Antiviral Restriction Factors Research Articles

Determining the function of proteins degraded by Human Cytomegalovirus

Human Cytomegalovirus (HCMV) is associated with significant morbidity and mortality in the immunocompromised, and is a leading cause of congenital infection. Only three drugs are available for treatment, all with significant toxicities. New therapies and a vaccine are urgently required. Susceptibility to viral infection and disease is determined in part by antiviral restriction factors (ARFs) and the viral proteins that have evolved to degrade them. Small-molecule disruption of the interaction between an ARF and a viral antagonist can inhibit viral replication and may be utilised for antiviral therapies. To identify novel restriction factors against HCMV, we previously developed a multiplexed proteomic approach to identify proteins that are actively degraded early during HCMV infection. We reasoned that these would be enriched in known and novel ARFs that the virus must degrade in order to replicate. 35 proteins were shown to be degraded according to stringent statistical criteria, which included the known anti-HCMV restriction factors Sp100 and MORC3, and a novel ARF, HLTF. Here, we present preliminary results from a combination of viral replication assays to identify other novel ARFs. These include a new ‘two-colour’ approach to characterise HCMV restriction, which aims to eliminate variability in cell density by mixing populations of cells in a single cell culture well. Preliminary studies have identified a number of proteins that inhibit virus replication, as well as a novel dependency factor which the virus may degrade in order to attenuate cellular immune signalling or help establish latency.

Role for a Filamentous Nuclear Assembly of IFI16, DNA, and Host Factors in Restriction of Herpesviral Infection.

Several host cell nuclear factors are known to restrict herpes simplex virus 1 (HSV-1) replication, but their mechanisms of action remain to be defined. Interferon-inducible protein 16 (IFI16) and the nuclear domain 10-associated proteins, such as promyelocytic leukemia (PML) protein, localize to input viral genomes, but they are also capable of restricting progeny viral transcription. In this study, we used structured illumination microscopy to show that after HSV DNA replication, IFI16 forms nuclear filamentous structures on DNA within a subset of nuclear replication compartments in HSV-1 ICP0-null mutant virus-infected human cells. The ability to form filaments in different cell types correlates with the efficiency of restriction, and the kinetics of filament formation and epigenetic changes are similar. Thus, both are consistent with the filamentous structures being involved in epigenetic silencing of viral progeny DNA. IFI16 filaments recruit other restriction factors, including PML, Sp100, and ATRX, to aid in the restriction. Although the filaments are only in a subset of the replication compartments, IFI16 reduces the levels of elongation-competent RNA polymerase II (Pol II) in all replication compartments. Therefore, we propose that IFI16 filaments with associated restriction factors that form in replication compartments constitute a "restrictosome" structure that signals in cis and trans to silence the progeny viral DNA throughout the infected cell nucleus. The IFI16 filamentous structure may constitute the first known nuclear supramolecular organizing center for signaling in the cell nucleus.IMPORTANCE Mammalian cells exhibit numerous strategies to recognize and contain viral infections. The best-characterized antiviral responses are those that are induced within the cytosol by receptors that activate interferon responses or shut down translation. Antiviral responses also occur in the nucleus, yet these intranuclear innate immune responses are poorly defined at the receptor-proximal level. In this study, we explored the ability of cells to restrict infection by assembling viral DNA into transcriptionally silent heterochromatin within the nucleus. We found that the IFI16 restriction factor forms filaments on DNA within infected cells. These filaments recruit antiviral restriction factors to prevent viral replication in various cell types. Mechanistically, IFI16 filaments inhibit the recruitment of RNA polymerase II to viral genes. We propose that IFI16 filaments with associated restriction factors constitute a "restrictosome" structure that can signal to other parts of the nucleus where foreign DNA is located that it should be silenced.

Functional Mapping of Regions Involved in the Negative Imprinting of Virion Particle Infectivity and in Target Cell Protection by Interferon-Induced Transmembrane Protein 3 against HIV-1.

