Giada Mattiuzzo

Simian immunodeficiency virus envelope glycoprotein counteracts tetherin/BST-2/CD317 by intracellular sequestration

Tetherin is an IFN-inducible restriction factor that inhibits HIV-1 particle release in the absence of the HIV-1 countermeasure, viral protein U (Vpu). Although ubiquitous in HIV-1 and simian immunodeficiency viruses from chimpanzees, greater spot nosed monkeys, mustached monkeys, and Mona monkeys, other primate lentiviruses do not encode a Vpu protein. Here we demonstrate that SIV from Tantalus monkeys (SIVtan) encodes an envelope glycoprotein (SIVtan Env) able to counteract tetherin from Tantalus monkeys, rhesus monkeys, sooty mangabeys, and humans, but not from pigs. We show that sensitivity to Vpu but not SIVtan Env can be transferred with the human tetherin transmembrane region. We also identify a mutation in the tetherin extracellular domain, which almost completely abolishes sensitivity of human tetherin to SIVtan Env without compromising antiviral activity or sensitivity to Vpu. SIVtan Env expression results in a reduction of surface tetherin, as well as reduction in tetherin co-localization with mature surface-associated virus. Immuno-electron microscopy reveals co-localization of SIVtan Env with tetherin in intracellular tubulo-vesicular structures, suggesting that tetherin is sequestered away from budding virions at the cell surface. Along with HIV-1 Vpu and SIV Nef, envelope glycoprotein is the third and most broadly active lentiviral-encoded tetherin countermeasure to be described. Our observations emphasize the importance of tetherin in protecting mammals against viral infection and suggest that HIV-1 Vpu inhibitors may select active envelope mutants.

Regulation of Porcine Endogenous Retrovirus Release by Porcine and Human Tetherins

ABSTRACT The risk of transmission of porcine endogenous retrovirus (PERV) is one of the major safety issues in xenotransplantation. Human tetherin, recently described as an antiviral protein able to inhibit the release of enveloped viruses, and its porcine homologue were shown to inhibit PERV release from producer cells, establishing themselves as candidate molecules to suppress PERV production in porcine xenografts by animal engineering.

Microbial safety in xenotransplantation.

Purpose of reviewAs clinical trials are in progress involving porcine islet cell transplantation, microbial safety remains a key issue. Therefore, in the context of pig-to-human xenotransplantation, we provide an overview of the recent progress in the studies of relevant viruses including well known problematic viruses, such as herpesviruses and porcine endogenous retroviruses (PERV) in addition to some emerging issues regarding other pathogens. Recent findingsThe ability of herpesvirus to infect across species barriers is probably underestimated and requires monitoring and control of both xenograft donors and recipients for latent infection. Exclusion from donors and recipient monitoring for other exogenous pathogens including newly identified Parvovirus-4 are warranted. The availability of the swine whole genome sequence may help to characterize and select donor animals with less PERV infectivity. Rigorous PERV monitoring in both clinical and preclinical xenotransplantation experiments must be included in clinical protocols. SummaryA wide range of pathogens, both viruses and bacteria, pose potential safety problems in xenotransplantation, highlighting the importance of prescreening of the donor animals, and careful monitoring and follow-up of the patients.

Suboptimal Porcine Endogenous Retrovirus Infection in Non-Human Primate Cells: Implication for Preclinical Xenotransplantation

Background Porcine endogenous retrovirus (PERV) poses a potential risk of zoonotic infection in xenotransplantation. Preclinical transplantation trials using non-human primates (NHP) as recipients of porcine xenografts present the opportunity to assess the zoonosis risk in vivo. However, PERV poorly infects NHP cells for unclear reasons and therefore NHP may represent a suboptimal animal model to assess the risk of PERV zoonoses. We investigated the mechanism responsible for the low efficiency of PERV-A infection in NHP cells. Principal Findings Two steps, cell entry and exit, were inefficient for the replication of high-titer, human-tropic A/C recombinant PERV. A restriction factor, tetherin, is likely to be responsible for the block to matured virion release, supported by the correlation between the levels of inhibition and tetherin expression. In rhesus macaque, cynomolgus macaque and baboon the main receptor for PERV entry, PERV-A receptor 1 (PAR-1), was found to be genetically deficient: PAR-1 genes in these species encode serine at amino acid 109 in place of the leucine in human PAR-1. This genetic defect inevitably impacts in vivo sensitivity to PERV infection of these species. In contrast, African green monkey (AGM) PAR-1 is functional, but PERV infection is still poor. Although the mechanism is unclear, tunicamycin treatment, which removes N-glycosylated sugar chains, increases PERV infection, suggesting a possible role for the glycosylation of the receptors. Conclusions Since cynomolgus macaque and baboon, species often used in pig-to-NHP xenotransplantation experiments, have a defective PAR-1, they hardly represent an ideal animal model to assess the risk of PERV transmission in xenotransplantation. Alternatively, NHP species, like AGM, whose both PARs are functional may represent a better model than baboon and cynomolgus macaque for PERV zoonosis in vivo studies.

