Background The NF-κB family of transcription factors and associated signalling pathways are abundant and ubiquitous in human immune responses. Activation of NF-κB transcription factors by viral pathogen-associated molecular patterns, such as viral RNA and DNA, is fundamental to anti-viral innate immune defences and pro-inflammatory cytokine production that steers adaptive immune responses. Diverse non-viral stimuli, such as lipopolysaccharide and cytokines, also activate NF-κB and the same anti-pathogen gene networks. Viruses adapted to human cells often encode multiple proteins targeting the NF-κB pathway to mitigate the anti-viral effects of NF-κB-dependent host immunity. Results In this study we have demonstrated using a variety of assays, in a number of different cell types including primary cells, that plasmid-encoded or virus-delivered simian immunodeficiency virus (SIV) accessory protein Vpx is a broad antagonist of NF-κB signalling active against diverse innate NF-κB agonists. Using targeted Vpx mutagenesis, we showed that this novel Vpx phenotype is independent of known Vpx cofactor DCAF1 and other cellular binding partners, including SAMHD1, STING and the HUSH complex. We found that Vpx co-immunoprecipitated with canonical NF-κB transcription factor p65, but not NF-κB family members p50 or p100, preventing nuclear translocation of p65. We found that broad antagonism of NF-κB activation by Vpx was conserved across distantly related lentiviruses as well as for Vpr from SIV Mona monkey (SIVmon), which has Vpx-like SAMHD1-degradation activity. Conclusions We have discovered a novel mechanism by which lentiviruses antagonise NF-κB activation by targeting p65. These findings extend our knowledge of how lentiviruses manipulate universal regulators of immunity to avoid the anti-viral sequelae of pro-inflammatory gene expression stimulated by both viral and extra-viral agonists. Importantly our findings are also relevant to the gene therapy field where virus-like particle associated Vpx is routinely used to enhance vector transduction through antagonism of SAMHD1, and perhaps also through manipulation of NF-κB. Graphical Abstract

SUMMARYPandemic viruses remain a global threat to health and economics but how they adapt to become pandemic remains poorly understood. Here we compare pandemic HIV-1(M) and non-pandemic HIV-(O) and HIV-2 strains finding that non-pandemic HIV replicate poorly in myeloid cell models due to activation of cGAS and TRIM5, and ensuing antiviral responses. We use phylogenetics and viral capsid structural biology to define specific differences between pandemic and non-pandemic HIV capsids and demonstrate that their genetic reversal in HIV-1(M) mutants causes TRIM5, cGAS and innate immune activation. We propose a model in which the parental lineage of pandemic HIV-1(M) has uniquely evolved a dynamic capsid that avoids activation of cGAS and TRIM5 to establish cloaked replication in myeloid cells. The unique adaptations of the pandemic virus lineage suggests a role in effective human-to-human transmissibility and highlight the importance of avoiding innate immune activation during pandemic human-to-human viral transmission.

Of the 13 known independent zoonoses of simian immunodeficiency viruses to humans, only one, leading to human immunodeficiency virus (HIV) type 1(M) has become pandemic, causing over 80 million human infections. To understand the specific features associated with pandemic human-to-human HIV spread, we compared replication of HIV-1(M) with non-pandemic HIV-(O) and HIV-2 strains in myeloid cell models. We found that non-pandemic HIV lineages replicate less well than HIV-1(M) owing to activation of cGAS and TRIM5-mediated antiviral responses. We applied phylogenetic and X-ray crystallography structural analyses to identify differences between pandemic and non-pandemic HIV capsids. We found that genetic reversal of two specific amino acid adaptations in HIV-1(M) enables activation of TRIM5, cGAS and innate immune responses. We propose a model in which the parental lineage of pandemic HIV-1(M) evolved a capsid that prevents cGAS and TRIM5 triggering, thereby allowing silent replication in myeloid cells. We hypothesize that this capsid adaptation promotes human-to-human spread through avoidance of innate immune response activation.

