Apobec 3G Efficiently Reduces Infectivity of the Human Exogenous Gammaretrovirus XMRV

The human exogenous gammaretrovirus XMRV is thought to be implicated in prostate cancer and chronic fatigue syndrome. Besides pressing epidemiologic questions, the elucidation of the tissue and cell tropism of the virus, as well as its sensitivity to retroviral restriction factors is of fundamental importance. The Apobec3 (A3) proteins, a family of cytidine deaminases, are one important group of host proteins that control primary infection and efficient viral spread.Here we demonstrate that XMRV is resistant to human Apobec 3B, 3C and 3F, while being highly susceptible to the human A3G protein, a factor which is known to confer antiviral activity against most retroviruses. We show that XMRV as well as MoMLV virions package Apobec proteins independent of their specific restriction activity. hA3G was found to be a potent inhibitor of XMRV as well as of MoMLV infectivity. In contrast to MoMLV, XMRV infection can also be partially reduced by low concentrations of mA3. Interestingly, established prostate cancer cell lines, which are highly susceptible to XMRV infection, do not or only weakly express hA3G.Our findings confirm and extend recently published data that show restriction of XMRV infection by hA3G. The results will be of value to explore which cells are infected with XMRV and efficiently support viral spread in vivo. Furthermore, the observation that XMRV infection can be reduced by mA3 is of interest with regard to the current natural reservoir of XMRV infection

Persistent prion infection disturbs the function of Oct-1, resulting in the down-regulation of murine interferon regulatory factor-3

As a prompt response against invasion of various viruses, interferon regulatory factor-3 (IRF-3) is initially phosphorylated to become activated and upregulates mainly Type I Interferons (IFN-I) in most cell types. We previously reported that IRF-3-dependent host innate immune responses partially interfere in infection of prions. Here, we found that stable infection of prion suppressed IRF-3 gene-expression. The decreased promoter activity of IRF-3 was significantly restored along with treatment of anti-prion drugs in the prion-infected cells, suggesting that infection of prion directly influence the regulation of IRF-3 transcription. We further investigated promoter activity of 5\u27- flanking region of murine IRF-3 using a luciferase reporter system and found that the nucleotides -119 to -1 were indispensable for the promoter activity. Within this region, mutations in the Oct-1 binding site significantly reduced the promoter activity and chromatin immunoprecipitation (ChIP) assay revealed that Oct-1 indeed binds to the region. In addition, overexpression of Oct-1 increased the promoter activity of IRF-3. Intriguingly, Oct-1 protein was significantly reduced in prion-infected cells and mice brains compared with uninfected groups. Taken together, we concluded that prion infection could interfere in the function of Oct-1, resulting in the down-regulation of IRF-3

Demonstration of a Novel HIV-1 Restriction Phenotype from a Human T Cell Line

Although retroviruses may invade host cells, a productive infection can be established only after the virus counteracts inhibition from different types of host restriction factors. Fv1, APOBEC3G/F, TRIM5alpha, ZAP, and CD317 inhibit the replication of different retroviruses by interfering with viral uncoating, reverse transcription, nuclear import, RNA stability, and release. In humans, although APOBEC3G/3F and CD317 block HIV-1 replication, their antiviral activities are neutralized by viral proteins Vif and Vpu. So far, no human gene has been found to effectively block wild type HIV-1 replication under natural condition. Thus, identification of such a gene product would be of great medical importance for the development of HIV therapies.In this study, we discovered a new type of host restriction against the wild type HIV-1 from a CD4/CXCR4 double-positive human T cell line. We identified a CEM-derived cell line (CEM.NKR) that is highly resistant to productive HIV-1 infection. Viral production was reduced by at least 1000-fold when compared to the other permissive human T cell lines such as H9, A3.01, and CEM-T4. Importantly, this resistance was evident at extremely high multiplicity of infection. Further analyses demonstrated that HIV-1 could finish the first round of replication in CEM.NKR cells, but the released virions were poorly infectious. These virions could enter the target cells, but failed to initiate reverse transcription. Notably, this restriction phenotype was also present in CEM.NKR and 293T heterokaryons.These results clearly indicate that CEM.NKR cells express a HIV inhibitory gene(s). Further characterization of this novel gene product(s) will reveal a new antiretroviral mechanism that directly inactivates wild type HIV-1