Differential resistance to cell entry by porcine endogenous retrovirus subgroup A in rodent species

BackgroundThe risk of zoonotic infection by porcine endogenous retroviruses (PERV) has been highlighted in the context of pig-to-human xenotransplantation. The use of receptors for cell entry often determines the host range of retroviruses. A human-tropic PERV subgroup, PERV-A, can enter human cells through either of two homologous multitransmembrane proteins, huPAR-1 and huPAR-2. Here, we characterised human PARs and their homologues in the PERV-A resistant rodent species, mouse and rat (muPAR and ratPAR, respectively).ResultsUpon exogenous expression in PERV-A resistant cells, human and rat PARs, but not muPAR, conferred PERV-A sensitivity. Exogenously expressed ratPAR binds PERV-A Env and allows PERV-A infection with equivalent efficiency to that of huPAR-1. Endogenous ratPAR expression in rat cell lines appeared to be too low for PERV-A infection. In contrast, the presence of Pro at position 109 in muPAR was identified to be the determinant for PERV-A resistance. Pro109. was shown to be located in the second extracellular loop (ECL2) and affected PERV-A Env binding to PAR molecules.ConclusionThe basis of resistance to PERV-A infection in two rodent species is different. Identification of a single a.a. mutation in muPAR, which is responsible for mouse cell resistance to PERV-A highlighted the importance of ECL-2 for the viral receptor function.

Early Potent Protection against Heterologous SIVsmE660 Challenge Following Live Attenuated SIV Vaccination in Mauritian Cynomolgus Macaques

Background Live attenuated simian immunodeficiency virus (SIV) vaccines represent the most effective means of vaccinating macaques against pathogenic SIV challenge. However, thus far, protection has been demonstrated to be more effective against homologous than heterologous strains. Immune correlates of vaccine-induced protection have also been difficult to identify, particularly those measurable in the peripheral circulation. Methodology/Principal Findings Here we describe potent protection in 6 out of 8 Mauritian-derived cynomolgus macaques (MCM) against heterologous virus challenge with the pathogenic, uncloned SIVsmE660 viral stock following vaccination with live attenuated SIVmac251/C8. MCM provided a characterised host genetic background with limited Major Histocompatibility Complex (MHC) and TRIM5α allelic diversity. Early protection, observed as soon as 3 weeks post-vaccination, was comparable to that of 20 weeks vaccination. Recrudescence of vaccine virus was most pronounced in breakthrough cases where simultaneous identification of vaccine and challenge viruses by virus-specific PCR was indicative of active co-infection. Persistence of the vaccine virus in a range of lymphoid tissues was typified by a consistent level of SIV RNA positive cells in protected vaccinates. However, no association between MHC class I /II haplotype or TRIM5α polymorphism and study outcome was identified. Conclusion/Significance This SIV vaccine study, conducted in MHC-characterised MCM, demonstrated potent protection against the pathogenic, heterologous SIVsmE660 challenge stock after only 3 weeks vaccination. This level of protection against this viral stock by intravenous challenge has not been hitherto observed. The mechanism(s) of protection by vaccination with live attenuated SIV must account for the heterologous and early protection data described in this study, including those which relate to the innate immune system.