The control of pandemic pathogens depends on early prediction of pandemic variants and, more generally, understanding origins of such variants and factors that drive their global spread. This is especially important for GII.4 norovirus, where vaccines under development offer promise to prevent hundreds of millions of annual gastroenteritis cases. Previous studies have suggested that new GII.4 pandemic viruses evolve from previous pandemic variants through substitutions in the antigenic region of the VP1 protein that enable evasion of host population immunity, leading to global spread. In contrast, we show here that the acquisition of new genetic and antigenic characteristics is not the proximal driver of new pandemics. Instead, pandemic GII.4 viruses circulate undetected for years before causing a new pandemic, during which time they diversify and spread over wide geographical areas. Serological data demonstrate that by 2003, some nine years before it emerged as a new pandemic, the ancestral 2012 pandemic strain had already acquired the antigenic characteristics that allowed it to evade prevailing population immunity against the previous 2009 pandemic variant. These results provide strong evidence that viral genetic changes are necessary but not sufficient for GII.4 pandemic spread. Instead, we suggest that it is changes in host population immunity that enable pandemic spread of an antigenically-preadapted GII.4 variant. These results indicate that predicting future GII.4 pandemic variants will require surveillance of currently unsampled reservoir populations. Furthermore, a broadly acting GII.4 vaccine will be critical to prevent future pandemics. Significance Norovirus pandemics and their associated public health and economic costs could be prevented by effective vaccines. However, vaccine development and distribution will require identification of the sources and drivers of new pandemics. We here use phylogenetics and serological experiments to develop and test a new hypothesis of pandemic norovirus emergence. We find that pandemic noroviruses preadapt, diversify and spread worldwide years prior to emergence, strongly indicating that genetic changes are necessary but not sufficient to drive a new pandemic. We instead suggest that changes in population immunity enable pandemic emergence of a pre-adapted low-level variant. These findings indicate that prediction of new pandemics will require surveillance of under-sampled virus reservoirs and that norovirus vaccines will need to elicit broad immunity.

The type one interferon induced restriction factor Myxovirus resistance B (MxB) restricts HIV-1 nuclear entry evidenced by inhibition of 2-LTR but not linear forms of viral DNA. The HIV-1 capsid is the key determinant of MxB sensitivity and cofactor binding defective HIV-1 capsid mutants P90A (defective for cyclophilin A and Nup358 recruitment) and N74D (defective for CPSF6 recruitment) have reduced dependency on nuclear transport associated cofactors, altered integration targeting preferences and are not restricted by MxB expression. This has suggested that nuclear import mechanism may determine MxB sensitivity. Here we have use genetics to separate HIV-1 nuclear import cofactor dependence from MxB sensitivity. We provide evidence that MxB sensitivity depends on HIV-1 capsid conformation, rather than cofactor recruitment. We show that depleting CPSF6 to change nuclear import pathway does not impact MxB sensitivity, but mutants that recapitulate the effect of Cyclophilin A binding on capsid conformation and dynamics strongly impact MxB sensitivity. We demonstrate that HIV-1 primary isolates have different MxB sensitivities due to cytotoxic T lymphocyte (CTL) selected differences in Gag sequence but similar cofactor dependencies. Overall our work demonstrates a complex relationship between cyclophilin dependence and MxB sensitivity likely driven by CTL escape. We propose that cyclophilin binding provides conformational flexibility to HIV-1 capsid facilitating simultaneous evasion of capsid-targeting restriction factors including TRIM5 as well as MxB.

Background Studying site-specific amino acid frequencies by eye can reveal biologically significant variability and lineage-specific adaptation. This so-called ‘sequence gazing’ often informs bioinformatics and experimental research. But it is important to also account for the underlying phylogeny, since similarities may be due to common descent rather than selection pressure, and because it is important to distinguish between founder effects and convergent evolution. We set out to combine phylogenetic and sequence data to produce evolutionarily insightful visualisations. Results We present ChromaClade, a convenient tool with a graphical user-interface that works in concert with popular tree viewers to produce colour-annotated phylogenies highlighting residues found in each taxon and at each site in a sequence alignment. Colouring branches according to residues found at descendent tips also quickly identifies lineage-specific residues and those internal branches where key substitutions have occurred. We demonstrate applications of ChromaClade to human immunodeficiency virus and influenza A virus datasets, illustrating cases of conservative, adaptive and convergent evolution. Conclusions We find this to be a powerful approach for visualising site-wise residue distributions and detecting evolutionary patterns, especially in large datasets. ChromaClade is available for Windows, macOS and Unix or Linux; program executables and source code are available at github.com/chrismonit/chroma_clade .

