Both TRIM5α and TRIMCyp have only weak antiviral activity in canine D17 cells

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TRIM5α and TRIMCyp form apparent hexamers and their multimeric state is not affected by exposure to restriction-sensitive viruses or by treatment with pharmacological inhibitors

Proteins of the TRIM5 family, such as TRIM5α and the related TRIMCyp, are cytoplasmic factors that can inhibit incoming retroviruses. This type of restriction requires a direct interaction between TRIM5 proteins and capsid proteins that are part of mature, intact retroviral cores. In such cores, capsids are arranged as hexameric units. Multiple lines of evidence imply that TRIM5 proteins themselves interact with retroviral cores as multimers. Accordingly, stabilization by crosslinking agents has revealed that TRIM5α and TRIMCyp are present as trimers in mammalian cells. We report here that TRIM5 proteins seem to form dimers, trimers, hexamers and multimers of higher complexity in mammalian cells. The hexameric form in particular seems to be the most abundant multimer. Multimerization did not involve disulfide bridges and was not affected by infection with restriction-sensitive viruses or by treatment with the known TRIM5 inhibitors arsenic trioxide, MG132 and cyclosporine A. We conclude that TRIM5 multimerization results from more than one protein-protein interface and that it is seemingly not triggered by contact with retroviral cores

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

Cytotoxicity and Antiviral Properties of Alkaloids Isolated from Pancratium maritimum

Ten Amaryllidaceae alkaloids (AAs) were isolated for the first time from Pancratium maritimum collected in Calabria region, Italy. They belong to different subgroups of this family and were identi-fied as lycorine, which is the main alkaloid, 9-O-demethyllycorine, haemanthidine, haemanthamine, 11-hydroxyvittatine, homolycorine, pancracine, obliquine, tazettine and vittatine. Haemanthidine was isolated as a scalar mixture of two 6-epimers, as already known also for other 6-hydroxycrinine alkaloids, but for the first time they were separated as 6,11-O,O′-di-p-bromobenzoyl esters. The evaluation of the cytotoxic and antiviral potentials of all isolated compounds was undertaken. Ly-corine and haemanthidine showed cytotoxic activity on Hacat cells and A431 and AGS cancer cells while, pancracine exhibited selective cytotoxicity against A431 cells. We uncovered that in addition to lycorine and haemanthidine, haemanthamine and pancracine also possess antiretroviral abilities, inhibiting pseudotyped human immunodeficiency virus (HIV)−1 with EC50 of 25.3 µM and 18.5 µM respectively. Strikingly, all the AAs isolated from P. maritimum were able to impede dengue virus (DENV) replication (EC50 ranged from 0.34–73.59 µM) at low to non-cytotoxic concentrations (CC50 ranged from 6.25 µM to >100 µM). Haemanthamine (EC50 = 337 nM), pancracine (EC50 = 357 nM) and haemanthidine (EC50 = 476 nM) were the most potent anti-DENV inhibitors. Thus, this study uncovered new antiviral properties of P. maritimum isolated alkaloids, a significant finding that could lead to the development of new therapeutic strategies to fight viral infectious diseases

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

G2 Cell Cycle Arrest and Cyclophilin A in Lentiviral Gene Transfer

Lentiviral vectors derived from the human immunodeficiency virus-1 (HIV-1) have a higher propensity to transduce nondividing cells compared to vectors based on oncoretroviruses. We report here that genistein, a previously known protein tyrosine kinase (PTK) inhibitor and G2 cell cycle arrest inducer, significantly enhanced lentiviral transduction in a dose-dependent manner. Increased transduction, as measured by vector expression, was seen in a variety of human cell lines, murine primary lymphocytes, and primary human CD34+ peripheral blood progenitor cells as well. Increased vector expression was also associated with an increase in vector DNA copy number, as assessed by quantitative PCR. Genistein-mediated G2 cell cycle arrest, rather than PTK inhibition, appears to be the major factor responsible for increased gene transfer. Genistein also increases cyclophilin A (CypA) protein, a cellular protein important for efficient HIV-1 infection. While we show that CypA−/− Jurkat cells transduce poorly with lentiviral vectors, genistein does increase gene transfer in CypA-deficient cells. CypA and G2 cell cycle arrest appear to be two independent factors important for efficient lentiviral gene transfer. The role of genistein and other G2-arresting agents may be useful for improving the efficiency of lentiviral gene therapy