The interferon-induced transmembrane proteins (IFITMs) are a family of highly related antiviral factors that affect numerous viruses at two steps: in target cells by sequestering incoming viruses in endosomes and in producing cells by leading to the production of virions that package IFITMs and exhibit decreased infectivity. While most studies have focused on the former, little is known about the regulation of the negative imprinting of virion particle infectivity by IFITMs and about its relationship with target cell protection. Using a panel of IFITM3 mutants against HIV-1, we have explored these issues as well as others related to the biology of IFITM3, in particular virion packaging, stability, the relation to CD63/multivesicular bodies (MVBs), the modulation of cholesterol levels, and the relationship between negative imprinting of virions and target cell protection. The results that we have obtained exclude a role for cholesterol and indicate that CD63 accumulation does not directly relate to an antiviral behavior. We have defined regions that modulate the two antiviral properties of IFITM3 as well as novel domains that modulate protein stability and that, in so doing, influence the extent of its packaging into virions. The results that we have obtained, however, indicate that, even in the context of an IFITM-susceptible virus, IFITM3 packaging is not sufficient for negative imprinting. Finally, while most mutations concomitantly affect target cell protection and negative imprinting, a region in the C-terminal domain (CTD) exhibits a differential behavior, potentially highlighting the regulatory role that this domain may play in the two antiviral activities of IFITM3.IMPORTANCE IFITM proteins have been associated with the sequestration of incoming virions in endosomes (target cell protection) and with the production of virion particles that incorporate IFITMs and exhibit decreased infectivity (negative imprinting of virion infectivity). How the latter is regulated and whether these two antiviral properties are related remain unknown. By examining the behavior of a large panel of IFITM3 mutants against HIV-1, we determined that IFITM3 mutants are essentially packaged into virions proportionally to their intracellular levels of expression. However, even in the context of an IFITM-susceptible virus, IFITM3 packaging is not sufficient for the antiviral effects. Most mutations were found to concomitantly affect both antiviral properties of IFITM3, but one CTD mutant exhibited a divergent behavior, possibly highlighting a novel regulatory role for this domain. These findings thus advance our comprehension of how this class of broad antiviral restriction factors acts.

A Mass Spectrometry-Based Profiling of Interactomes of Viral DDB1- and Cullin Ubiquitin Ligase-Binding Proteins Reveals NF-κB Inhibitory Activity of the HIV-2-Encoded Vpx.

Viruses and hosts are situated in a molecular arms race. To avoid morbidity and mortality, hosts evolved antiviral restriction factors. These restriction factors exert selection pressure on the viruses and drive viral evolution toward increasingly efficient immune antagonists. Numerous viruses exploit cellular DNA damage-binding protein 1 (DDB1)-containing Cullin RocA ubiquitin ligases (CRLs) to induce the ubiquitination and subsequent proteasomal degradation of antiviral factors expressed by their hosts. To establish a comprehensive understanding of the underlying protein interaction networks, we performed immuno-affinity precipitations for a panel of DDB1-interacting proteins derived from viruses such as mouse cytomegalovirus (MCMV, Murid herpesvirus [MuHV] 1), rat cytomegalovirus Maastricht MuHV2, rat cytomegalovirus English MuHV8, human cytomegalovirus (HCMV), hepatitis B virus (HBV), and human immunodeficiency virus (HIV). Cellular interaction partners were identified and quantified by mass spectrometry (MS) and validated by classical biochemistry. The comparative approach enabled us to separate unspecific interactions from specific binding partners and revealed remarkable differences in the strength of interaction with DDB1. Our analysis confirmed several previously described interactions like the interaction of the MCMV-encoded interferon antagonist pM27 with STAT2. We extended known interactions to paralogous proteins like the interaction of the HBV-encoded HBx with different Spindlin proteins and documented interactions for the first time, which explain functional data like the interaction of the HIV-2-encoded Vpr with Bax. Additionally, several novel interactions were identified, such as the association of the HIV-2-encoded Vpx with the transcription factor RelA (also called p65). For the latter interaction, we documented a functional relevance in antagonizing NF-κB-driven gene expression. The mutation of the DDB1 binding interface of Vpx significantly impaired NF-κB inhibition, indicating that Vpx counteracts NF-κB signaling by a DDB1- and CRL-dependent mechanism. In summary, our findings improve the understanding of how viral pathogens hijack cellular DDB1 and CRLs to ensure efficient replication despite the expression of host restriction factors.

