Spatiotemporal binding of cyclophilin A and CPSF6 to capsid regulates HIV-1 nuclear entry and integration.

Human immunodeficiency virus type 1 (HIV-1) capsid, which is the target of the antiviral lenacapavir, protects the viral genome and binds multiple host proteins to influence intracellular trafficking, nuclear import, and integration. Previously, we showed that capsid binding to cleavage and polyadenylation specificity factor 6 (CPSF6) in the cytoplasm is competitively inhibited by cyclophilin A (CypA) binding and regulates capsid trafficking, nuclear import, and infection. Here we determined that a capsid mutant with increased CypA binding affinity had significantly reduced nuclear entry and mislocalized integration. However, disruption of CypA binding to the mutant capsid restored nuclear entry, integration, and infection in a CPSF6-dependent manner. Furthermore, relocalization of CypA expression from the cell cytoplasm to the nucleus failed to restore mutant HIV-1 infection. Our results clarify that sequential binding of CypA and CPSF6 to HIV-1 capsid is required for optimal nuclear entry and integration targeting, informing antiretroviral therapies that contain lenacapavir.

Virus specificity and nucleoporin requirements for MX2 activity are affected by GTPase function and capsid-CypA interactions.

Human myxovirus resistance 2 (MX2/MXB) is an interferon-induced GTPase that inhibits human immunodeficiency virus-1 (HIV-1) infection by preventing nuclear import of the viral preintegration complex. The HIV-1 capsid (CA) is the major viral determinant for sensitivity to MX2, and complex interactions between MX2, CA, nucleoporins (Nups), cyclophilin A (CypA), and other cellular proteins influence the outcome of viral infection. To explore the interactions between MX2, the viral CA, and CypA, we utilized a CRISPR-Cas9/AAV approach to generate CypA knock-out cell lines as well as cells that express CypA from its endogenous locus, but with specific point mutations that would abrogate CA binding but should not affect enzymatic activity or cellular function. We found that infection of CypA knock-out and point mutant cell lines with wild-type HIV-1 and CA mutants recapitulated the phenotypes observed upon cyclosporine A (CsA) addition, indicating that effects of CsA treatment are the direct result of blocking CA-CypA interactions and are therefore independent from potential interactions between CypA and MX2 or other cellular proteins. Notably, abrogation of GTP hydrolysis by MX2 conferred enhanced antiviral activity when CA-CypA interactions were abolished, and this effect was not mediated by the CA-binding residues in the GTPase domain, or by phosphorylation of MX2 at position T151. We additionally found that elimination of GTPase activity also altered the Nup requirements for MX2 activity. Our data demonstrate that the antiviral activity of MX2 is affected by CypA-CA interactions in a virus-specific and GTPase activity-dependent manner. These findings further highlight the importance of the GTPase domain of MX2 in regulation of substrate specificity and interaction with nucleocytoplasmic trafficking pathways.

Effect of osteopathic manipulative treatment and Bio-Electro-Magnetic Energy Regulation (BEMER) therapy on generalized musculoskeletal neck pain in adults.

