Lv4, an activity that restricts nuclear entry of SIVMAC/SIVSM in human blood cells

SIVSM is a lentivirus endemic to the West African sooty mangabey (Cercocebus atys). HIV-2 and SIVMAC are zoonoses that resulted from SIVSM transmission to humans and Asian rhesus macaques (Macaca mulatto), respectively. Human leukemia cell lines, human peripheral blood mononuclear cells and CD4+ T cells, were 4 to 50-fold less permissive for SIVMAC and SIVSM than for HIV-1. In contrast, SIVMAC transduction of human adherent cell lines was equivalent to that of HIV-1. Consistent with adaptation to human cells, HIV-2 was not restricted as potently as was SIVMAC. SIVMAC transduction of human blood cells was rescued up to the level of HIV-1 by As2O3, a compound that increases the infectivity of viruses in the context of TRIM5-mediated restriction. Nonetheless, efficient knockdown of TRIM5 or cyclophilin A, a cytoplasmic factor that sometimes regulates TRIM5 restriction activity, did not rescue SIVMAC tranduction of these cells. Substitution of HIV-1 CA with the CA from SIVMAC rendered HIV-1 poorly infectious for Jurkat T cells. The block occurred after completion of reverse transcription and the formation of 2-LTR circles, but before establishment of the provirus. Heterokaryons resulting from fusion of permissive with restrictive cells exhibited the restrictive phenotype, indicating that SIV transduction of human blood cells is inefficient due to a dominant-acting restriction factor. These results demonstrate that the nucleus of human blood cells possesses a TRIM5-like restriction factor specific for the SIVMAC/SIVSM capsid and that, by extension, cross-species transmission of SIVSM to human cells necessitated adaptation of HIV-2 to this restriction factor

Lv4 Is a Capsid-Specific Antiviral Activity in Human Blood Cells That Restricts Viruses of the SIVMAC/SIVSM/HIV-2 Lineage Prior to Integration

HIV-2 and SIVMAC are AIDS-causing, zoonotic lentiviruses that jumped to humans and rhesus macaques, respectively, from SIVSM-bearing sooty mangabey monkeys. Cross-species transmission events such as these sometimes necessitate virus adaptation to species-specific, host restriction factors such as TRIM5. Here, a new human restriction activity is described that blocks viruses of the SIVSM/SIVMAC/HIV-2 lineage. Human T, B, and myeloid cell lines, peripheral blood mononuclear cells and dendritic cells were 4 to \u3e 100-fold less transducible by VSV G-pseudotyped SIVMAC, HIV-2, or SIVSM than by HIV-1. In contrast, transduction of six epithelial cell lines was equivalent to that by HIV-1. Substitution of HIV-1 CA with the SIVMAC or HIV-2 CA was sufficient to reduce HIV-1 transduction to the level of the respective vectors. Among such CA chimeras there was a general trend such that CAs from epidemic HIV-2 Group A and B isolates were the most infectious on human T cells, CA from a 1 degrees sooty mangabey isolate was the least infectious, and non-epidemic HIV-2 Group D, E, F, and G CAs were in the middle. The CA-specific decrease in infectivity was observed with either HIV-1, HIV-2, ecotropic MLV, or ALV Env pseudotypes, indicating that it was independent of the virus entry pathway. As2O3, a drug that suppresses TRIM5-mediated restriction, increased human blood cell transduction by SIVMAC but not by HIV-1. Nonetheless, elimination of TRIM5 restriction activity did not rescue SIVMAC transduction. Also, in contrast to TRIM5-mediated restriction, the SIVMAC CA-specific block occurred after completion of reverse transcription and the formation of 2-LTR circles, but before establishment of the provirus. Transduction efficiency in heterokaryons generated by fusing epithelial cells with T cells resembled that in the T cells, indicative of a dominant-acting SIVMAC restriction activity in the latter. These results suggest that the nucleus of human blood cells possesses a restriction factor specific for the CA of HIV-2/SIVMAC/SIVSM and that cross-species transmission of SIVSM to human T cells necessitated adaptation of HIV-2 to this putative restriction factor

How to Warrant Microbiological Food Safety in Food Companies: Problems and a Possible Solution