Restriction Factors: From Intrinsic Viral Restriction to Shaping Cellular Immunity Against HIV-1.

Antiviral restriction factors are host cellular proteins that constitute a first line of defense blocking viral replication and propagation. In addition to interfering at critical steps of the viral replication cycle, some restriction factors also act as innate sensors triggering innate responses against infections. Accumulating evidence suggests an additional role for restriction factors in promoting antiviral cellular immunity to combat viruses. Here, we review the recent progress in our understanding on how restriction factors, particularly APOBEC3G, SAMHD1, Tetherin, and TRIM5α have the cell-autonomous potential to induce cellular resistance against HIV-1 while promoting antiviral innate and adaptive immune responses. Also, we provide an overview of how these restriction factors may connect with protein degradation pathways to modulate anti-HIV-1 cellular immune responses, and we summarize the potential of restriction factors-based therapeutics. This review brings a global perspective on the influence of restrictions factors in intrinsic, innate, and also adaptive antiviral immunity opening up novel research avenues for therapeutic strategies in the fields of drug discovery, gene therapy, and vaccines to control viral infections.

Deception through Mimicry: A Cellular Antiviral Strategy

Viruses use mimicry to hijack cellular machinery, but can human cells use mimicry as an antiviral approach? Batra etal. identify a novel antiviral restriction factor, RBBP6, by characterizing the cellular interactome of Ebola virus. RBBP6 targets the Ebola virus transcription factor VP30 by mimicking the binding of Ebola virus nucleoprotein.

Restriction of HIV-1 Infection in Sickle Cell Disease and Trait

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) replication is controlled by host intrinsic antiviral restriction factors some of which are counteracted by HIV-1 accessory proteins. We recently showed that ex vivo HIV-1 infection is suppressed in PBMCs obtained from Sickle Cell Disease (SCD) patients. The inhibition was mediated in part by SAMHD1 and NF-κB inhibitor, IkBα and triggered by ferroportin, an iron export protein. Ferroportin expression reduced intracellular iron levels and inhibited cellular CDK2 activity leading to reduction of SAMHD1 phosphorylation and increased expression of IkBα.OBJECTIVES: The study was designed to further clarify the mechanism of HIV-1 inhibition in SCD and identify additional HIV-1 restriction factors that may contribute to the restriction mechanism.METHODS: A customized array was utilized to determine the expression of restriction factors in SCD PBMCs. The shRNA-mediated knockdowns of the identified genes were used to further validate the role of these factors in HIV-1 replication in cultured and primary cells. HIV-1(IIIB strain) and VSVG-pseudotyped pNL4-3.Luc.R-E-virus was used to analyze HIV-1 replication.RESULTS: Overexpression of ferroportin in THP-1 cells led to increased expression of HO-1 and p21 and reduced phosphorylation of SAMHD1. Inhibition of HO-1 and p21 by small molecules increased HIV-1 replication in SCD PBMCs suggesting that these factors contributed to the restriction mechanism. Knocking down SAMHD1 in SCD PBMC did not fully restore HIV-1 replication pointing to additional restriction factors. Analysis of HIV-1 restriction factors in SCD PBMCs using customized array showed increased expression of APOBECs, TRIMs, CH25H, CPSF6, CTR9, EIF2ak2, IFI16, MX2, PML and RTF1 mRNAs. We next tested the effect of SCD trait on HIV-1 infection ex vivo and in vivo. PBMCs obtained from SCD trait individuals showed restricted HIV-1 infection with HIV-1(IIIB strain). To further validate these findings, we compared 9 trait (HbAS) HIV-1 infected individuals with 107 non-SCD (HbAA or HbAC) HIV-1 infected individuals from Howard University HIV clinic. HIV-1 viral load and levels of HIV-1 env and gag were significantly lower in HIV-1 infected SCD trait subjects. Expression of HO-1, p21 were increased and RNR2 expression was reduced in SCD trait PBMCs. Small molecule inhibitors of HO-1 and p21 induced HIV-1 replication in SCD trait PBMCs.CONCLUSIONS: Our findings point to HO-1 and p21 as factors that, in addition to SAMHD1 and IKBα, mediate HIV-1 restriction in SCD. In SCD trait, HO-1, p21 and RNR2 play a key role in ex vivo HIV-1 restriction. Thus HIV-1 infection is deregulated not only in SCD patients but also in SCD trait individuals. The HIV-1 restriction is mediated by iron-activated antiviral restriction factors.ACKNOWLEDGMENTS: This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005, 5G12MD007597, 1UM1AI26617 and P30AI087714). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. DisclosuresNekhai:NIMHD, NIH: Research Funding; NHLBI, NIH: Research Funding; NIAID, NIH: Research Funding.

