combining assessments of stability with assessments of predictive performance can improve the true positive rate; and (iii) correlations between biomarkers can have adverse effects on their stability and hence must be carefully taken into account when undertaking biomarker discovery. We then apply our strategies in a proteomics context to identify a number of robust candidate biomarkers for the human disease HTLV1-associated myelopathy/tropical spastic paraparesis (HAM/TSP)

HTLV-1 Integration into Transcriptionally Active Genomic Regions Is Associated with Proviral Expression and with HAM/TSP

Human T-lymphotropic virus type 1 (HTLV-1) causes leukaemia or chronic inflammatory disease in ∼5% of infected hosts. The level of proviral expression of HTLV-1 differs significantly among infected people, even at the same proviral load (proportion of infected mononuclear cells in the circulation). A high level of expression of the HTLV-1 provirus is associated with a high proviral load and a high risk of the inflammatory disease of the central nervous system known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). But the factors that control the rate of HTLV-1 proviral expression remain unknown. Here we show that proviral integration sites of HTLV-1 in vivo are not randomly distributed within the human genome but are associated with transcriptionally active regions. Comparison of proviral integration sites between individuals with high and low levels of proviral expression, and between provirus-expressing and provirus non-expressing cells from within an individual, demonstrated that frequent integration into transcription units was associated with an increased rate of proviral expression. An increased frequency of integration sites in transcription units in individuals with high proviral expression was also associated with the inflammatory disease HAM/TSP. By comparing the distribution of integration sites in human lymphocytes infected in short-term cell culture with those from persistent infection in vivo, we infer the action of two selective forces that shape the distribution of integration sites in vivo: positive selection for cells containing proviral integration sites in transcriptionally active regions of the genome, and negative selection against cells with proviral integration sites within transcription units

Human T-Lymphotropic Virus-1 Visualized at the Virological Synapse by Electron Tomography

Human T-lymphotropic virus 1 (HTLV-1) is transmitted directly between cells via an organized cell-cell contact called a virological synapse (VS) [1], [2]. The VS has been studied by light microscopy, but the ultrastructure of the VS and the nature of the transmitted viral particle have remained unknown. Cell-free enveloped virions of HTLV-1 are undetectable in the serum of individuals infected with the human T-lymphotropic virus 1 (HTLV-1) and during in vitro culture of naturally infected lymphocytes. However, the viral envelope protein is required for infectivity of HTLV-1, suggesting that complete, enveloped HTLV-1 virions are transferred across the synapse. Here, we use electron tomography combined with immunostaining of viral protein to demonstrate the presence of enveloped HTLV-1 particles within the VS formed between naturally infected lymphocytes. We show in 3D that HTLV-1 particles can be detected in multiple synaptic clefts at different locations simultaneously within the same VS. The synaptic clefts are surrounded by the tightly apposed plasma membranes of the two cells. HTLV-1 virions can contact the recipient cell membrane before detaching from the infected cell. The results show that the HTLV-1 virological synapse that forms spontaneously between lymphocytes of HTLV-1 infected individuals allows direct cell-cell transmission of the virus by triggered, directional release of enveloped HTLV-1 particles into confined intercellular spaces