The rising prevalence of HIV drug resistance (HIVDR) could threaten gains made in combating the HIV epidemic and compromise the 90-90-90 target proposed by United Nations Programme on HIV/AIDS (UNAIDS) to have achieved virological suppression in 90% of all persons receiving antiretroviral therapy (ART) by the year 2020. HIVDR has implications for the persistence of HIV, the selection of current and future ART drug regimens, and strategies of vaccine and cure development. Focusing on drug classes that are in clinical use, this Review critically summarizes what is known about the mechanisms the virus utilizes to escape drug control. Armed with this knowledge, strategies to limit the expansion of HIVDR are proposed.

A cure for HIV-1 remains unattainable as only one case has been reported, a decade ago1,2. The individual—who is known as the ‘Berlin patient’—underwent two allogeneic haematopoietic stem-cell transplantation (HSCT) procedures using a donor with a homozygous mutation in the HIV coreceptor CCR5 (CCR5Δ32/Δ32) to treat his acute myeloid leukaemia. Total body irradiation was given with each HSCT. Notably, it is unclear which treatment or patient parameters contributed to this case of long-term HIV remission. Here we show that HIV-1 remission may be possible with a less aggressive and toxic approach. An adult infected with HIV-1 underwent allogeneic HSCT for Hodgkin’s lymphoma using cells from a CCR5Δ32/Δ32 donor. He experienced mild gut graft-versus-host disease. Antiretroviral therapy was interrupted 16 months after transplantation. HIV-1 remission has been maintained over a further 18 months. Plasma HIV-1 RNA has been undetectable at less than one copy per millilitre along with undetectable HIV-1 DNA in peripheral CD4 T lymphocytes. Quantitative viral outgrowth assays from peripheral CD4 T lymphocytes show no reactivatable virus using a total of 24 million resting CD4 T cells. CCR5-tropic, but not CXCR4-tropic, viruses were identified in HIV-1 DNA from CD4 T cells of the patient before the transplant. CD4 T cells isolated from peripheral blood after transplantation did not express CCR5 and were susceptible only to CXCR4-tropic virus ex vivo. HIV-1 Gag-specific CD4 and CD8 T cell responses were lost after transplantation, whereas cytomegalovirus-specific responses were detectable. Similarly, HIV-1-specific antibodies and avidities fell to levels comparable to those in the Berlin patient following transplantation. Although at 18 months after the interruption of treatment it is premature to conclude that this patient has been cured, these data suggest that a single allogeneic HSCT with homozygous CCR5Δ32 donor cells may be sufficient to achieve HIV-1 remission with reduced intensity conditioning and no irradiation, and the findings provide further support for the development of HIV-1 remission strategies based on preventing CCR5 expression. An adult infected with HIV-1 who underwent allogeneic haematopoietic stem-cell transplantation for Hodgkin’s lymphoma using cells from a CCR5Δ32/Δ32 donor achieved full remission of HIV-1 for 18 months after transplantation and 16 months after cessation of antiretroviral therapy.

The vertebrate protein SAMHD1 is highly unusual in having roles in cellular metabolic regulation, antiviral restriction, and regulation of innate immunity. Its deoxynucleoside triphosphohydrolase activity regulates cellular dNTP concentration, reducing levels below those required by lentiviruses and other viruses to replicate. To counter this threat, some primate lentiviruses encode accessory proteins that bind SAMHD1 and induce its degradation; in turn, positive diversifying selection has been observed in regions bound by these lentiviral proteins, suggesting that primate SAMHD1 has coevolved to evade these countermeasures. Moreover, deleterious polymorphisms in human SAMHD1 are associated with autoimmune disease linked to uncontrolled DNA synthesis of endogenous retroelements. Little is known about how evolutionary pressures affect these different SAMHD1 functions. Here, we examine the deeper history of these interactions by testing whether evolutionary signatures in SAMHD1 extend to other mammalian groups and exploring the molecular basis of this coevolution. Using codon-based likelihood models, we find positive selection in SAMHD1 within each mammal lineage for which sequence data are available. We observe positive selection at sites clustered around T592, a residue that is phosphorylated to regulate SAMHD1 activity. We verify experimentally that mutations within this cluster affect catalytic rate and lentiviral restriction, suggesting that virus-host coevolution has required adaptations of enzymatic function. Thus, persistent positive selection may have involved the adaptation of SAMHD1 regulation to balance antiviral, metabolic, and innate immunity functions.