Host Restriction Factors and Human Immunodeficiency Virus (HIV-1): A Dynamic Interplay Involving All Phases of the Viral Life Cycle.

Mammalian cells have evolved several mechanisms to prevent or block lentiviral infection and spread. Among the innate immune mechanisms, the signaling cascade triggered by type I interferon (IFN) plays a pivotal role in limiting the burden of HIV-1. In the presence of IFN, human cells upregulate the expression of a number of genes, referred to as IFN-stimulated genes (ISGs), many of them acting as antiviral restriction factors (RFs). RFs are dominant proteins that target different essential steps of the viral cycle, thereby providing an early line of defense against the virus. The identification and characterization of RFs have provided unique insights into the molecular biology of HIV-1, further revealing the complex host-pathogen interplay that characterizes the infection. The presence of RFs drove viral evolution, forcing the virus to develop specific proteins to counteract their activity. The knowledge of the mechanisms that prevent viral infection and their viral counterparts may offer new insights to improve current antiviral strategies. This review provides an overview of the RFs targeting HIV-1 replication and the mechanisms that regulate their expression as well as their impact on viral replication and the clinical course of the disease.

Host Restriction Factors APOBEC3G/3F and Other Interferon-Related Gene Expressions Affect Early HIV-1 Infection in Northern Pig-Tailed Macaque (Macaca leonina).

The northern pig-tailed macaques (NPMs) lack TRIM5α, an antiviral restriction factor, and instead have TRIM5-CypA. In our previous study, we demonstrated that HIV-1NL4−3 successfully infected NPMs and formed a long-term viral reservoir in vivo. However, the HIV-1-infected NPMs showed relatively high viremia in the first 6 weeks of infection, which declined thereafter suggesting that HIV-1 NL4−3 infection in these animals was only partly permissive. To optimize HIV-1 infection in NPMs therefore, we generated HIV-1NL4−R3A and stHIV-1sv, and infected NPMs with these viruses. HIV-1NL4−R3A and stHIV-1sv can replicate persistently in NPMs during 41 weeks of acute infection stage. Compared to the HIV-1NL4−R3A, stHIV-1sv showed a notably higher level of replication, and the NPMs infected with the latter induced a more robust neutralizing antibody but a weaker cellular immune response. In addition, IFN-I signaling was significantly up-regulated with the viral replication, and was higher in the stHIV-1sv infected macaques. Consequently, the sequences of pro-viral env showed fewer G-A hyper-mutations in stHIV-1sv, suggesting that vif gene of SIV could antagonize the antiviral effects of APOBEC3 proteins in NPMs. Taken together, NPMs infected with HIV-1NL4−R3A and stHIV-1sv show distinct virological and immunological features. Furthermore, interferon-related gene expression might play a role in controlling primary HIV-1NL4−R3A and stHIV-1sv replication in NPMs. This result suggests NPM is a potential HIV/AIDS animal model.