Effects of prostratin on Cyclin T1/P-TEFb function and the gene expression profile in primary resting CD4(+ )T cells

BACKGROUND: The latent reservoir of human immunodeficiency virus type 1 (HIV-1) in resting CD4(+ )T cells is a major obstacle to the clearance of infection by highly active antiretroviral therapy (HAART). Recent studies have focused on searches for adjuvant therapies to activate this reservoir under conditions of HAART. Prostratin, a non tumor-promoting phorbol ester, is a candidate for such a strategy. Prostratin has been shown to reactivate latent HIV-1 and Tat-mediated transactivation may play an important role in this process. We examined resting CD4(+ )T cells from healthy donors to determine if prostratin induces Cyclin T1/P-TEFb, a cellular kinase composed of Cyclin T1 and Cyclin-dependent kinase-9 (CDK9) that mediates Tat function. We also examined effects of prostratin on Cyclin T2a, an alternative regulatory subunit for CDK9, and 7SK snRNA and the HEXIM1 protein, two factors that associate with P-TEFb and repress its kinase activity. RESULTS: Prostratin up-regulated Cyclin T1 protein expression, modestly induced CDK9 protein expression, and did not affect Cyclin T2a protein expression. Although the kinase activity of CDK9 in vitro was up-regulated by prostratin, we observed a large increase in the association of 7SK snRNA and the HEXIM1 protein with CDK9. Using HIV-1 reporter viruses with and without a functional Tat protein, we found that prostratin stimulation of HIV-1 gene expression appears to require a functional Tat protein. Microarray analyses were performed and several genes related to HIV biology, including APOBEC3B, DEFA1, and S100 calcium-binding protein genes, were found to be regulated by prostratin. CONCLUSION: Prostratin induces Cyclin T1 expression and P-TEFb function and this is likely to be involved in prostratin reactivation of latent HIV-1 proviruses. The large increase in association of 7SK and HEXIM1 with P-TEFb following prostratin treatment may reflect a requirement in CD4(+ )T cells for a precise balance between active and catalytically inactive P-TEFb. Additionally, genes regulated by prostratin were identified that have the potential to regulate HIV-1 replication both positively and negatively

Vpr14-88-Apobec3G Fusion Protein Is Efficiently Incorporated into Vif-Positive HIV-1 Particles and Inhibits Viral Infection

APOBEC3G (A3G), a deoxycytidine deaminase, is a potent host antiviral factor that can restrict HIV-1 infection. During Vif-negative HIV-1 replication, A3G is incorporated into HIV-1 particles, induces mutations in reverse transcribed viral DNA and inhibits reverse transcription. However, HIV-1 Vif counteracts A3G's activities by inducing its degradation and by blocking its incorporation into HIV-1 particles. Thus, it is interesting to elucidate a mechanism that would allow A3G to escape the effects of Vif in order to rescue its potent antiviral activity and to provide a possible novel therapeutic strategy for treating HIV-1 infection.In this study, we generated an R88-A3G fusion protein by fusing A3G to a virion-targeting polypeptide (R14-88) derived from HIV-1 Vpr protein and compared its antiviral effects relative to those of HA-tagged native A3G (HA-A3G). Our study showed that transient expression of the R88-A3G fusion protein in both Vif(-) and Vif(+) HIV-1 producing cells drastically inhibited viral infection in HeLa-CD4-CCR5-cells, CD4(+) C8166 T cells and human primary PBMCs. Moreover, we established CD4(+) C8166 T cell lines that stably express either R88-A3G or HA-A3G by transduction with VSV-G-pseudotyped lentiviral vector that harbor expression cassettes for R88-A3G or HA-A3G, respectively, and tested their susceptibility to Vif(+) HIV-1 infection. Our results clearly reveal that expression of R88-A3G in transduced CD4(+) C8166 cells significantly blocked Vif(+) HIV-1 infection. In an attempt to understand the mechanism underlying the antiviral activity of R88-A3G, we demonstrated that R88-A3G was efficiently incorporated into viral particles in the presence of Vif. Moreover, PCR analysis revealed that R88-A3G significantly inhibited viral cDNA synthesis during the early stage of Vif(+) virus infection.Our results clearly indicate that R88 delivers A3G into Vif(+) HIV-1 particles and inhibits infectivity and spread of the virions among CD4(+) T cells. This study provides evidence for an effective strategy to modify a host protein with innate anti-HIV-1 activity and rescue its potent anti-HIV potential in the presence of Vif. Further characterization and optimization of this system may lead to the development of an effective therapeutic approach against HIV-1 infection

