HIV-1 Restriction Research Articles

HIV-1 inhibits IFITM3 expression to promote the infection of megakaryocytes.

Despite an undetectable plasma viral load as a result of antiretroviral therapy, HIV-1-infected individuals with poor immune reconstitution harbor infectious HIV-1 within their platelets. Megakaryocytes, as platelet precursors, are the likely cellular origin of these HIV-1-containing platelets. To investigate the mechanisms that allow megakaryocytes to support HIV-1 infection, we established in vitro models of viral infection using hematopoietic stem cell-derived megakaryocytes and the megakaryocytic MEG-01 cell line. We observed HIV-1 DNA provirus integration into the megakaryocyte cell genome, self-limiting virus production, and HIV-1 protein and RNA compartmentalization, which are hallmarks of HIV-1 infection in myeloid cells. In addition, following HIV-1 infection of megakaryocyte precursors, the expression of interferon-induced transmembrane protein 3 (IFITM3), an antiviral factor constitutively expressed in megakaryocytes, was inhibited in terminally differentiated HIV-1-infected megakaryocytes. IFITM3 knockdown in MEG-01 cells prior to infection led to enhanced HIV-1 infection, indicating that IFITM3 acts as an HIV-1 restriction factor in megakaryocytes. Together, these findings indicate that megakaryocyte precursors are susceptible to HIV-1 infection, leading to terminally differentiated megakaryocytes harboring virus in a process regulated by IFITM3. Megakaryocytes may thus constitute a neglected HIV-1 reservoir that warrants further study in order to develop improved antiretroviral therapies and to facilitate HIV-1 eradication.

Interaction between ESCRT-III proteins and the yeast SERINC homolog Tms1.

The endosomal sorting complex required for transport (ESCRT)-III is involved in membrane remodeling and abscission during intraluminal vesicle (ILV) formation at endosomes. Our data now suggest that ESCRT-III function could be connected to lipid remodeling of the endosomal membrane. This notion is based on our finding that ESCRT-III proteins bind to the yeast serine incorporator (SERINC) homolog Tms1. Human SERINC3 and SERINC5 are HIV-1 restriction factors and have been shown to act as scramblases, flipping phospholipids between membrane leaflets. Due to the extraordinarily high sequence conservation between Tms1 and human SERINCs, it is likely that Tms1 is also a scramblase. While deletion of TMS1 had only a moderate effect on the sorting of multivesicular body (MVB) cargo proteins, the simultaneous deletion of a component of the Vps55/Vps68 complex led to a strong synergistic phenotype. This pronounced synergism suggests that Tms1 and Vps55/Vps68 perform a parallel function at endosomes. Vps55/Vps68 loosely resembles Tms1 in its overall structure. Thus, it is possible that Vps55/Vps68 is also a scramblase. Since both Vps55 and Tms1 physically interact with ESCRT-III proteins, we propose that the recruitment of a scramblase plays a crucial role in ESCRT-III-dependent membrane remodeling at endosomes.

Altered memory CCR6+ Th17-polarised T-cell function and biology in people with HIV under successful antiretroviral therapy and HIV elite controllers

Despite successful antiretroviral therapy (ART), frequencies and immunological functions of memory CCR6+ Th17-polarised CD4+ T-cells are not fully restored in people with HIV (PWH). Moreover, long-lived Th17cells contribute to HIV persistence under ART. However, the molecular mechanisms underlying these observations remain understudied. mRNA-sequencing was performed using Illumina technology on freshly FACS-sorted memory CCR6+CD4+ T-cells from successfully ART-treated (ST), elite controllers (EC), and uninfected donors (HD). Gene expression validation was performed by RT-PCR, flow cytometry, and invitro functional assays. Decreased Th17cell frequencies in STs and ECs versus HDs coincided with reduced Th17-lineage cytokine production invitro. Accordingly, the RORγt/RORC2 repressor NR1D1 was upregulated, while the RORγt/RORC2 inducer Semaphorin 4D was decreased in memory CCR6+ T-cells of STs and ECs versus HDs. The presence of HIV-DNA in memory CCR6+ T-cells of ST and EC corresponded with the downregulation of HIV restriction factors (SERINC3, KLF3, and RNF125) and HIV inhibitors (tetraspanins), along with increased expression of the HIV-dependency factor MRE11, indicative of higher susceptibility/permissiveness to HIV-1 infection. Furthermore, markers of DNA damage/modification were elevated in memory CCR6+ T-cells of STs and ECs versus HDs, in line with their increased activation (CD38/HLA-DR), senescence/exhaustion phenotype (CTLA-4/PD-1/CD57) and their decreased expression of proliferation marker Ki-67. These results reveal new molecular mechanisms of Th17 cell deficit in ST and EC PWH despite a successful control of HIV-1 replication. This knowledge points to potential therapeutic interventions to limit HIV-1 infection and restore frequencies, effector functions, and senescence/exhaustion in Th17cells. This study was funded by the Canadian Institutes of Health Research (CIHR, operating grant MOP 142294, and the Canadian HIV Cure Enterprise [CanCURE 2.0] Team Grant HB2 164064), and in part, by the Réseau SIDA et maladies infectieuses du Fonds de recherche du Québec-Santé (FRQ-S).