High-Definition Analysis of Host Protein Stability during Human Cytomegalovirus Infection Reveals Antiviral Factors and Viral Evasion Mechanisms

SummaryHuman cytomegalovirus (HCMV) is an important pathogen with multiple immune evasion strategies, including virally facilitated degradation of host antiviral restriction factors. Here, we describe a multiplexed approach to discover proteins with innate immune function on the basis of active degradation by the proteasome or lysosome during early-phase HCMV infection. Using three orthogonal proteomic/transcriptomic screens to quantify protein degradation, with high confidence we identified 35 proteins enriched in antiviral restriction factors. A final screen employed a comprehensive panel of viral mutants to predict viral genes that target >250 human proteins. This approach revealed that helicase-like transcription factor (HLTF), a DNA helicase important in DNA repair, potently inhibits early viral gene expression but is rapidly degraded during infection. The functionally unknown HCMV protein UL145 facilitates HLTF degradation by recruiting the Cullin4 E3 ligase complex. Our approach and data will enable further identifications of innate pathways targeted by HCMV and other viruses.

Antiviral activity of Tripartite-Motif 2 protein against New World Arenaviruses

Background: New World Arenaviruses (NWA) are single-stranded bisegmented RNA viruses that cause viral hemorrhagic fevers in humans. NWAs are endemic in New World rodents and are transmitted to humans by aerosol. Host factors that alter entry and infection by Junín virus (JUNV), a NWA found in Argentina, were identified through a high throughput RNAi screen. Among them was an antiviral restriction factor, TRIM2, a member of the tripartite-motif (TRIM) family, many of which have effector roles responses to viral infections. TRIM proteins share a RING finger-B box-Coiled coil (RBCC) motif at the N-terminus but have diverse C-terminal domains; TRIM2 contains FIL/NHL domain. TRIMs are E3 ubiquitin ligases whose activity is conferred by the RING domain. Our data suggests that TRIM2 acts at the viral entry step, which would be unique for TRIM proteins. Methods & Materials: Using CRISPR/Cas9 technology we generated different TRIM2 mutant mice with different deletions. Bone Marrow-derived macrophages (BMDMs) were isolated from the mutant mice, and infected with the JUNV vaccine strain Candid 1 for 24 hours. RNA was extracted from the cells and subjected to RT-qPCR to assess the levels of infection. JUNV was also detected by immunoblotting using an anti NP monoclonal antibody. Eight old weeks mice were infected intracranially with 1 × 104 PFU of Candid 1 for 5 days and the RNA was assayed as described above. Constructs containing different domains of TRIM2 were cloned into expression vectors and transfected into 293T cells followed by JUNV infection to assess the domain involved in the antiviral activity. TRIM2-interacting proteins were identified by co-immunoprecipitation from BMDMs and JUNV-infected brains using an anti-TRIM2 monoclonal antibody. Results: We observed higher levels of JUNV infection in the TRIM2 mutant cells and mice in comparison to wild-type. We found that the FIL domain, but not the RING domain, was involved in the antiviral activity and that TRIM2 interacted with signal regulatory protein alpha (SIRPA), which had anti-NWA activity. Conclusion: TRIM2 is an anti-arenaviral host factor and might work through SIRPA by a mechanism not fully elucidated yet. This type of restriction has not been previously described for any TRIM protein.

Vpr Targets TET2 for Degradation by CRL4VprBP E3 Ligase to Sustain IL-6 Expression and Enhance HIV-1 Replication

HIV-1 expresses several accessory proteins to counteract host anti-viral restriction factors to facilitate viral replication and disease progression. One such protein, Vpr, has been implicated in affecting multiple cellular processes, but its mechanism remains elusive. Here we report that Vpr targets TET2 for polyubiquitylation by the VprBP-DDB1-CUL4-ROC1 E3 ligase and subsequent degradation. Genetic inactivation or Vpr-mediated degradation of TET2 enhances HIV-1 replication and substantially sustains expression of the pro-inflammatory cytokine interleukin-6 (IL-6). This process correlates with reduced recruitment of histone deacetylase 1 and 2 to the IL-6 promoter, thus enhancing its histone H3 acetylation level during resolution phase. Blocking IL-6 signaling reduced the ability of Vpr to enhance HIV-1 replication. We conclude that HIV-1 Vpr degrades TET2 to sustain IL-6 expression to enhance viral replication and disease progression. These results suggest that disrupting the Vpr-TET2-IL6 axis may prove clinically beneficial to reduce both viral replication and inflammation during HIV-1 infection.