Traditional methods for food microbiological analysis are mainly culture-based, but also combine biochemical, molecular, and immunological principles in order to provide specific and unequivocal identification of contaminants. They are considered as gold standards by international regulations and are the most popular and widely used. These methods, however, received several criticisms, which led to the development of new alternative analytical methods. Their main focus is to provide accurate and reliable results, also aiming to reduce the time of analysis and facilitate procedures and interpretation of data. Despite these strengths, every method, both traditional and alternative, has intrinsic weaknesses that may affect results and stem from various factors, such as specific food matrices’ characteristics, operating procedures, and sample treatment. In this work, we review and compare the analytical performances of several methods for microbiological analysis of food that are currently available on the market. We take into account their relative features (e.g. user-friendliness, costs, reliability) in order to highlight which ones may fit best in daily monitoring of food safety and quality. Among these alternative methods, the Micro Biological Survey (MBS) method is based on a colorimetric system that can easily analyze liquids and solids via selective counting bacteria in food samples, allowing even the smallest food companies to carry out all microbiological tests required by regulations “in house.

A Conserved Acidic Residue in the C-Terminal Flexible Loop of HIV-1 Nef Contributes to the Activity of SERINC5 and CD4 Downregulation

The host transmembrane protein SERINC5 is incorporated into retrovirus particles and inhibits HIV-1 infectivity. The lentiviral Nef protein counteracts SERINC5 by downregulating it from the cell surface and preventing its incorporation into virions. The ability of Nef to antagonize the host factor varies in magnitude between different HIV-1 isolates. After having identified a subtype H nef allele unable to promote HIV-1 infectivity in the presence of SERINC5, we investigated the molecular determinants responsible for the defective counteraction of the host factor. Chimeric molecules with a subtype C Nef highly active against SERINC5 were constructed to locate Nef residues crucial for the activity against SERINC5. An Asn at the base of the C-terminal loop of the defective nef allele was found in place of a highly conserved acidic residue (D/E 150). The conversion of Asn to Asp restored the ability of the defective Nef to downregulate SERINC5 and promote HIV-1 infectivity. The substitution was also found to be crucial for the ability of Nef to downregulate CD4, but not for Nef activities that do not rely on the internalization of receptors from the cell surface, suggesting a general implication in promoting clathrin-mediated endocytosis. Accordingly, bimolecular fluorescence complementation revealed that the conserved acidic residue contributes to the recruitment of AP2 by Nef. Altogether, our results confirm that Nef downregulates SERINC5 and CD4 by engaging a similar machinery and indicates that, in addition to the di-leucine motif, other residues in the C-terminal flexible loop are important for the ability of the protein to sustain clathrin-mediated endocytosis

Exosomes surf on filopodia to enter cells at endocytic hot spots and shuttle within endosomes to scan the ER

Exosomes are nanovesicles released by virtually all cells which act as intercellular messengers by transfer of protein, lipid and RNA cargo. Their quantitative efficiency, routes of cell uptake and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake which saturates with dose and time and reaches near 100 % ‘transduction’ efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia, as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. Following internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction and suggest unanticipated routes of subcellular cargo delivery

As₂O₃ specifically increases SIV_MAC infectivity in human blood cells.

<p>TE671 cells (A), Jurkat T cells (B), human PBMC (C), or human CD4⁺ T cells (D) were transduced with two-part, VSV G-pseudotyped HIV-1_<b>NL4-3</b>-GFP or SIV_<b>MAC</b>GFP vectors using a predetermined quantity of virus such that 1% of cells were infected. As_<b>2</b>O_<b>3</b> was added 1 hr prior to vector challenge and maintained for 12 hrs post-infection, at the concentrations indicated on the X axis. 48 hrs post-challenge the percentage of GFP-expressing cells was determined. The Y axis shows the fold increase relative to infection without As_<b>2</b>O_<b>3</b>.</p

The block to SIV_MAC infection of Jurkat T cells occurs after formation of 2-LTR circles.

<p>CRFK and Jurkat (A), or Hela and Jurkat (B), or PBMCs (C) were infected with VSV G-pseudotyped HIV-1_NL4-3-GFP, or with isogenic vector bearing the SIV_MAC239 CA residues 1 to 202. 24 hrs post-infection, DNA was collected from the cells and subjected to qPCR using primers specific for full-length linear viral cDNA, 2-LTR circles, or proviral DNA, as indicated. Shown is the abundance of signal from vector bearing the SIV_MAC239 CA^1-202, relative to the amount of signal from HIV-1_NL4-3-GFP. In each case, infection was performed in the presence of an RT inhibitor to control for background levels of signal.</p