CONTEXT: General neck pain is a prevalent complaint made by patients to their physicians and is often of a suspected musculoskeletal origin. Osteopathic manipulative treatment (OMT) is a form of manual therapy utilized by osteopathic physicians and some allopathic physicians to treat a broad variety of musculoskeletal ailments, including neck pain. Bio-Electro-Magnetic Energy Regulation (BEMER) is an emerging therapeutic modality that deploys a biorhythmically defined stimulus through a pulsed electromagnetic field and has been shown to reduce musculoskeletal pain. Studies on these treatments have independently yielded promising results. Therefore, it is possible that the utility of OMT and BEMER can produce an additive improvement in the treatment of neck pain. OBJECTIVES: The objectives of this study are to investigate the individual and combined effects of OMT and BEMER therapy on neck pain in adults. METHODS: Adults with nonspecific neck pain were recruited for the study. A total of 44 participants met the study inclusion criteria and were randomized into one of four study groups: OMT-only, BEMER-only, OMT+BEMER, or CONTROL (light touch and sham). Forty subjects completed the study, and data for 38 participants were included in our analyses. An OMT and BEMER protocol were specifically designed for this study under the guidance of a licensed osteopathic physician. Participants underwent intervention for a duration of 3 weeks. Data were obtained through baseline and postintervention assessments utilizing three surveys: Neck Disability Index (NDI), Visual Analog Scale (VAS), and Short Form 12-item Health Survey (SF-12, divided into Mental and Physical). One-way analysis of variance (ANOVA) analysis was performed retrospectively on pre- and postintervention absolute means between study groups. Significance was set at p<0.05. RESULTS: One-way ANOVA analysis demonstrated a statistically significant difference in pre- vs. postintervention mean scores between BEMER and CONTROL (p<0.05), BEMER compared to OMT (p<0.005), and BEMER compared to BEMER+OMT (p<0.05), in the NDI. The OMT+BEMER group reported an average reduction in pain on the VAS of 21.3 (±29.3) points, or a 65.0 % reduction of pain. A similarly substantial decrease in pain was reported in the BEMER study group, which showed a 46.2 % reduction in pain from baseline. The OMT and CONTROL study groups only reported a 2.9 and 23.9 % decrease, respectively. The BEMER and OMT+BEMER study groups also demonstrated a reduction in subjective reporting on the NDI, by 53.8 and 26.3 %, respectively. The BEMER study group also achieved the most substantial improvement in mental and physical well-being as reported by the SF-12. CONCLUSIONS: Study arms that incorporated BEMER yielded improvements on the NDI, VAS, and SF-12, indicating benefits to BEMER regarding improved overall functionality in routine daily activities as well as a reduction in nonspecific neck pain. Perceived pain, as demonstrated on the VAS, was seemingly improved in an additive fashion from the BEMER group to the OMT+BEMER group, although the results did not achieve statistical significance. Further study with greater participation could provide additional insight.

Virus specificity and nucleoporin requirements for MX2 activity are affected by GTPase function and capsid-CypA interactions.

Human myxovirus resistance 2 (MX2/MXB) is an interferon-induced GTPase that inhibits human immunodeficiency virus-1 (HIV-1) infection by preventing nuclear import of the viral preintegration complex. The HIV-1 capsid (CA) is the major viral determinant for sensitivity to MX2, and complex interactions between MX2, CA, nucleoporins (Nups), cyclophilin A (CypA), and other cellular proteins influence the outcome of viral infection. To explore the interactions between MX2, the viral CA, and CypA, we utilized a CRISPR-Cas9/AAV approach to generate CypA knock-out cell lines as well as cells that express CypA from its endogenous locus, but with specific point mutations that would abrogate CA binding but should not affect enzymatic activity or cellular function. We found that infection of CypA knock-out and point mutant cell lines with wild-type HIV-1 and CA mutants recapitulated the phenotypes observed upon cyclosporine A (CsA) addition, indicating that effects of CsA treatment are the direct result of blocking CA-CypA interactions and are therefore independent from potential interactions between CypA and MX2 or other cellular proteins. Notably, abrogation of GTP hydrolysis by MX2 conferred enhanced antiviral activity when CA-CypA interactions were abolished, and this effect was not mediated by the CA-binding residues in the GTPase domain, or by phosphorylation of MX2 at position T151. We additionally found that elimination of GTPase activity also altered the Nup requirements for MX2 activity. Our data demonstrate that the antiviral activity of MX2 is affected by CypA-CA interactions in a virus-specific and GTPase activity-dependent manner. These findings further highlight the importance of the GTPase domain of MX2 in regulation of substrate specificity and interaction with nucleocytoplasmic trafficking pathways.

Insights into the role of HIV-1 Vpu in modulation of NF-ĸB signaling pathways.

HIV-1 inhibits the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) to prevent the induction of a proinflammatory state but also activates the NF-κB pathway to promote viral transcription. Thus, optimal regulation of this pathway is important for the viral life cycle. In recent work, Pickering et al. (3) demonstrate that HIV-1 viral protein U has contrasting effects on the two distinct paralogs of ß-transducin repeat-containing protein (ß-TrCP1 and ß-TrCP2) and that this interaction has important implications for the regulation of both the canonical and non-canonical NF-κB pathways. Additionally, the authors identified the viral requirements for the dysregulation of ß-TrCP. In this commentary, we discuss how these findings further our understanding of how the NF-κB pathway functions during viral infection.

