The intriguing Cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding

<p>Abstract</p> <p>Background</p> <p>Cyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr) with the human peptidyl prolyl isomerase CypA is known to occur <it>in vitro </it>and <it>in vivo</it>. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined.</p> <p>Results</p> <p>Characterization of the interactions of human CypA with N-terminal peptides of HIV-1 Vpr has been achieved using a combination of nuclear magnetic resonace (NMR) exchange spectroscopy and surface plasmon resonance spectroscopy (SPR). NMR data at atomic resolution indicate prolyl <it>cis</it>/<it>trans </it>isomerisation of the highly conserved proline residues Pro-5, -10, -14 and -35 of Vpr are catalyzed by human CypA and require only very low concentrations of the isomerase relative to that of the peptide substrates. Of the N-terminal peptides of Vpr only those containing Pro-35 bind to CypA in a biosensor assay. SPR studies of specific N-terminal peptides with decreasing numbers of residues revealed that a seven-residue motif centred at Pro-35 consisting of RHFPRIW, which under membrane-like solution conditions comprises the loop region connecting helix 1 and 2 of Vpr and the two terminal residues of helix 1, is sufficient to maintain strong specific binding.</p> <p>Conclusions</p> <p>Only N-terminal peptides of Vpr containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in a biosensor assay. This indicates that Pro-35 is essential for a specific CypA-Vpr binding interaction, in contrast to the general prolyl <it>cis</it>/<it>trans </it>isomerisation observed for all proline residues of Vpr, which only involve transient enzyme-substrate interactions. Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are now supported by our data. In detail the SPR data of this interaction were compatible with a two-state binding interaction model that involves a conformational change during binding. This is in accord with the structural changes observed by NMR suggesting CypA catalyzes the prolyl <it>cis/trans </it>interconversion during binding to the RHFP³⁵RIW motif of N-terminal Vpr.</p

The ability of multimerized cyclophilin A to restrict retrovirus infection

AbstractIn owl monkeys, the typical retroviral restriction factor of primates, TRIM5α, is replaced by TRIMCyp. TRIMCyp consists of the TRIM5 RING, B-box 2 and coiled-coil domains, as well as the intervening linker regions, fused with cyclophilin A. TRIMCyp restricts infection of retroviruses, such as human immunodeficiency virus (HIV-1) and feline immunodeficiency virus (FIV), with capsids that can bind cyclophilin A. The TRIM5 coiled coil promotes the trimerization of TRIMCyp. Here we show that cyclophilin A that is oligomeric as a result of fusion with a heterologous multimer exhibits substantial antiretroviral activity. The addition of the TRIM5 RING, B-box 2 and Linker 2 to oligomeric cyclophilin A generated a protein with antiretroviral activity approaching that of wild-type TRIMCyp. Multimerization increased the binding of cyclophilin A to the HIV-1 capsid, promoting accelerated uncoating of the capsid and restriction of infection

The control of viral infection by tripartite motif proteins and cyclophilin A

The control of retroviral infection by antiviral factors referred to as restriction factors has become an exciting area in infectious disease research. TRIM5α has emerged as an important restriction factor impacting on retroviral replication including HIV-1 replication in primates. TRIM5α has a tripartite motif comprising RING, B-Box and coiled coil domains. The antiviral α splice variant additionally encodes a B30.2 domain which is recruited to incoming viral cores and determines antiviral specificity. TRIM5 is ubiquitinylated and rapidly turned over by the proteasome in a RING dependent way. Protecting restricted virus from degradation, by inhibiting the proteasome, rescues DNA synthesis, but not infectivity, indicating that restriction of infectivity by TRIM5α does not depend on the proteasome but the early block to DNA synthesis is likely to be mediated by rapid degradation of the restricted cores. The peptidyl prolyl isomerase enzyme cyclophilin A isomerises a peptide bond on the surface of the HIV-1 capsid and impacts on sensitivity to restriction by TRIM5α from Old World monkeys. This suggests that TRIM5α from Old World monkeys might have a preference for a particular capsid isomer and suggests a role for cyclophilin A in innate immunity in general. Whether there are more human antiviral TRIMs remains uncertain although the evidence for TRIM19's (PML) antiviral properties continues to grow. A TRIM5-like molecule with broad antiviral activity in cattle suggests that TRIM mediated innate immunity might be common in mammals. Certainly the continued study of restriction of viral infectivity by antiviral host factors will remain of interest to a broad audience and impact on a variety of areas including development of animal models for infection, development of viral vectors for gene therapy and the search for novel antiviral drug targets