IFITM3 is cardioprotective during influenza virus infection

Abstract Influenza virus annually infects more than one million individuals worldwide and causes severe pandemic outbreaks. Myocarditis and cardiac dysfunction can be observed in particularly severe cases of influenza. Humans with polymorphisms in the gene encoding interferon-induced transmembrane protein 3 (IFITM3), an antiviral restriction factor, experience severe influenza virus infections. Thus, we hypothesized that IFITM3 deficiency leads to cardiac dysfunction during influenza virus infection. We found that Ifitm3−/− mice generated in our laboratory exhibited increased morbidity, mortality, and viral lung titers compared to WT mice when infected with influenza A virus. Ifitm3−/− and WT mice showed identical cardiac function and electrical activity prior to infection as confirmed by echocardiogram and electrocardiogram. While major alterations were not observed early in infection for WT mice, Ifitm3−/− mice exhibited aberrant electrical activity, including decreased heart rate, decreased QRS interval, and irregular R/R interval. These changes were further exacerbated as infection progressed, likely contributing to the complete lethality observed in knockout mice. In contrast, WT mice exhibited only mild cardiac dysfunction late in infection that resolved following recovery. Minimal viral replication was observed in the hearts of WT and Ifitm3−/− mice. Heart sectioning and staining suggests a role for lymphocyte infiltration and tissue damage underlying the cardiac dysfunction observed in Ifitm3−/− mice. These data identify a novel cardioprotective role for IFITM3 during influenza virus infection and establish Ifitm3−/− mice as a tool for studying molecular events leading to virus-induced cardiac dysfunction.

Differential Evolution of Antiretroviral Restriction Factors in Pteropid Bats as Revealed by APOBEC3 Gene Complexity.

Bats have attracted attention in recent years as important reservoirs of viruses deadly to humans and other mammals. These infections are typically nonpathogenic in bats raising questions about innate immune differences that might exist between bats and other mammals. The APOBEC3 gene family encodes antiviral DNA cytosine deaminases with important roles in the suppression of diverse viruses and genomic parasites. Here, we characterize pteropid APOBEC3 genes and show that species within the genus Pteropus possess the largest and most diverse array of APOBEC3 genes identified in any mammal reported to date. Several bat APOBEC3 proteins are antiviral as demonstrated by restriction of retroviral infectivity using HIV-1 as a model, and recombinant A3Z1 subtypes possess strong DNA deaminase activity. These genes represent the first group of antiviral restriction factors identified in bats with extensive diversification relative to homologues in other mammals.

Identification of a Conserved Interface of Human Immunodeficiency Virus Type 1 and Feline Immunodeficiency Virus Vifs with Cullin 5.

Members of the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3 [A3]) family of DNA cytidine deaminases are intrinsic restriction factors against retroviruses. In felids such as the domestic cat (Felis catus), the A3 genes encode the A3Z2, A3Z3, and A3Z2Z3 antiviral cytidine deaminases. Only A3Z3 and A3Z2Z3 inhibit viral infectivity factor (Vif)-deficient feline immunodeficiency virus (FIV). The FIV Vif protein interacts with Cullin (CUL), Elongin B (ELOB), and Elongin C (ELOC) to form an E3 ubiquitination complex to induce the degradation of feline A3s. However, the functional domains in FIV Vif for the interaction with Cullin are poorly understood. Here, we found that the expression of dominant negative CUL5 prevented the degradation of feline A3s by FIV Vif, while dominant negative CUL2 had no influence on the degradation of A3. In coimmunoprecipitation assays, FIV Vif bound to CUL5 but not CUL2. To identify the CUL5 interaction site in FIV Vif, the conserved amino acids from positions 47 to 160 of FIV Vif were mutated, but these mutations did not impair the binding of Vif to CUL5. By focusing on a potential zinc-binding motif (K175-C161-C184-C187) of FIV Vif, we found a conserved hydrophobic region (174IR175) that is important for the CUL5 interaction. Mutation of this region also impaired the FIV Vif-induced degradation of feline A3s. Based on a structural model of the FIV Vif-CUL5 interaction, the 52LW53 region in CUL5 was identified as mediating binding to FIV Vif. By comparing our results to the human immunodeficiency virus type 1 (HIV-1) Vif-CUL5 interaction surface (120IR121, a hydrophobic region that is localized in the zinc-binding motif), we suggest that the CUL5 interaction surface in the diverse HIV-1 and FIV Vifs is evolutionarily conserved, indicating a strong structural constraint. However, the FIV Vif-CUL5 interaction is zinc independent, which contrasts with the zinc dependence of HIV-1 Vif.IMPORTANCE Feline immunodeficiency virus (FIV), which is similar to human immunodeficiency virus type 1 (HIV-1), replicates in its natural host in T cells and macrophages that express the antiviral restriction factor APOBEC3 (A3). To escape A3s, FIV and HIV induce the degradation of these proteins by building a ubiquitin ligase complex using the viral protein Vif to connect to cellular proteins, including Cullin 5. Here, we identified the protein residues that regulate this interaction in FIV Vif and Cullin 5. While our structural model suggests that the diverse FIV and HIV-1 Vifs use conserved residues for Cullin 5 binding, FIV Vif binds Cullin 5 independently of zinc, in contrast to HIV-1 Vif.