Genetic Differences between 129S Substrains Affect Antiretroviral Immune Responses.

Inbred mouse lines vary in their ability to mount protective antiretroviral immune responses, and even closely related strains can exhibit opposing phenotypes upon retroviral infection. Here, we found that 129S mice inherit a previously unknown mechanism for the production of anti-murine leukemia virus (MLV) antibodies and control of infection. The resistant phenotype in 129S1 mice is controlled by two dominant loci that are independent from known MLV resistance genes. We also show that production of anti-MLV antibodies in 129S7 mice, but not 129S1 mice, is independent of interferon gamma signaling. Thus, our data indicate that 129S mice inherit an unknown mechanism for control of MLV infection and demonstrate that there is genetic variability in 129S substrains that affects their ability to mount antiviral immune responses. IMPORTANCE Understanding the genetic basis for production of protective antiviral immune responses is crucial for the development of novel vaccines and adjuvants. Additionally, characterizing the genetic and phenotypic variability in inbred mice has implications for the selection of strains for targeted mutagenesis, choice of controls, and for broader understanding of the requirements for protective immunity.

Genetic Control of Neonatal Immune Tolerance to an Exogenous Retrovirus.

Viruses, including retroviruses, can be passed from mothers to their progeny during birth and breastfeeding. It is assumed that newborns may develop immune tolerance to milk-transmitted pathogens similarly to food antigens. I/LnJ mice are uniquely resistant to retroviruses acquired as newborns or as adults as they produce virus-neutralizing antibodies (Abs). A loss-of-function allele of H2-Ob (Ob), originally mapped within the virus infectivity controller 1 (vic1) locus, is responsible for production of antiretrovirus Abs in I/LnJ mice. Importantly, Ob-deficient and vic1 I/LnJ congenic mice on other genetic backgrounds produce antivirus Abs when infected as adults, but not as newborns. We report here that I/LnJ mice carry an additional genetic locus, virus infectivity controller 2 (vic2), that abrogates neonatal immune tolerance to retroviruses. Further genetic analysis mapped the vic2 locus to the telomeric end of chromosome 15. Identification of the vic2 gene and understanding of the related signaling pathways would make blocking of neonatal immune tolerance to retroviruses an achievable goal.IMPORTANCE This work describes a previously unknown genetic mechanism that allows neonates to respond to infections as efficiently as adults.

Inhibition of spumavirus gene expression by PHF11.

The foamy viruses (FV) or spumaviruses are an ancient subfamily of retroviruses that infect a variety of vertebrates. FVs are endemic, but apparently apathogenic, in modern non-human primates. Like other retroviruses, FV replication is inhibited by type-I interferon (IFN). In a previously described screen of IFN-stimulated genes (ISGs), we identified the macaque PHD finger domain protein-11 (PHF11) as an inhibitor of prototype foamy virus (PFV) replication. Here, we show that human and macaque PHF11 inhibit the replication of multiple spumaviruses, but are inactive against several orthoretroviruses. Analysis of other mammalian PHF11 proteins revealed that antiviral activity is host species dependent. Using multiple reporter viruses and cell lines, we determined that PHF11 specifically inhibits a step in the replication cycle that is unique to FVs, namely basal transcription from the FV internal promoter (IP). In so doing, PHF11 prevents expression of the viral transactivator Tas and subsequent activation of the viral LTR promoter. These studies reveal a previously unreported inhibitory mechanism in mammalian cells, that targets a family of ancient viruses and may promote viral latency.

Mapping Viral Susceptibility Loci in Mice.

Genetic alleles that contribute to enhanced susceptibility or resistance to viral infections and virally induced diseases have often been first identified in mice before humans due to the significant advantages of the murine system for genetic studies. Herein we review multiple discoveries that have revealed significant insights into virus-host interactions, all made using genetic mapping tools in mice. Factors that have been identified include innate and adaptive immunity genes that contribute to host defense against pathogenic viruses such as herpes viruses, flaviviruses, retroviruses, and coronaviruses. Understanding the genetic mechanisms that affect infectious disease outcomes will aid the development of personalized treatment and preventive strategies for pathogenic infections.