Acetylation of SAMHD1 at lysine 580 is crucial for blocking HIV-1 infection.

In humans, sterile alpha motif (SAM) domain- and histidine-aspartic acid (HD) domain-containing protein 1 (SAMHD1) is a dNTPase enzyme that prevents HIV-1 infection in non-cycling cells, such as differentiated THP-1 cells and human primary macrophages. Although phosphorylation of threonine 592 (T592) in SAMHD1 is recognized as the primary regulator of the ability to prevent HIV-1 infection, the contributions of SAMHD1 acetylation to this ability remain unknown. Mass spectrometry analysis of SAMHD1 proteins derived from cycling and non-cycling THP-1 cells, primary cycling B cells, and primary macrophages revealed that SAMHD1 is preferentially acetylated at lysine residues 354, 494, and 580 (K354, K494, and K580). In non-cycling cells, SAMHD1 is preferentially acetylated at K580, suggesting that this post-translational modification may contribute to the ability of SAMHD1 to block HIV-1 infection. Consistent with this finding, we found that mutations in K580 disrupted the ability of SAMHD1 to block HIV-1 infection without affecting the ability of SAMHD1 to deplete cellular dNTP levels. Gene editing of SAMHD1 in macrophage-like cells revealed that an intact K580 is required for HIV-1 restriction. This finding suggests that K580 acetylation in SAMHD1 is essential for blocking HIV-1 infection. More importantly, we found that a larger proportion of SAMHD1 featuring K580 acetylation could be detected in human primary macrophages when compared to human primary monocytes. In agreement, we found that SAMHD1 is acetylated during the monocyte-to-macrophage differentiation process. This finding agrees with the idea that the blockade of HIV-1 infection in macrophages requires SAMHD1 acetylation.IMPORTANCEThe natural inhibitor of HIV-1, sterile alpha motif (SAM) domain- and histidine-aspartic acid (HD) domain-containing protein 1 (SAMHD1), plays a pivotal role in preventing HIV-1 infection of macrophages and dendritic cells, which are vital components of the immune system. This study unveils that SAMHD1 undergoes post-translational modifications, specifically acetylation at lysines 354, 494, and 580. Our research underscores the significance of these modifications, demonstrating that acetylation at residue K580 is indispensable for SAMHD1's efficacy in blocking HIV-1 infection. Notably, K580 is found in a critical regulatory domain of SAMHD1, highlighting acetylation as a novel layer of SAMHD1 regulation for HIV-1 restriction in humans. A comprehensive understanding of the regulation mechanisms governing this anti-HIV-1 protein is crucial for leveraging nature's defense mechanisms against HIV-1 and could pave the way for innovative therapeutic strategies.

SAMD9L acts as an antiviral factor against HIV-1 and primate lentiviruses by restricting viral and cellular translation.

Sterile alpha motif domain-containing proteins 9 and 9-like (SAMD9/9L) are associated with life-threatening genetic diseases in humans and are restriction factors of poxviruses. Yet, their cellular function and the extent of their antiviral role are poorly known. Here, we found that interferon-stimulated human SAMD9L restricts HIV-1 in the late phases of replication, at the posttranscriptional and prematuration steps, impacting viral translation and, possibly, endosomal trafficking. Surprisingly, the paralog SAMD9 exerted an opposite effect, enhancing HIV-1. More broadly, we showed that SAMD9L restricts primate lentiviruses, but not a gammaretrovirus (MLV), nor 2 RNA viruses (arenavirus MOPV and rhabdovirus VSV). Using structural modeling and mutagenesis of SAMD9L, we identified a conserved Schlafen-like active site necessary for HIV-1 restriction by human and a rodent SAMD9L. By testing a gain-of-function constitutively active variant from patients with SAMD9L-associated autoinflammatory disease, we determined that SAMD9L pathogenic functions also depend on the Schlafen-like active site. Finally, we found that the constitutively active SAMD9L strongly inhibited HIV, MLV, and, to a lesser extent, MOPV. This suggests that the virus-specific effect of SAMD9L may involve its differential activation/sensing and the virus ability to evade from SAMD9L restriction. Overall, our study identifies SAMD9L as an HIV-1 antiviral factor from the cell autonomous immunity and deciphers host determinants underlying the translational repression. This provides novel links and therapeutic avenues against viral infections and genetic diseases.