The Host-Pathogen interaction of human cyclophilin A and HIV-1 Vpr requires specific N-terminal and novel C-terminal domains

<p>Abstract</p> <p>Background</p> <p>Cyclophilin A (CypA) represents a potential key molecule in future antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication. CypA interacts with the virus proteins Capsid (CA) and Vpr, however, the mechanism through which CypA influences HIV-1 infectivity still remains unclear.</p> <p>Results</p> <p>Here the interaction of full-length HIV-1 Vpr with the host cellular factor CypA has been characterized and quantified by surface plasmon resonance spectroscopy. A C-terminal region of Vpr, comprising the 16 residues ⁷⁵GCRHSRIGVTRQRRAR⁹⁰, with high binding affinity for CypA has been identified. This region of Vpr does not contain any proline residues but binds much more strongly to CypA than the previously characterized N-terminal binding domain of Vpr, and is thus the first protein binding domain to CypA described involving no proline residues. The fact that the mutant peptide Vpr^75-90R80A binds more weakly to CypA than the wild-type peptide confirms that Arg-80 is a key residue in the C-terminal binding domain. The N- and C-terminal binding regions of full-length Vpr bind cooperatively to CypA and have allowed a model of the complex to be created. The dissociation constant of full-length Vpr to CypA was determined to be approximately 320 nM, indicating that the binding may be stronger than that of the well characterized interaction of HIV-1 CA with CypA.</p> <p>Conclusions</p> <p>For the first time the interaction of full-length Vpr and CypA has been characterized and quantified. A non-proline-containing 16-residue region of C-terminal Vpr which binds specifically to CypA with similar high affinity as full-length Vpr has been identified. The fact that this is the first non-proline containing binding motif of any protein found to bind to CypA, changes the view on how CypA is able to interact with other proteins. It is interesting to note that several previously reported key functions of HIV-1 Vpr are associated with the identified N- and C-terminal binding domains of the protein to CypA.</p

Host Cell Peptidylprolyl cis-trans Isomerases as Immune Modulators of HIV-1 Infection