Derivation of simian tropic HIV-1 infectious clone reveals virus adaptation to a new host.

To replicate in a new host, lentiviruses must adapt to exploit required host factors and evade species-specific antiviral proteins. Understanding how host protein variation drives lentivirus adaptation allowed us to expand the host range of HIV-1 to pigtail macaques. We have previously derived a viral swarm (in the blood of infected animals) that can cause AIDS in this new host. To further exploit this reagent, we generated infectious molecular clones (IMCs) from the viral swarm. We identified clones with high replicative capacity in pigtail peripheral blood mononuclear cells (PBMC) in vitro and used in vivo replication to select an individual IMC, named stHIV-A19 (for simian tropic HIV-1 clone A19), which recapitulated the phenotype obtained with the viral swarm. Adaptation of HIV-1 in macaques led to the acquisition of amino acid changes in viral proteins, such as capsid (CA), that are rarely seen in HIV-1-infected humans. Using stHIV-A19, we show that these CA changes confer a partial resistance to the host cell inhibitor Mx2 from pigtail macaques, but that complete resistance is associated with a fitness defect. Adaptation of HIV-1 to a new host will lead to a more accurate animal model and a better understanding of virus-host interactions.

Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2.

HIV-1 accesses the nuclear DNA of interphase cells via a poorly defined process involving functional interactions between the capsid protein (CA) and nucleoporins (Nups). Here, we show that HIV-1 CA can bind multiple Nups, and that both natural and manipulated variation in Nup levels impacts HIV-1 infection in a manner that is strikingly dependent on cell-type, cell-cycle, and cyclophilin A (CypA). We also show that Nups mediate the function of the antiviral protein MX2, and that MX2 can variably inhibit non-viral NLS function. Remarkably, both enhancing and inhibiting effects of cyclophilin A and MX2 on various HIV-1 CA mutants could be induced or abolished by manipulating levels of the Nup93 subcomplex, the Nup62 subcomplex, NUP88, NUP214, RANBP2, or NUP153. Our findings suggest that several Nup-dependent 'pathways' are variably exploited by HIV-1 to target host DNA in a cell-type, cell-cycle, CypA and CA-sequence dependent manner, and are differentially inhibited by MX2.

A Single Locus Controls Interferon Gamma-Independent Antiretroviral Neutralizing Antibody Responses.

An essential step in the development of effective antiviral humoral responses is cytokine-triggered class switch recombination resulting in the production of antibodies of a specific isotype. Most viral and parasitic infections in mice induce predominantly IgG2a-specific antibody responses that are stimulated by interferon gamma (IFN-γ). However, in some mice deficient in IFN-γ, class switching to IgG2a antibodies is relatively unaffected, indicating that another signal(s) can be generated upon viral or parasitic infections that trigger this response. Here, we found that a single recessive locus, provisionally called IFN-γ-independent IgG2a (Igii), confers the ability to produce IFN-γ-independent production of IgG2a antibodies upon retroviral infection. The Igii locus was mapped to chromosome 9 and was found to function in the radiation-resistant compartment. Thus, our data implicate nonhematopoietic cells in activation of antiviral antibody responses in the absence of IFN-γ.IMPORTANCE Understanding the signals that stimulate antibody production and class switch recombination to specific antibody isotypes is crucial for the development of novel vaccines and adjuvants. While an interferon gamma-mediated switch to the IgG2a isotype upon viral infection in mice has been well established, this investigation reveals a noncanonical, interferon gamma-independent pathway for antiretroviral antibody production and IgG2a class switch recombination that is controlled by a single recessive locus. Furthermore, this study indicates that the radiation-resistant compartment can direct antiviral antibody responses, suggesting that detection of infection by nonhematopoietic cells is involved is stimulating adaptive immunity.

Neutralizing Antibody Responses to Viral Infections Are Linked to the Non-classical MHC Class II Gene H2-Ob.