MARCH2, a T cell specific factor that restricts HIV-1 infection.

Membrane-associated RING-CH (MARCH) 2 is a member of the MARCH protein family of RING-CH finger E3 ubiquitin ligases that play important roles in regulating the levels of proteins found on the cell surface. MARCH1, 2 and 8 inhibit HIV-1 infection by preventing the incorporation of the envelope glycoproteins into nascent virions. However, a better understanding of the mechanism utilized by MARCH proteins to restrict HIV-1 infection is needed. In this report, we identify an amino acid in human MARCH2, absent in mouse MARCH2, critical for its antiretroviral function. Moreover, we map the domains of human MARCH2 critical for restricting as well as binding to the HIV-1 envelope glycoproteins. In addition, we demonstrate that MARCH2 is present inside nascent virions and reduces particle infectivity by blocking virus entry in a RING-CH-independent manner. Finally, we show that MARCH2 acts as an HIV-1 restriction factor only in primary CD4+ T cells and can prevent cell-to-cell transmission of HIV-1. Our findings reveal important new aspects of the antiviral mechanism utilized by human MARCH2 to restrict HIV-1 that have potential implications to all MARCH proteins with antiviral functions and their viral targets.

Induction of the antiviral factors APOBEC3A and RSAD2 upon CCL2 neutralization in primary human macrophages involves NF-κB, JAK/STAT, and gp130 signaling.

The CCL2/CC chemokine receptor 2 axis plays key roles in the pathogenesis of HIV-1 infection. We previously reported that exposure of monocyte-derived macrophages to CCL2 neutralizing antibody (αCCL2 Ab) restricted HIV-1 replication at postentry steps of the viral life cycle. This effect was associated with induction of transcripts coding for innate antiviral proteins, including APOBEC3A and RSAD2. This study aimed at identifying the signaling pathways involved in induction of these factors by CCL2 blocking in monocyte-derived macrophages. Through a combination of pharmacologic inhibition, quantitative reverse transcription polymerase chain reaction, Western blotting, and confocal laser-scanning microscopy, we demonstrated that CCL2 neutralization activates the canonical NF-κB and JAK/STAT pathways, as assessed by time-dependent phosphorylation of IκB, STAT1, and STAT3 and p65 nuclear translocation. Furthermore, pharmacologic inhibition of IκB kinase and JAKs strongly reduced APOBEC3A and RSAD2 transcript accumulation elicited by αCCL2 Ab treatment. Interestingly, exposure of monocyte-derived macrophages to αCCL2 Ab resulted in induction of IL-6 family cytokines, and interference with glycoprotein 130, the common signal-transducing receptor subunit shared by these cytokines, inhibited APOBEC3A and RSAD2 upregulation triggered by CCL2 neutralization. These results provide novel insights into the signal transduction pathways underlying the activation of innate responses triggered by CCL2 neutralization in macrophages. Since this response was found to be associated with protective antiviral effects, the new findings may help design innovative therapeutic approaches targeting CCL2 to strengthen host innate immunity.

Disruption of Transmembrane Phosphatidylserine Asymmetry by HIV-1 Incorporated SERINC5 Is Not Responsible for Virus Restriction.