Since the 1980s, the human immunodeficiency virus 1 (HIV-1) has been acknowledged as the trigger for AIDS, the acquired immunodeficiency syndrome. Every year, worldwide approximately 700,000 people die from late effects of HIV-1 infection and AIDS (UNAIDS, 2018). Thus, continuous research is important to better understand the interaction of the virus with the human host and to develop a cure. Host cell proteins that promote or fight infection are referred to as co- and restriction factors, respectively. Innate immunity restriction factors are, for example, TRIM5 or tetherin and some of them are induced by interferons (IFNs). Cyclophilin A (CypA), a small protein that influences the folding and thus the function of several cellular proteins, is a co-factor for HIV-1 infection. According to current knowledge, CypA shields cellular HIV-1 capsid cores form restriction factors after viral cell entry and thus ensures the safe transport of the virus genome into the cell nucleus. There, the HIV-1 genome can integrate into the host genome. In addition to CypA, the protein family of cyclophilins (Cyps) contains at least 16 other proteins in humans, all of which have similar cyclophilin domain structures, but have diverse cellular functions. While the role of CypA during HIV-1 infection is reasonably well characterized, almost no information is available for the other Cyps. This work investigated the influence of Cyps on early HIV-1 infection events in connection with the antiviral effects of type I IFNs. In general, some type I IFN-stimulated cells exert a significantly reduced HIV-1 infection. Interestingly, this early block to infection is amplified in CypA deficient cells. This indicates a role of CypA in the immune defense against HIV-1. Furthermore, an increase in infection after treatment with a cyclophilin inhibitor, Cyclosporin A (CsA) was observed. This can also be observed in the absence of CypA, the supposedly main target of CsA inhibition. This suggested the presence of CsA-sensitive factors that affect HIV-1 infection in type I IFN treated cells. Since both, CsA and type I IFNs have been proposed and tested as possible therapy strategies, however with little success, this observation warrants further investigation to reveal the underlying mechanisms, which could lead towards an adapted therapeutic strategy. The most obvious candidate targets are other members of the cyclophilin family. Therefore, CypB, CypC, CypD, CypE and CypH deficient THP-1 cells were generated using CRISPR/Cas9, and the effect of type I IFN treatment and CsA stimulation on HIV-1 infection was examined. While knockout of CypB, CypC and CypD modulated infection but showed no effect in response to IFN or CsA, CypE and CypH knockout cells showed a significantly increased sensitivity of HIV-1 infection to type I IFN-induced blocks. Due to these different phenotypes, the interplay of several Cyps was examined by generating double knockout cell lines. Depletion of CypB alone had no effect on HIV-1 infection, but a significantly increased sensitivity to type I IFN-induced post entry blocks was observed in the absence of both, CypA and CypB. The same could be observed for the double knockout of CypA and CypE. This suggests that Cyp functions on early HIV-1 infection events are complex and that some functions may depend on other members of this protein family. Furthermore, the results of this study show that CypA is not the only member of this family that has a function during early HIV-1 infection. The results from this study suggest that knockout of single cyclophilin genes was insufficient to render THP-1 cells insensitive to the CsA-induced increase in HIV-1 infection in type I IFN-induced cells, i.e. the phenotype could not be explained by one the candidates tested. To conduct a more unbiased approach, a mass spectrometry screen based on thermal protein stability was carried out covering the entire cellular proteome. In addition to known CsA targets, several new factors could be identified for which protein stability was sometimes dramatically altered upon CsA treatment of cells, indicating possible functional sensitivity to CsA. Some of these were IFN-induced proteins, such as members of the Retinoic Acid inducible gene I (RIG-I) signaling pathway. RIG-I recognizes viral RNA and induces an antiviral signaling cascade within the cell, which among other things leads to type I IFN production. Since this signaling pathway has been already associated with HIV-1 infection in the literature, some members of this signaling pathway were examined in more detail regarding HIV-1 infection and sensitivity to type I IFN and CsA. Knockout of RIG-I, MDA5, MAVS, TRADD or IRF3 in CypA deficient THP-1 cells showed increased sensitivity to type I IFN-induced early infection blocks. In addition, an increased infection in IRF3 knockout cells was observed, which indicates a function of IRF3 in the restriction of HIV-1. It was also seen that the CsA-induced effects in CypA knockout cells were no longer observable when either RIG-I, MDA5, MAVS or IRF3 were knocked out on top. While a mechanism of action of CsA on the RIG-I signaling pathway could unfortunately not be identified due to time limitations, the generated cell lines in this study are excellent tools for future studies that will aim to reveal mechanistic insights. The complex interplay between HIV-1 co-factors and type I IFN-induced cellular restriction factors in early infection events may yet again underline how perfectly well HIV-1 has adapted to exploit cellular pathways

Multiple sites in the N-terminal half of simian immunodeficiency virus capsid protein contribute to evasion from rhesus monkey TRIM5α-mediated restriction