Human Intestinal Epithelial Cells Release Antiviral Factors That Inhibit HIV Infection of Macrophages.

As a rich source of CD4+ T cells and macrophages, the gastrointestinal (GI) tract is a major target site for HIV infection. The interplay between GI-resident macrophages and intestinal epithelial cells (IECs) constitutes an important element of GI innate immunity against pathogens. In this study, we investigated whether human IECs have the ability to produce antiviral factors that can inhibit HIV infection of macrophages. We demonstrated that IECs possess functional toll-like receptor 3 (TLR3), the activation of which resulted in induction of key interferon (IFN) regulatory factors (IRF3 and IRF7), IFN-β, IFN-λ, and CC chemokines (MIP-1α, MIP-1β, RANTES), the ligands of HIV entry co-receptor CCR5. In addition, TLR3-activated IECs release exosomes that contained the anti-HIV factors, including IFN-stimulated genes (ISGs: ISG15, ISG56, MxB, OAS-1, GBP5, and Viperin) and HIV restriction miRNAs (miRNA-17, miRNA-20, miRNA-28, miRNA-29 family members, and miRNA-125b). Importantly, treatment of macrophages with supernatant (SN) from the activated IEC cultures inhibited HIV replication. Further studies showed that IEC SN could also induce the expression of antiviral ISGs and cellular HIV restriction factors (Tetherin and APOBEC3G/3F) in HIV-infected macrophages. These findings indicated that IECs might act as an important element in GI innate immunity against HIV infection/replication.

SMARCA2-regulated host cell factors are required for MxA restriction of influenza A viruses

The human interferon (IFN)-induced MxA protein is a key antiviral host restriction factor exhibiting broad antiviral activity against many RNA viruses, including highly pathogenic avian influenza A viruses (IAV) of the H5N1 and H7N7 subtype. To date the mechanism for how MxA exerts its antiviral activity is unclear, however, additional cellular factors are believed to be essential for this activity. To identify MxA cofactors we performed a genome-wide siRNA-based screen in human airway epithelial cells (A549) constitutively expressing MxA using an H5N1 reporter virus. These data were complemented with a proteomic screen to identify MxA-interacting proteins. The combined data identified SMARCA2, the ATPase subunit of the BAF chromatin remodeling complex, as a crucial factor required for the antiviral activity of MxA against IAV. Intriguingly, our data demonstrate that although SMARCA2 is essential for expression of some IFN-stimulated genes (ISGs), and the establishment of an antiviral state, it is not required for expression of MxA, suggesting an indirect effect on MxA activity. Transcriptome analysis of SMARCA2-depleted A549-MxA cells identified a small set of SMARCA2-regulated factors required for activity of MxA, in particular IFITM2 and IGFBP3. These findings reveal that several virus-inducible factors work in concert to enable MxA restriction of IAV.