Host restriction factor SERINC5 (SER5) incorporates into the HIV-1 membrane and inhibits infectivity by a poorly understood mechanism. Recently, SER5 was found to exhibit scramblase-like activity leading to the externalization of phosphatidylserine (PS) on the viral surface, which has been proposed to be responsible for SER5's antiviral activity. This and other reports that document modulation of HIV-1 infectivity by viral lipid composition prompted us to investigate the role of PS in regulating SER5-mediated HIV-1 restriction. First, we show that the level of SER5 incorporation into virions correlates with an increase in PS levels in the outer leaflet of the viral membrane. We developed an assay to estimate the PS distribution across the viral membrane and found that SER5, but not SER2, which lacks antiviral activity, abrogates PS asymmetry by externalizing this lipid. Second, SER5 incorporation diminished the infectivity of pseudoviruses produced from cells lacking a flippase subunit CDC50a and, therefore, exhibited a higher baseline level of surface-accessible PS. Finally, exogenous manipulation of the viral PS levels utilizing methyl-alpha-cyclodextrin revealed a lack of correlation between external PS and virion infectivity. Taken together, our study implies that the increased PS exposure to SER5-containing virions itself is not directly linked to HIV-1 restriction.

Inhibition of caspase pathways limits CD4+ T cell loss and restores host anti-retroviral function in HIV-1 infected humanized mice with augmented lymphoid tissue.

The study of HIV infection and pathogenicity in physical reservoirs requires a biologically relevant model. The human immune system (HIS) mouse is an established model of HIV infection, but defects in immune tissue reconstitution remain a challenge for examining pathology in tissues. We utilized exogenous injection of the human recombinant FMS-like tyrosine kinase 3 ligand (rFLT-3L) into the hematopoietic stem cell (HSC) cord blood HIS mouse model to significantly expand the total area of lymph node (LN) and the number of circulating human T cells. The results enabled visualization and quantification of HIV infectivity, CD4 T cell depletion and other measures of pathogenesis in the secondary lymphoid tissues of the spleen and LN. Treatment with the Caspase-1/4 inhibitor VX-765 limited CD4+ T cell loss in the spleen and reduced viral load in both the spleen and axillary LN. In situ hybridization further demonstrated a decrease in viral RNA in both the spleen and LN. Transcriptomic analysis revealed that in vivo inhibition of caspase-1/4 led to an upregulation in host HIV restriction factors including SAMHD1 and APOBEC3A. These findings highlight the use of rFLT-3L to augment human immune system characteristics in HIS mice to support investigations of HIV pathogenesis and test host directed therapies, though further refinements are needed to further augment LN architecture and cellular populations. The results further provide in vivo evidence of the potential to target inflammasome pathways as an avenue of host-directed therapy to limit immune dysfunction and virus replication in tissue compartments of HIV+ persons.

APOBEC3D excludes APOBEC3F from HIV-1 virions by competitive binding of RNA

The human family of APOBEC3 enzymes are primarily studied as single-stranded DNA deoxycytidine deaminases that act as host restriction factors for a number of viruses and retroelements. The deamination of deoxycytidine to deoxyuridine causes inactivating mutations in target DNA and the nucleic acid binding ability may also cause deamination independent restriction. There are seven APOBEC3 enzymes in humans, named A-H, excluding E, each of which has restriction activity against a subset of viruses or retroelements. There are primarily four, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H that have been found to restrict replication of HIV-1, however their restriction activity varies and they have primarily been studied individually despite co-expression in the cells that HIV-1 infects. It is known that APOBEC3F hetero-oligomerizes with APOBEC3G and APOBEC3H and that this influences host restriction outcomes during HIV-1 infection in tissue culture. Here, we examined if APOBEC3F interacts with APOBEC3D and the functional outcomes. We found that APOBEC3D mRNA expression was similar to or higher than APOBEC3F mRNA in multiple donors, suggesting that the proteins would be co-expressed, allowing for interactions to occur. We determined that APOBEC3F and APOBEC3D interacted primarily through an RNA intermediate; however, this interaction resulted in APOBEC3D competitively excluding APOBEC3F from virions. Although HIV-1 restriction still occurred when APOBEC3F and APOBEC3D were co-expressed, it was due to primarily APOBEC3D-mediated deamination-independent restriction. The APOBEC3D-mediated exclusion of APOBEC3F from HIV-1 encapsidation could be recapitulated in vitro through RNA capture experiments in which APOBEC3D decreased or abrogated the ability of APOBEC3F to bind to HIV-1 protease or 5’UTR RNA, respectively. Overall, the data suggest that there are mechanisms at the protein level that segregate APOBEC3s into different virus particles.