Select humans and animals control persistent viral infections via adaptive immune responses that include production of neutralizing antibodies. The precise genetic basis for the control remains enigmatic. Here, we report positional cloning of the gene responsible for production of retrovirus-neutralizing antibodies in mice of the I/LnJ strain. It encodes the beta subunit of the non-classical major histocompatibility complex class II (MHC-II)-like molecule H2-O, a negative regulator of antigen presentation. The recessive and functionally null I/LnJ H2-Ob allele supported the production of virus-neutralizing antibodies independently of the classical MHC haplotype. Subsequent bioinformatics and functional analyses of the human H2-Ob homolog, HLA-DOB, revealed both loss- and gain-of-function alleles, which could affect the ability of their carriers to control infections with human hepatitis B (HBV) and C (HCV) viruses. Thus, understanding of the previously unappreciated role of H2-O (HLA-DO) in immunity to infections may suggest new approaches in achieving neutralizing immunity to viruses.

Identification of Interferon-Stimulated Genes with Antiretroviral Activity.

Interferons (IFNs) exert their anti-viral effects by inducing the expression of hundreds of IFN-stimulated genes (ISGs). The activity of known ISGs is insufficient to account for the antiretroviral effects of IFN, suggesting that ISGs with antiretroviral activity are yet to be described. We constructed an arrayed library of ISGs from rhesus macaques and tested the ability of hundreds of individual macaque and human ISGs to inhibit early and late replication steps for 11 members of the retroviridae from various host species. These screens uncovered numerous ISGs with antiretroviral activity at both the early and late stages of virus replication. Detailed analyses of two antiretroviral ISGs indicate that indoleamine 2,3-dioxygenase 1 (IDO1) can inhibit retroviral replication by metabolite depletion while tripartite motif-56 (TRIM56) accentuates ISG induction by IFNα and inhibits the expression of late HIV-1 genes. Overall, these studies reveal numerous host proteins that mediate the antiretroviral activity of IFNs.

Host and viral determinants of Mx2 antiretroviral activity.

Myxovirus resistance 2 (Mx2/MxB) has recently been uncovered as an effector of the anti-HIV-1 activity of type I interferons (IFNs) that inhibits HIV-1 at an early stage postinfection, after reverse transcription but prior to proviral integration into host DNA. The mechanistic details of Mx2 antiviral activity are not yet understood, but a few substitutions in the HIV-1 capsid have been shown to confer resistance to Mx2. Through a combination of in vitro evolution and unbiased mutagenesis, we further map the determinants of sensitivity to Mx2 and reveal that multiple capsid (CA) surfaces define sensitivity to Mx2. Intriguingly, we reveal an unanticipated sensitivity determinant within the C-terminal domain of capsid. We also report that Mx2s derived from multiple primate species share the capacity to potently inhibit HIV-1, whereas selected nonprimate orthologs have no such activity. Like TRIM5α, another CA targeting antiretroviral protein, primate Mx2s exhibit species-dependent variation in antiviral specificity against at least one extant virus and multiple HIV-1 capsid mutants. Using a combination of chimeric Mx2 proteins and evolution-guided approaches, we reveal that a single residue close to the N terminus that has evolved under positive selection can determine antiviral specificity. Thus, the variable N-terminal region can define the spectrum of viruses inhibited by Mx2. Importance: Type I interferons (IFNs) inhibit the replication of most mammalian viruses. IFN stimulation upregulates hundreds of different IFN-stimulated genes (ISGs), but it is often unclear which ISGs are responsible for inhibition of a given virus. Recently, Mx2 was identified as an ISG that contributes to the inhibition of HIV-1 replication by type I IFN. Thus, Mx2 might inhibit HIV-1 replication in patients, and this inhibitory action might have therapeutic potential. The mechanistic details of how Mx2 inhibits HIV-1 are currently unclear, but the HIV-1 capsid protein is the likely viral target. Here, we determine the regions of capsid that specify sensitivity to Mx2. We demonstrate that Mx2 from multiple primates can inhibit HIV-1, whereas Mx2 from other mammals (dogs and sheep) cannot. We also show that primate variants of Mx2 differ in the spectrum of lentiviruses they inhibit and that a single residue in Mx2 can determine this antiviral specificity.

