Characterization of the HIV-1 RNA associated proteome identifies Matrin 3 as a nuclear cofactor of Rev function

<p>Abstract</p> <p>Background</p> <p>Central to the fully competent replication cycle of the human immunodeficiency virus type 1 (HIV-1) is the nuclear export of unspliced and partially spliced RNAs mediated by the Rev posttranscriptional activator and the Rev response element (RRE).</p> <p>Results</p> <p>Here, we introduce a novel method to explore the proteome associated with the nuclear HIV-1 RNAs. At the core of the method is the generation of cell lines harboring an integrated provirus carrying RNA binding sites for the MS2 bacteriophage protein. Flag-tagged MS2 is then used for affinity purification of the viral RNA. By this approach we found that the viral RNA is associated with the host nuclear matrix component MATR3 (Matrin 3) and that its modulation affected Rev activity. Knockdown of MATR3 suppressed Rev/RRE function in the export of unspliced HIV-1 RNAs. However, MATR3 was able to associate with Rev only through the presence of RRE-containing viral RNA.</p> <p>Conclusions</p> <p>In this work, we exploited a novel proteomic method to identify MATR3 as a cellular cofactor of Rev activity. MATR3 binds viral RNA and is required for the Rev/RRE mediated nuclear export of unspliced HIV-1 RNAs.</p

Intragenic transcriptional cis-activation of the human immunodeficiency virus 1 does not result in allele-specific inhibition of the endogenous gene

<p>Abstract</p> <p>Background</p> <p>The human immunodeficiency virus type 1 (HIV-1) favors integration in active genes of host chromatin. It is believed that transcriptional interference of the viral promoter over the endogenous gene or vice versa might occur with implications in HIV-1 post-integrative transcriptional latency.</p> <p>Results</p> <p>In this work a cell line has been transduced with a HIV-based vector and selected for Tat-inducible expression. These cells were found to carry a single silent integration in sense orientation within the second intron of the <it>HMBOX1 </it>gene. The HIV-1 Tat transactivator induced the viral LTR and repressed <it>HMBOX1 </it>expression independently of vector integration. Instead, single-cell quantitative <it>in situ </it>hybridization revealed that allele-specific transcription of <it>HMBOX1 </it>carrying the integrated provirus was not affected by the transactivation of the viral LTR in <it>cis</it>.</p> <p>Conclusion</p> <p>A major observation of the work is that the HIV-1 genome has inserted in genes that are also repressed by Tat and this could be an advantage for the virus during transcriptional reactivation. In addition, it has also been observed that transcription of the provirus and of the endogenous gene in which it is integrated may coexist at the same time in the same genomic location.</p

A real-time view of the TAR:Tat:P-TEFb complex at HIV-1 transcription sites

HIV-1 transcription is tightly regulated: silent in long-term latency and highly active in acutely-infected cells. Transcription is activated by the viral protein Tat, which recruits the elongation factor P-TEFb by binding the TAR sequence present in nascent HIV-1 RNAs. In this study, we analyzed the dynamic of the TAR:Tat:P-TEFb complex in living cells, by performing FRAP experiments at HIV-1 transcription sites. Our results indicate that a large fraction of Tat present at these sites is recruited by Cyclin T1. We found that in the presence of Tat, Cdk9 remained bound to nascent HIV-1 RNAs for 71s. In contrast, when transcription was activated by PMA/ionomycin, in the absence of Tat, Cdk9 turned-over rapidly and resided on the HIV-1 promoter for only 11s. Thus, the mechanism of trans-activation determines the residency time of P-TEFb at the HIV-1 gene, possibly explaining why Tat is such a potent transcriptional activator. In addition, we observed that Tat occupied HIV-1 transcription sites for 55s, suggesting that the TAR:Tat:P-TEFb complex dissociates from the polymerase following transcription initiation, and undergoes subsequent cycles of association/dissociation

pKBuS13, a KPC-2-Encoding Plasmid from Klebsiella pneumoniae Sequence Type 833, Carrying Tn4401b Inserted into an Xer Site-Specific Recombination Locus