HIV-1 reservoir size after neonatal antiretroviral therapy and the potential to evaluate antiretroviral-therapy-free remission (IMPAACT P1115): a phase 1/2 proof-of-concept study

Infants born with HIV-1 require lifelong antiretroviral therapy (ART). We aimed to assess whether very early ART in neonates might restrict HIV-1 reservoirs, an important step towards ART-free remission. IMPAACT P1115 is an ongoing, phase 1/2, proof-of-concept study in which infants were enrolled at 30 research clinics in 11 countries (Brazil, Haiti, Kenya, Malawi, South Africa, Tanzania, Thailand, Uganda, the USA, Zambia, and Zimbabwe) into two cohorts. Infants at least 34 weeks' gestational age at high risk for in-utero HIV-1 with either untreated maternal HIV-1 (cohort 1) or who were receiving pre-emptive triple antiretroviral prophylaxis outside of the study (maternal ART permissible; cohort 2) were included. All infants initiated treatment within 48 h of life. Cohort 1 initiated three-drug nevirapine-based ART, and cohort 2 initiated three-drug nevirapine-based prophylaxis then three-drug nevirapine-based ART following HIV diagnosis by age 10 days. We added twice-daily coformulated oral ritonavir 75 mg/m2 and lopinavir 300 mg/m2 from 14 days of life and 42 weeks postmenstrual age. We discontinued nevirapine 12 weeks after two consecutive plasma HIV-1 RNA levels below limit of detection. We tracked virological suppression, safety outcomes, and meeting a predetermined biomarker profile at age 2 years (undetectable RNA since week 48, HIV-1 antibody-negative, HIV-1 DNA not detected, and normal CD4 count and CD4 percentage) to assess qualification for analytical treatment interruption. This study is registered with ClinicalTrials.gov, NCT02140255. Between Jan 23, 2015, and Dec 14, 2017, 440 infants were included in cohort 1 and 20 were included in cohort 2. 54 of these infants (34 from cohort 1 and 20 from cohort 2) had confirmed in-utero HIV-1 and were enrolled to receive study ART. 33 (61%) of 54 infants were female and 21 (39%) were male. The estimated probability of maintaining undetectable plasma RNA through to 2 years was 33% (95% CI 17-49) in cohort 1 and 57% (28-78) in cohort 2. Among infants maintaining protocol-defined virological control criteria through to study week 108, seven of 11 (64%, 95% CI 31-89) in cohort 1 and five of seven (71%, 29-96) in cohort 2 had no detected HIV-1 DNA. Ten of 12 (83%, 52-100) in cohort 1 and all seven (100%, 59-100) in cohort 2 tested HIV-1 antibody-negative at week 108. Among 54 infants initiated on very early ART, ten (19%; six in cohort 1 and four in cohort 2) met all criteria for possible analytical treatment interruption. Reversible grade 3 or 4 adverse events occurred in 15 (44%) of 34 infants in cohort 1 and seven (35%) of 20 infants in cohort 2. Very early ART for in-utero HIV-1 can achieve sustained virological suppression in association with biomarkers indicating restricted HIV-1 reservoirs by age 2 years, which might enable potential ART-free remission. National Institute of Allergy and Infectious Diseases, the Eunice Kennedy Shriver National Institute of Child Health and Human Development, and the National Institute of Mental Health.