Differentiation-dependent antiviral capacities of amphibian (Xenopus laevis) macrophages

Infections by ranaviruses such as Frog virus 3 (Fv3), are significantly contributing to worldwide amphibian population declines. Notably, amphibian macrophages (Mφs) are important to both the Fv3 infection strategies and the immune defense against this pathogen. However, the mechanisms underlying amphibian Mφ Fv3 susceptibility and resistance remain unknown. Mφ differentiation is mediated by signaling through the colony-stimulating factor-1 receptor (CSF-1R) which is now known to be bound not only by CSF-1, but also by the unrelated interleukin-34 (IL-34) cytokine. Pertinently, amphibian (Xenopus laevis) Mφs differentiated by CSF-1 and IL-34 are highly susceptible and resistant to Fv3, respectively. Accordingly, in the present work, we elucidate the facets of this Mφ Fv3 susceptibility and resistance. Because cellular resistance to viral replication is marked by expression of antiviral restriction factors, it was intuitive to find that IL-34-Mφs possess significantly greater mRNA levels of select restriction factor genes than CSF-1-Mφs. Xenopodinae amphibians have highly expanded repertoires of antiviral interferon (IFN) cytokine gene families, and our results indicated that in comparison with the X. laevis CSF-1-Mφs, the IL-34-Mφs express substantially greater transcripts of representative IFN genes, belonging to distinct gene family clades, as well as their cognate receptor genes. Finally, we demonstrate that IL-34-Mφ-conditioned supernatants confer IFN-mediated anti-Fv3 protection to the virally susceptible X. laevis kidney (A6) cell line. Together, this work underlines the differentiation pathways leading to Fv3-susceptible and -resistant amphibian Mφ populations and defines the molecular mechanisms responsible for these differences.

Regulation of CD4 Receptor and HIV-1 Entry by MicroRNAs-221 and -222 during Differentiation of THP-1 Cells.

Human immunodeficiency virus type-1 (HIV-1) infection of monocyte/macrophages is modulated by the levels of entry receptors cluster of differentiation 4 (CD4) and C-C chemokine receptor type 5 (CCR5), as well as by host antiviral restriction factors, which mediate several post-entry blocks. We recently identified two microRNAs, miR-221 and miR-222, which limit HIV-1 entry during infection of monocyte-derived macrophages (MDMs) by down-regulating CD4 expression. Interestingly, CD4 is also down-regulated during the differentiation of monocytes into macrophages. In this study, we compared microRNA expression profiles in primary monocytes and macrophages by RNAseq and found that miR-221/miR-222 are enhanced in macrophages. We took advantage of the monocytic THP-1 cell line that, once differentiated, is poorly susceptible to HIV-1. Accordingly, we found that CD4 levels are very low in THP-1 differentiated cells and that this down-regulation of the virus receptor is the result of miR-221/miR-222 up-regulation during differentiation. We thus established a THP-1 cell line stably expressing a modified CD4 (THP-1-CD4R) that is not modulated by miR-221/miR-222. We show that in contrast to parental THP-1, this line is productively infected by HIV-1 following differentiation, sustaining efficient HIV-1 CD4-dependent replication and spread. This new THP-1-CD4R cell line represents a useful tool for the study of HIV-1-macrophage interactions particularly in contexts where spreading of viral infection is necessary.

Interferons and beyond: Induction of antiretroviral restriction factors.

Antiviral restriction factors are structurally and functionally diverse cellular proteins that play a key role in the first line of defense against viral pathogens. Although many cell types constitutively express restriction factors at low levels, their induction in response to viral exposure and replication is often required for potent control and repulse of the invading pathogens. It is well established that type I IFNs efficiently induce antiviral restriction factors. Accumulating evidence suggests that other types of IFN, as well as specific cytokines, such as IL-27, and other activators of the cell are also capable of enhancing the expression of restriction factors and hence to establish an antiviral cellular state. Agents that efficiently induce restriction factors, increase their activity, and/or render them resistant against viral antagonists without causing general inflammation and significant side effects hold some promise for novel therapeutic or preventive strategies. In the present review, we summarize some of the current knowledge on the induction of antiretroviral restriction factors and perspectives for therapeutic application.