<p>Abstract</p> <p>Background</p> <p>We previously reported that cynomolgus monkey (CM) TRIM5α could restrict human immunodeficiency virus type 2 (HIV-2) strains carrying a proline at the 120^thposition of the capsid protein (CA), but it failed to restrict those with a glutamine or an alanine. In contrast, rhesus monkey (Rh) TRIM5α could restrict all HIV-2 strains tested but not simian immunodeficiency virus isolated from macaque (SIVmac), despite its genetic similarity to HIV-2.</p> <p>Results</p> <p>We attempted to identify the viral determinant of SIVmac evasion from Rh TRIM5α-mediated restriction using chimeric viruses formed between SIVmac239 and HIV-2 GH123 strains. Consistent with a previous study, chimeric viruses carrying the loop between α-helices 4 and 5 (L4/5) (from the 82^ndto 99^thamino acid residues) of HIV-2 CA were efficiently restricted by Rh TRIM5α. However, the corresponding loop of SIVmac239 CA alone (from the 81^stto 97^thamino acid residues) was not sufficient to evade Rh TRIM5α restriction in the HIV-2 background. A single glutamine-to-proline substitution at the 118^thamino acid of SIVmac239 CA, corresponding to the 120^thamino acid of HIV-2 GH123, also increased susceptibility to Rh TRIM5α, indicating that glutamine at the 118^thof SIVmac239 CA is necessary to evade Rh TRIM5α. In addition, the N-terminal portion (from the 5^thto 12^thamino acid residues) and the 107^thand 109^thamino acid residues in α-helix 6 of SIVmac CA are necessary for complete evasion from Rh TRIM5α-mediated restriction. A three-dimensional model of hexameric GH123 CA showed that these multiple regions are located on the CA surface, suggesting their direct interaction with TRIM5α.</p> <p>Conclusion</p> <p>We found that multiple regions of the SIVmac CA are necessary for complete evasion from Rh TRIM5α restriction.</p

Contribution of RING domain to retrovirus restriction by TRIM5α depends on combination of host and virus

AbstractThe anti-retroviral restriction factor TRIM5α contains the RING domain, which is frequently observed in E3 ubiquitin ligases. It was previously proposed that TRIM5α restricts human immunodeficiency virus type 1 (HIV-1) via proteasome-dependent and -independent pathways. Here we examined the effects of RING domain mutations on retrovirus restriction by TRIM5α in various combinations of virus and host species. Simian immunodeficiency virus isolated from macaque (SIVmac) successfully avoided attacks by RING mutants of African green monkey (AGM)-TRIM5α that could still restrict HIV-1. Addition of proteasome inhibitor did not affect the anti-HIV-1 activity of AGM-TRIM5α, whereas it disrupted at least partly its anti-SIVmac activity. In the case of mutant human TRIM5α carrying proline at the position 332, however, both HIV-1 and SIVmac restrictions were eliminated as a result of RING domain mutations. These results suggested that the mechanisms of retrovirus restriction by TRIM5α vary depending on the combination of host and virus

PLoS One

Mature HIV-1 viral particles assemble as a fullerene configuration comprising p24 capsid hexamers, pentamers and dimers. In this paper, we report the X-ray crystal structures of the p24 protein from natural HIV-1 strain (BMJ4) in complex with Fab A10F9, which recognizes a conserved epitope in the C-terminal domain of the BMJ4 p24 protein. Our structures reveal a novel shoulder-to-shoulder p24 dimerization mode that is mediated by an S-S bridge at C177. Consistent with these structures, the shoulder-to-shoulder dimer that was obtained from the BMJ4 strain was also observed in p24 proteins from other strains by the introduction of a cysteine residue at position 177. The potential biological significance was further validated by the introduction of a C177A mutation in the BMJ4 strain, which then displays a low infectivity. Our data suggest that this novel shoulder-to-shoulder dimer interface trapped by this unique S-S bridge could represent a physiologically relevant mode of HIV-1 capsid assembly during virus maturation, although Cys residue itself may not be critical for HIV-I replication