HIV-1 Restriction in Systemic Lupus Erythematosus Involves Iron Homeostasis

INTRODUCTION: Over the past two decades, there has been a growing emphasis on host restriction factors. However, a comprehensive understanding of their role in modulating HIV-1 infection in vivo requires further detailed investigation. Intracellular iron levels can affect HIV-1 replication by influencing expression of host cell viral restriction factors. Systemic Lupus Erythematosus (SLE) is characterized by upregulation of interferon response and the coexistence of SLE and HIV infection is rare. In lupus nephritis, deregulation of proteins involved in iron homeostasis have also been identified, with hepcidin suggested to play a role in disease severity . Hif1α, a protein regulated by hypoxia and under iron depletion, and reported to block HIV transcription, is upregulated in SLE. High levels of IL-10 and IL-16 which inhibits HIV infection are upregulated in SLE. APOBECs and oxysterols and Interferon Regulating Factors (IRFs) are also known to be upregulated in SLE. The current study is aimed to investigate ex vivo HIV-1 infection in peripheral blood mononuclear cells (PBMCs) derived from individuals SLE and identify factors that contributed to this inhibition. METHODS: We utilized replication deficient VSVg-pseudotype HIV-1 and replication competent HIV-IIIB to infect SLE PBMCs. Real-time PCR was conducted to determine the upregulation of genes involved in HIV-1 replication. RNA-seq of PBMCs were done to identify altered restriction factors. RESULTS: We investigated one round of HIV-1 infection in peripheral blood mononuclear cells (PBMCs) obtained from individuals with Systemic Lupus Erythematosus (SLE) and compared them to control PBMCs. We found that the HIV-1 infection in SLE-derived PBMCs was significantly reduced, as indicated by lower mRNA levels of Gag and Nef, two essential viral proteins. Our ongoing study, which focused on the inhibition of HIV-1 replication ex vivo in Sickle Cell Disease (SCD), revealed interesting findings. RNA sequencing of PBMCs from individuals with SCD showed an upregulation of APOBEC3A, APOBEC3B, and CH25H, which are known restriction factors against HIV-1. Given these intriguing results, we decided to analyze the RNA expression of these restriction factors in SLE-derived PBMCs. Surprisingly, we found a significant increase in the mRNA levels of APOBEC3A, APOBEC3B, and CH25H in SLE-derived PBMCs as well. This suggests that the enhanced expression of these restriction factors could be contributing to the reduced HIV-1 infection observed in SLE-derived PBMCs. Additionally, we explored the RNA expression of HIF-1α and TFR1, which are iron-regulated factors, and found that their mRNA levels were upregulated in SLE-derived PBMCs. Furthermore, we observed an upregulation of interferon regulatory factors, including IL-10, IFN-β, and IRF-7, which are involved in immune responses and antiviral defense mechanisms. Interestingly IRF-7 was upregulated in SCD PBMCs too. DISSUCION: The ongoing study in our lab on SCD and the study reported here are known for deregulated iron metabolism. Interestingly on one hand PBMCs derived from both inhibit HIV-1 and on other have some common restriction factors, iron regulated genes and IRF upregulated. Iron-regulated factors, such as TFR1, HIF-1α and ferroportin have been associated with antiviral effects. Modulating the expression or activity of these factors could influence HIV infection. CONCLUSIONs: Taken together, our findings indicate that SLE-derived PBMCs exhibit a distinct gene expression profile that may confer protection against HIV-1 infection. The upregulation of APOBEC3A, APOBEC3B, CH25H, HIF-1α, TFR1, and interferon regulatory factors could collectively contribute to the observed reduction in HIV-1 replication in SLE-derived PBMCs. Further investigation is needed to fully understand the mechanisms underlying these observations and their implications for HIV-1 pathogenesis under altered intracellular iron levels influencing HIV-1 replication by modulating host cell factors. Acknowledgments This work was supported by NIH Research Grants 1R01HL125005, 5U54MD007597, P30AI087714 and P50AI150476. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Patterns of Evolution of TRIM Genes Highlight the Evolutionary Plasticity of Antiviral Effectors in Mammals.

The innate immune system of mammals is formed by a complex web of interacting proteins, which together constitute the first barrier of entry for infectious pathogens. Genes from the E3-ubiquitin ligase tripartite motif (TRIM) family have been shown to play an important role in the innate immune system by restricting the activity of different retrovirus species. For example, TRIM5 and TRIM22 have both been associated with HIV restriction and are regarded as crucial parts of the antiretroviral machinery of mammals. Our analyses of positive selection corroborate the great significance of these genes for some groups of mammals. However, we also show that many species lack TRIM5 and TRIM22 altogether. By analyzing a large number of mammalian genomes, here we provide the first comprehensive view of the evolution of these genes in eutherians, showcasing that the pattern of accumulation of TRIM genes has been dissimilar across mammalian orders. Our data suggest that these differences are caused by the evolutionary plasticity of the immune system of eutherians, which have adapted to use different strategies to combat retrovirus infections. Altogether, our results provide insights into the dissimilar evolution of a representative family of restriction factors, highlighting an example of adaptive and idiosyncratic evolution in the innate immune system.

The innate immune factor RPRD2/REAF and its role in the Lv2 restriction of HIV.