Summary:Existing ancestral sequence reconstruction techniques are ill-suited to investigating substitutions on a single branch of interest. We present SubRecon, an implementation of a hybrid technique integrating joint and marginal reconstruction for protein sequence data. SubRecon calculates the joint probability of states at adjacent internal nodes in a phylogeny, i.e. how the state has changed along a branch. This does not condition on states at other internal nodes and includes site rate variation. Simulation experiments show the technique to be accurate and powerful. SubRecon has a user-friendly command line interface and produces concise output that is intuitive yet suitable for subsequent parsing in an automated pipeline. Availability and implementation:SubRecon is platform independent, requiring Java v1.8 or above. Source code, installation instructions and an example dataset are freely available under the Apache 2.0 license at https://github.com/chrismonit/SubRecon.

Using both novel and established molecular evolutionary modelling techniques, we have investigated the evolution of primate lentiviruses and interactions with their hosts. Firstly, we studied SAMHD1, a restriction factor of HIV-1 which is neutralised by lentiviral proteins. SAMHD1 has previously been shown to be under positive selection in primates, ostensibly due to pressure to escape recognition by lentiviral antagonists. We show positive selection is not unique to primates but has occurred throughout chordate evolution. In mammals, we unexpectedly find SAMHD1 sites under positive selection are clustered in the domain controlling enzymatic activation. We hypothesise that positive selection is driven by undiscovered animal viruses and/or precise regulation of SAMHD1 activity. Secondly, we analysed the capsid proteins of pandemic HIV-1 and its chimpanzee progenitor, SIVcpz. We looked for sites evolving under different selective constraints with the aim of discovering host specific adaptation. We identify sites in the domain bound by host cofactors, which govern crucial events in virus replication and prevent immune sensing, suggesting host specific responses to cofactor interaction. Thirdly, we apply this same approach to pandemic HIV-1 and SIVcpz accessory proteins, which mitigate host immunity. Surprisingly, we identify sites in regions of nef and vpr involved in putatively conserved interactions with host proteins, suggesting unexpected host specific adaptation. In vpu, we identify sites involved in antagonism of the restriction factor tetherin - a function acquired by pandemic HIV-1 on adaptation to humans - together with sites which we hypothesise are similarly involved. Finally, lentiviruses and other organisms possess overlapping coding sequences, for which existing codon selection models are unsuitable. We propose a novel approach which models nucleotide substitution. In synthetic data tests, one of four candidate models was accurate and we developed a mixture model for identifying positive selection at codon sites, which we also tested with synthetic data.

AIDS Research and Human RetrovirusesVol. 31, No. 10 Letter to the EditorPositive Selection Analysis of Overlapping Reading Frames Is InvalidChristopher Monit, Richard A. Goldstein, Greg Towers, and Stéphane HuéChristopher MonitDivision of Infection and Immunity, University College London, London, United Kingdom.Search for more papers by this author, Richard A. GoldsteinDivision of Infection and Immunity, University College London, London, United Kingdom.Search for more papers by this author, Greg TowersDivision of Infection and Immunity, University College London, London, United Kingdom.Search for more papers by this author, and Stéphane HuéDepartment of Infectious Disease Epidemiology, London School of Hygiene and Tropical Medicine, London, United Kingdom.Search for more papers by this authorPublished Online:18 Sep 2015https://doi.org/10.1089/aid.2015.0150AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Positive Selection Analysis of Overlapping Reading Frames Is Invalid." AIDS Research and Human Retroviruses, 31(10), p. 947FiguresReferencesRelatedDetailsCited byA CRISPR-Cas Cure for HIV/AIDS13 January 2023 | International Journal of Molecular Sciences, Vol. 24, No. 2Molecular epidemiology of peste des petits ruminants virus emergence in critically endangered Mongolian saiga antelope and other wild ungulates25 June 2021 | Virus Evolution, Vol. 7, No. 2Selection on protein structure, interaction, and sequence11 February 2016 | Protein Science, Vol. 25, No. 7 Volume 31Issue 10Oct 2015 InformationCopyright 2015, Mary Ann Liebert, Inc.To cite this article:Christopher Monit, Richard A. Goldstein, Greg Towers, and Stéphane Hué.Positive Selection Analysis of Overlapping Reading Frames Is Invalid.AIDS Research and Human Retroviruses.Oct 2015.947-947.http://doi.org/10.1089/aid.2015.0150Published in Volume: 31 Issue 10: September 18, 2015Online Ahead of Print:July 22, 2015Online Ahead of Editing: June 30, 2015PDF download