MX2 is an interferon-induced inhibitor of HIV-1 infection.

HIV-1 replication can be inhibited by type I interferon (IFN), and the expression of a number of gene products with anti-HIV-1 activity is induced by type I IFN. However, none of the known antiretroviral proteins can account for the ability of type I IFN to inhibit early, preintegration phases of the HIV-1 replication cycle in human cells. Here, by comparing gene expression profiles in cell lines that differ in their ability to support the inhibitory action of IFN-α at early steps of the HIV-1 replication cycle, we identify myxovirus resistance 2 (MX2) as an interferon-induced inhibitor of HIV-1 infection. Expression of MX2 reduces permissiveness to a variety of lentiviruses, whereas depletion of MX2 using RNA interference reduces the anti-HIV-1 potency of IFN-α. HIV-1 reverse transcription proceeds normally in MX2-expressing cells, but 2-long terminal repeat circular forms of HIV-1 DNA are less abundant, suggesting that MX2 inhibits HIV-1 nuclear import, or destabilizes nuclear HIV-1 DNA. Consistent with this notion, mutations in the HIV-1 capsid protein that are known, or suspected, to alter the nuclear import pathways used by HIV-1 confer resistance to MX2, whereas preventing cell division increases MX2 potency. Overall, these findings indicate that MX2 is an effector of the anti-HIV-1 activity of type-I IFN, and suggest that MX2 inhibits HIV-1 infection by inhibiting capsid-dependent nuclear import of subviral complexes.

Nucleolar trafficking of the mouse mammary tumor virus gag protein induced by interaction with ribosomal protein L9.

The mouse mammary tumor virus (MMTV) Gag protein directs the assembly in the cytoplasm of immature viral capsids, which subsequently bud from the plasma membranes of infected cells. MMTV Gag localizes to discrete cytoplasmic foci in mouse mammary epithelial cells, consistent with the formation of cytosolic capsids. Unexpectedly, we also observed an accumulation of Gag in the nucleoli of infected cells derived from mammary gland tumors. To detect Gag-interacting proteins that might influence its subcellular localization, a yeast two-hybrid screen was performed. Ribosomal protein L9 (RPL9 or L9), an essential component of the large ribosomal subunit and a putative tumor suppressor, was identified as a Gag binding partner. Overexpression of L9 in cells expressing the MMTV(C3H) provirus resulted in specific, robust accumulation of Gag in nucleoli. Förster resonance energy transfer (FRET) and coimmunoprecipitation analyses demonstrated that Gag and L9 interact within the nucleolus, and the CA domain was the major site of interaction. In addition, the isolated NC domain of Gag localized to the nucleolus, suggesting that it contains a nucleolar localization signal (NoLS). To determine whether L9 plays a role in virus assembly, small interfering RNA (siRNA)-mediated knockdown was performed. Although Gag expression was not reduced with L9 knockdown, virus production was significantly impaired. Thus, our data support the hypothesis that efficient MMTV particle assembly is dependent upon the interaction of Gag and L9 in the nucleoli of infected cells.

Realities of virus sensing.

Detection of viral infections by the innate immune system is essential for the subsequent upregulation of host protective responses. This review will focus on the relevance of innate immune pathways in the induction of protective adaptive immune responses and will discuss the discrepancies often found between in vitro and in vivo investigations.

Successful transmission of a retrovirus depends on the commensal microbiota.

To establish chronic infections, viruses must develop strategies to evade the host's immune responses. Many retroviruses, including mouse mammary tumor virus (MMTV), are transmitted most efficiently through mucosal surfaces rich in microbiota. We found that MMTV, when ingested by newborn mice, stimulates a state of unresponsiveness toward viral antigens. This process required the intestinal microbiota, as antibiotic-treated mice or germ-free mice did not transmit infectious virus to their offspring. MMTV-bound bacterial lipopolysaccharide triggered Toll-like receptor 4 and subsequent interleukin-6 (IL-6)-dependent induction of the inhibitory cytokine IL-10. Thus, MMTV has evolved to rely on the interaction with the microbiota to induce an immune evasion pathway. Together, these findings reveal the fundamental importance of commensal microbiota in viral infections.