Intracellular innate immunity involves co-evolved antiviral restriction factors that specifically inhibit infecting viruses. Studying these restrictions has increased our understanding of viral replication, host-pathogen interactions, and pathogenesis, and represent potential targets for novel antiviral therapies. Lentiviral restriction 2 (Lv2) was identified as an unmapped early-phase restriction of HIV-2 and later shown to also restrict HIV-1 and simian immunodeficiency virus. The viral determinants of Lv2 susceptibility have been mapped to the envelope and capsid proteins in both HIV-1 and HIV-2, and also viral protein R (Vpr) in HIV-1, and appears dependent on cellular entry mechanism. A genome-wide screen identified several likely contributing host factors including members of the polymerase-associated factor 1 (PAF1) and human silencing hub (HUSH) complexes, and the newly characterized regulation of nuclear pre-mRNA domain containing 2 (RPRD2). Subsequently, RPRD2 (or RNA-associated early-stage antiviral factor) has been shown to be upregulated upon T cell activation, is highly expressed in myeloid cells, binds viral reverse transcripts, and potently restricts HIV-1 infection. RPRD2 is also bound by HIV-1 Vpr and targeted for degradation by the proteasome upon reverse transcription, suggesting RPRD2 impedes reverse transcription and Vpr targeting overcomes this block. RPRD2 is mainly localized to the nucleus and binds RNA, DNA, and DNA:RNA hybrids. More recently, RPRD2 has been shown to negatively regulate genome-wide transcription and interact with the HUSH and PAF1 complexes which repress HIV transcription and are implicated in maintenance of HIV latency. In this review, we examine Lv2 restriction and the antiviral role of RPRD2 and consider potential mechanism(s) of action.

Gene editing of SAMHD1 in macrophage-like cells reveals complex relationships between SAMHD1 phospho-regulation, HIV-1 restriction, and cellular dNTP levels.

We introduce BLaER1 cells as an alternative myeloid cell model in combination with CRISPR/Cas9-mediated gene editing to study the influence of sterile α motif and HD domain-containing protein 1 (SAMHD1) T592 phosphorylation on anti-viral restriction and the control of cellular dNTP levels in an endogenous, physiologically relevant context. A proper understanding of the mechanism of the anti-viral function of SAMHD1 will provide attractive strategies aiming at selectively manipulating SAMHD1 without affecting other cellular functions. Even more, our toolkit may inspire further genetic analysis and investigation of restriction factors inhibiting retroviruses and their cellular function and regulation, leading to a deeper understanding of intrinsic anti-viral immunity.

Independent loss events of a functional tetherin gene in galliform birds.

Birds represent important hosts for numerous viruses, including zoonotic viruses and pathogens with the potential to cause major economic losses to the poultry industry. Viral replication and transmission can be inhibited or blocked by the action of antiviral restriction factors (RFs) encoded by the host. One well-characterized RF is tetherin, a protein that directly blocks the release of newly formed viral particles from infected cells. Here, we describe the evolutionary loss of a functional tetherin gene in two galliform birds, turkey (Meleagris gallopavo) and Mikado pheasant (Syrmaticus mikado). Moreover, we demonstrate that the structurally related protein TMCC(aT) exerts antiviral activity in several birds, albeit by a mechanism different from that of tetherin. The evolutionary scenario described here represents the first documented loss-of-tetherin cases in vertebrates.

APOBEC3 degradation is the primary function of HIV-1 Vif determining virion infectivity in the myeloid cell line THP-1.

HIV-1 must overcome multiple innate antiviral mechanisms to replicate in CD4+ T lymphocytes and macrophages. Previous studies have demonstrated that the apolipoprotein B mRNA editing enzyme polypeptide-like 3 (APOBEC3, A3) family of proteins (at least A3D, A3F, A3G, and stable A3H haplotypes) contribute to HIV-1 restriction in CD4+ T lymphocytes. Virus-encoded virion infectivity factor (Vif) counteracts this antiviral activity by degrading A3 enzymes allowing HIV-1 replication in infected cells. In addition to A3 proteins, Vif also targets other cellular proteins in CD4+ T lymphocytes, including PPP2R5 proteins. However, whether Vif primarily degrades only A3 proteins during viral replication is currently unknown. Herein, we describe the development and characterization of A3F-, A3F/A3G-, and A3A-to-A3G-null THP-1 cells. In comparison to Vif-proficient HIV-1, Vif-deficient viruses have substantially reduced infectivity in parental and A3F-null THP-1 cells, and a more modest decrease in infectivity in A3F/A3G-null cells. Remarkably, disruption of A3A-A3G protein expression completely restores the infectivity of Vif-deficient viruses in THP-1 cells. These results indicate that the primary function of Vif during infectious HIV-1 production from THP-1 cells is the targeting and degradation of A3 enzymes. IMPORTANCE HIV-1 Vif neutralizes the HIV-1 restriction activity of A3 proteins. However, it is currently unclear whether Vif has additional essential cellular targets. To address this question, we disrupted A3A to A3G genes in the THP-1 myeloid cell line using CRISPR and compared the infectivity of wild-type HIV-1 and Vif mutants with the selective A3 neutralization activities. Our results demonstrate that the infectivity of Vif-deficient HIV-1 and the other Vif mutants is fully restored by ablating the expression of cellular A3A to A3G proteins. These results indicate that A3 proteins are the only essential target of Vif that is required for fully infectious HIV-1 production from THP-1 cells.