Strategies to enhance the safety profile of xenotransplantation: minimizing the risk of viral zoonoses

Purpose of reviewPig-to-human xenotransplantation has taken steps closer to reality through advances in animal engineering to address immunological as well as microbial problems. The most highlighted problem in xenotransplantation safety has been the potential risk for zoonotic infection mediated by porcine endogenous retroviruses. Safety issues regarding viral zoonosis, particularly porcine endogenous retroviruses, are summarized and commented upon. Recent findingsSeveral molecular, transgenic strategies to provide safer transplant source animals with less porcine endogenous retrovirus infectivity have been developed. A genomics approach by selective breeding and porcine endogenous retrovirus loci knockout is at least theoretically possible. For other viruses, advances have been made in technologies for virus discovery and identification. SummaryThe consequences of possible zoonoses in xenotransplantation are largely unknown. Further work to identify and control potential zoonotic agents based on recent progress will improve the safety profile of xenotransplantation. Advances made should be subjected to cautious testing in well controlled, preclinical and clinical experiments.

Potential zoonotic infection of porcine endogenous retrovirus in xenotransplantation.

Porcine endogenous retrovirus (PERV) is considered the major biosafety issue in xenotransplantation. Several techniques have been employed for the analysis of the PERV status in the animal donor and for the assessment of PERV transmission/infection in the xenograft recipient. In this chapter, methods to assess the expression of PERV and the potential for PERV transmission from a donor animal are described in addition to the identification of relevant loci within the porcine genome.PERV detection can be carried out using several techniques of which quantitative polymerase chain reaction (PCR) and RT-PCR are the most sensitive. However, other procedures can be employed such as detection of reverse transcriptase activity (i.e. viral replication) in the sample or immunostaining of the infected cells using an anti-PERV antibody. The PERV transmission assay has been described to identify the transmission phenotype of the pig donor, and subsequent risk from a donor. This assay can, therefore, direct the selection of the most suitable animal. Finally, it is important to determine the presence of critical PERV loci involved in transmission in the pig genome and compare between different animals. One of the methods for the analysis of these PERV integration sites is described.

Comparison of platform technologies for assaying antibody to Ebola virus

Background The recent Ebola outbreak in West Africa led to the use of a variety of different platform technologies for assaying antibodies because of the difficulties of handling the live virus. The same types of method could be applied rapidly to other infections when they emerge. There is a need to compare quantitative results of different assays, which means that the assays must measure similar parameters and give comparable results. Methods A collaborative study was carried out to establish an International Reference Reagent through WHO. Nine samples were sent to 16 laboratories and the results from 22 different assays compared to those obtained by neutralisation assays using the wild type virus. Findings Quantitative correlation with the wild type neutralisation assays was very variable but generally poor, with only five of the twenty-two assays giving a correlation coefficient of 0.7 or greater; the five best assays included methods based on wild type and VSV pseudotype neutralisation and ELISA. They could be applicable to other rapidly emerging diseases. The remaining assays including neutralisation of lentiviral pseudotypes need further development. Interpretation The assay platform should be chosen with care to ensure that it is fit for purpose. Many of the assays were not suitable for quantitation of antibody levels, a finding that is not surprising given the urgency with which they had to be implemented but some may be of generic value. Antibody titres in samples from a vaccine trial were comparable to those from convalescent patients or lower. Funding Funding was from the UK DoH and the Wellcome Tust.

Role of DNA Methylation in Expression and Transmission of Porcine Endogenous Retroviruses

ABSTRACT Porcine endogenous retroviruses (PERV) represent a major safety concern in pig-to-human xenotransplantation. To date, no PERV infection of a xenograft recipient has been recorded; however, PERVs are transmissible to human cells in vitro. Some recombinants of the A and C PERV subgroups are particularly efficient in infection and replication in human cells. Transcription of PERVs has been described in most pig cells, but their sequence and insertion polymorphism in the pig genome impede identification of transcriptionally active or silenced proviral copies. Furthermore, little is known about the epigenetic regulation of PERV transcription. Here, we report on the transcriptional suppression of PERV by DNA methylation in vitro and describe heavy methylation in the majority of PERV 5′ long terminal repeats (LTR) in porcine tissues. In contrast, we have detected sparsely methylated or nonmethylated proviruses in the porcine PK15 cells, which express human cell-tropic PERVs. We also demonstrate the resistance of PERV DNA methylation to inhibitors of methylation and deacetylation. Finally, we show that the high permissiveness of various human cell lines to PERV infection coincides with the inability to efficiently silence the PERV proviruses by 5′LTR methylation. In conclusion, we suggest that DNA methylation is involved in PERV regulation, and that only a minor fraction of proviruses are responsible for the PERV RNA expression and porcine cell infectivity.