Here, we report the first detection of a Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing Klebsiella pneumoniae strain belonging to sequence type 833 (ST833), collected in an Italian hospital from a patient coming from South America. Its bla KPC determinant was carried by a ColE1 plasmid, pKBuS13, that showed the Tn4401b::bla KPC-2 transposon inserted into the regulatory region of an Xer site-specific recombination locus. This interfered with the correct resolution of plasmid multimers into monomers, lowering plasmid stability and leading to overestimation of the number of plasmids harbored by a single host cell. Sequencing of the fragments adjacent to Tn4401b detected a region that did not have significant matches in databases other than the genome of a carbapenem-resistant Escherichia coli strain collected during the same year at a hospital in Boston. This is interesting in an epidemiologic context, as it suggests that despite the absence of tra genes and the instability under nonselective conditions, the circulation of pKBuS13 or of analogous plasmids might be wider than reported

Clinical management of non-faecium non-faecalis vancomycin-resistant enterococci infection. Focus on Enterococcus gallinarum and Enterococcus casseliflavus/flavescens

Enterococcus gallinarum and Enterococcus casseliflavus/flavescens are enterococci intrinsically resistant to vancomycin belonging to the E. gallinarum group. They are responsible mainly for healthcare-associated infections, in particular bloodstream, urinary tract and surgical wound infections. Diseases due to these bacteria are significantly increasing worldwide, as they are prone to cause infection in patients with concurrent hepatobiliary or oncohematological disorders. Along with their distinguishing vancomycin resistance, due to a chromosomally-encoded VanC operon, their additional intrinsic resistance to many antibiotics other than glycopeptides limits the therapeutic choices. In addition, their intrinsic vancomycin resistance, unlike the vancomycin resistance of Enterococcus faecalis and Enterococcus faecium caused by transmissible plasmids, poses different infection control issues. We focused on the therapeutic and infection control issues of clinical syndromes caused by E. gallinarum and E. casseliflavus/flavescens. We propose therapeutic algorithms on bloodstream infections, endocarditis, central nervous system infections, endophthalmitis and urinary tract infections. The implementation of infection control measures in cases of E. gallinarum and E. casseliflavus/flavescens infection or colonization should be evaluated on a case-by-case basis, especially for epidemic outbreaks or for isolates supposed to harbor a potential transmissible vancomycin-resistance phenotype

Cellulosimicrobium cellulans aortic prosthetic valve endocarditis

Introduction: Invasive infections due to Cellulosimicrobium spp. (a Gram-positive coryneform) are extremely rare. Only a few cases of bloodstream infections and endocarditis have been described, as bacteraemia due to coryneforms is usually discarded as blood culture contamination.Case presentation: A 66-year-old female, with a history of aortic valve replacement, presented with fever, left leg purpura and acute kidney injury. Multiple repeated blood cultures were positive for Cellulosimicrobium cellulans , and targeted therapy was started. At first, endocarditis was excluded by echocardiograms, and the acute nephritis was interpreted as an atypical presentation of Henoch-Shonlein purpura. High-dose prednisone was started, and after 10 weeks the patient presented again with fever, mental confusion and acute left arm ischaemia. A subsequent echocardiogram and radiolabelled leukocyte scintigraphic evaluation revealed aortic prosthetic valve endocarditis with periprosthetic abscess and arterial brachial thrombosis. The patient deceased, and the autoptic examination confirmed an aortic valve periprosthetic abscess and revealed multiple arterial thromboses and septic embolisms in the kidneys, brain, spleen and myocardium.Conclusion: Isolation of coryneform bacteria on blood culture should not always be discarded as blood culture contamination. In the case of endocarditis due to Cellulosimicrobium spp., the removal of any prosthetic material, along with prolonged in vitro active antimicrobial therapy, should be pursued in order to reduce persistence or relapses of infection