Biochemical functions and structure of Caenorhabditis elegans ZK177.8 protein: Aicardi–Goutières syndrome SAMHD1 dNTPase ortholog

Mutations in SAM domain and HD domain containing protein 1 (SAMHD1) are found in a neuro-developmental disorder, Aicardi-Goutières Syndrome (AGS) and cancers, and SAMHD1, which is a dNTP tri-phosphorylase, was identified as a myeloid specific HIV-1 restriction factor. Here, we characterized the enzymology and structure of a SAMHD1 ortholog of C. elegans, ZK177.8, which also reportedly induces developmental defects upon gene knockdown. We found ZK177.8 protein is a dNTPase allosterically regulated by dGTP. The active site of ZK177.8 recognizes both 2' OH and tri-phosphate moieties of dNTPs, but not base moiety. The dGTP activator induces the formation of the enzymatically active ZK177.8 tetramers, and ZK177.8 protein lowers cellular dNTP levels in a human monocytic cell line. Finally, ZK177.8 tetramers display very similar X-ray crystal structure with human and mouse SAMHD1s except its lack of the canonical SAM domain. This striking conservation in structure, function and allosteric regulatory mechanism for the hydrolysis of the DNA building blocks supports their host developmental roles.

Antiviral HIV-1 SERINC restriction factors disrupt virus membrane asymmetry

The host proteins SERINC3 and SERINC5 are HIV-1 restriction factors that reduce infectivity when incorporated into the viral envelope. The HIV-1 accessory protein Nef abrogates incorporation of SERINCs via binding to intracellular loop 4 (ICL4). Here, we determine cryoEM maps of full-length human SERINC3 and an ICL4 deletion construct, which reveal that hSERINC3 is comprised of two α-helical bundles connected by a ~ 40-residue, highly tilted, “crossmember” helix. The design resembles non-ATP-dependent lipid transporters. Consistently, purified hSERINCs reconstituted into proteoliposomes induce flipping of phosphatidylserine (PS), phosphatidylethanolamine and phosphatidylcholine. Furthermore, SERINC3, SERINC5 and the scramblase TMEM16F expose PS on the surface of HIV-1 and reduce infectivity, with similar results in MLV. SERINC effects in HIV-1 and MLV are counteracted by Nef and GlycoGag, respectively. Our results demonstrate that SERINCs are membrane transporters that flip lipids, resulting in a loss of membrane asymmetry that is strongly correlated with changes in Env conformation and loss of infectivity.

HSV-2 triggers upregulation of MALAT1 in CD4+ T cells and promotes HIV latency reversal.

Herpes simplex virus type 2 (HSV-2) coinfection is associated with increased HIV-1 viral loads and expanded tissue reservoirs, but the mechanisms are not well defined. HSV-2 recurrences result in an influx of activated CD4+ T cells to sites of viral replication and an increase in activated CD4+ T cells in peripheral blood. We hypothesized that HSV-2 induces changes in these cells that facilitate HIV-1 reactivation and replication and tested this hypothesis in human CD4+ T cells and 2D10 cells, a model of HIV-1 latency. HSV-2 promoted latency reversal in HSV-2-infected and bystander 2D10 cells. Bulk and single-cell RNA-Seq studies of activated primary human CD4+ T cells identified decreased expression of HIV-1 restriction factors and increased expression of transcripts including MALAT1 that could drive HIV replication in both the HSV-2-infected and bystander cells. Transfection of 2D10 cells with VP16, an HSV-2 protein that regulates transcription, significantly upregulated MALAT1 expression, decreased trimethylation of lysine 27 on histone H3 protein, and triggered HIV latency reversal. Knockout of MALAT1 from 2D10 cells abrogated the response to VP16 and reduced the response to HSV-2 infection. These results demonstrate that HSV-2 contributes to HIV-1 reactivation through diverse mechanisms, including upregulation of MALAT1 to release epigenetic silencing.