The V86M mutation in HIV-1 capsid confers resistance to TRIM5α by abrogation of cyclophilin A-dependent restriction and enhancement of viral nuclear import.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: HIV-1 is inhibited early after entry into cells expressing some simian orthologues of the tripartite motif protein family member TRIM5α. Mutants of the human orthologue (TRIM5αhu) can also provide protection against HIV-1. The host protein cyclophilin A (CypA) binds incoming HIV-1 capsid (CA) proteins and enhances early stages of HIV-1 replication by unknown mechanisms. On the other hand, the CA-CypA interaction is known to increase HIV-1 susceptibility to restriction by TRIM5α. Previously, the mutation V86M in the CypA-binding loop of HIV-1 CA was found to be selected upon serial passaging of HIV-1 in cells expressing Rhesus macaque TRIM5α (TRIM5αrh). The objectives of this study were (i) to analyze whether V86M CA allows HIV-1 to escape mutants of TRIM5αhu, and (ii) to characterize the role of CypA in the resistance to TRIM5α conferred by V86M. RESULTS: We find that in single-cycle HIV-1 vector transduction experiments, V86M confers partial resistance against R332G-R335G TRIM5αhu and other TRIM5αhu variable 1 region mutants previously isolated in mutagenic screens. However, V86M HIV-1 does not seem to be resistant to R332G-R335G TRIM5αhu in a spreading infection context. Strikingly, restriction of V86M HIV-1 vectors by TRIM5αhu mutants is mostly insensitive to the presence of CypA in infected cells. NMR experiments reveal that V86M alters CypA interactions with, and isomerisation of CA. On the other hand, V86M does not affect the CypA-mediated enhancement of HIV-1 replication in permissive human cells. Finally, qPCR experiments show that V86M increases HIV-1 transport to the nucleus of cells expressing restrictive TRIM5α. CONCLUSIONS: Our study shows that V86M de-couples the two functions associated with CA-CypA binding, i.e. the enhancement of restriction by TRIM5α and the enhancement of HIV-1 replication in permissive human cells. V86M enhances the early stages of HIV-1 replication in restrictive cells by improving nuclear import. In summary, our data suggest that HIV-1 escapes restriction by TRIM5α through the selective disruption of CypA-dependent, TRIM5α-mediated inhibition of nuclear import. However, V86M does not seem to relieve restriction of a spreading HIV-1 infection by TRIM5αhu mutants, underscoring context-specific restriction mechanisms

Cytoplasmic Dynein Promotes HIV-1 Uncoating

Retroviral capsid (CA) cores undergo uncoating during their retrograde transport (toward the nucleus), and/or after reaching the nuclear membrane. However, whether HIV-1 CA core uncoating is dependent upon its transport is not understood. There is some evidence that HIV-1 cores retrograde transport involves cytoplasmic dynein complexes translocating on microtubules. Here we investigate the role of dynein-dependent transport in HIV-1 uncoating. To interfere with dynein function, we depleted dynein heavy chain (DHC) using RNA interference, and we over-expressed p50/dynamitin. In immunofluorescence microscopy experiments, DHC depletion caused an accumulation of CA foci in HIV-1 infected cells. Using a biochemical assay to monitor HIV-1 CA core disassembly in infected cells, we observed an increase in amounts of intact (pelletable) CA cores upon DHC depletion or p50 over-expression. Results from these two complementary assays suggest that inhibiting dynein-mediated transport interferes with HIV-1 uncoating in infected cells, indicating the existence of a functional link between HIV-1 transport and uncoating

Cyclophilin A and TRIM5α Independently Regulate Human Immunodeficiency Virus Type 1 Infectivity in Human Cells

Cyclophilin A (CypA), a cytoplasmic, human immunodeficiency virus type 1 (HIV-1) CA-binding protein, acts after virion membrane fusion with human cells to increase HIV-1 infectivity. HIV-1 CA is similarly greeted by CypA soon after entry into rhesus macaque or African green monkey cells, where, paradoxically, the interaction decreases HIV-1 infectivity by facilitating TRIM5α-mediated restriction. These observations conjure a model in which CA recognition by the human TRIM5α orthologue is precluded by CypA. Consistent with the model, selection of a human cell line for decreased restriction of the TRIM5α-sensitive, N-tropic murine leukemia virus (N-MLV) rendered HIV-1 transduction of these cells independent of CypA. Additionally, HIV-1 virus-like particles (VLPs) saturate N-MLV restriction activity, particularly when the CA-CypA interaction is disrupted. Here the effects of CypA and TRIM5α on HIV-1 restriction were examined directly. RNA interference was used to show that endogenous human TRIM5α does indeed restrict HIV-1, but the magnitude of this antiviral activity was not altered by disruption of the CA-CypA interaction or by elimination of CypA protein. Conversely, the stimulatory effect of CypA on HIV-1 infectivity was completely independent of human TRIM5α. Together with previous reports, these data suggest that CypA protects HIV-1 from an unknown antiviral activity in human cells. Additionally, target cell permissivity increased after loading with heterologous VLPs, consistent with a common saturable target that is epistatic to both TRIM5α and the putative CypA-regulated restriction factor