APOBEC3G and APOBEC3F Require an Endogenous Cofactor to Block HIV-1 Replication

APOBEC3G (A3G)/APOBEC3F (A3F) are two members of APOBEC3 cytidine deaminase subfamily. Although they potently inhibit the replication of vif-deficient HIV-1, this mechanism is still poorly understood. Initially, A3G/A3F were thought to catalyze C-to-U transitions on the minus-strand viral cDNAs during reverse transcription to disrupt the viral life cycle. Recently, it was found more likely that A3G/A3F directly interrupts viral reverse transcription or integration. In addition, A3G/A3F are both found in the high-molecular-mass complex in immortalized cell lines, where they interact with a number of different cellular proteins. However, there has been no evidence to prove that these interactions are required for A3G/A3F function. Here, we studied A3G/A3F-restricted HIV-1 replication in six different human T cell lines by infecting them with wild-type or vif-deficient HIV-1. Interestingly, in a CEM-derived cell line CEM-T4, which expresses high levels of A3G/A3F proteins, the vif-deficient virus replicated as equally well as the wild-type virus, suggesting that these endogenous antiretroviral genes lost anti-HIV activities. It was confirmed that these A3G/A3F genes do not contain any mutation and are functionally normal. Consistently, overexpression of exogenous A3G/A3F in CEM-T4 cells still failed to restore their anti-HIV activities. However, this activity could be restored if CEM-T4 cells were fused to 293T cells to form heterokaryons. These results demonstrate that CEM-T4 cells lack a cellular cofactor, which is critical for A3G/A3F anti-HIV activity. We propose that a further study of this novel factor will provide another strategy for a complete understanding of the A3G/A3F antiretroviral mechanism

Genomic HIV RNA Induces Innate Immune Responses through RIG-I-Dependent Sensing of Secondary-Structured RNA

Contains fulltext : 108031.pdf (publisher's version ) (Open Access)BACKGROUND: Innate immune responses have recently been appreciated to play an important role in the pathogenesis of HIV infection. Whereas inadequate innate immune sensing of HIV during acute infection may contribute to failure to control and eradicate infection, persistent inflammatory responses later during infection contribute in driving chronic immune activation and development of immunodeficiency. However, knowledge on specific HIV PAMPs and cellular PRRs responsible for inducing innate immune responses remains sparse. METHODS/PRINCIPAL FINDINGS: Here we demonstrate a major role for RIG-I and the adaptor protein MAVS in induction of innate immune responses to HIV genomic RNA. We found that secondary structured HIV-derived RNAs induced a response similar to genomic RNA. In primary human peripheral blood mononuclear cells and primary human macrophages, HIV RNA induced expression of IFN-stimulated genes, whereas only low levels of type I IFN and tumor necrosis factor alpha were produced. Furthermore, secondary structured HIV-derived RNA activated pathways to NF-kappaB, MAP kinases, and IRF3 and co-localized with peroxisomes, suggesting a role for this organelle in RIG-I-mediated innate immune sensing of HIV RNA. CONCLUSIONS/SIGNIFICANCE: These results establish RIG-I as an innate immune sensor of cytosolic HIV genomic RNA with secondary structure, thereby expanding current knowledge on HIV molecules capable of stimulating the innate immune system

Innate Sensing of HIV-Infected Cells

Cell-free HIV-1 virions are poor stimulators of type I interferon (IFN) production. We examined here how HIV-infected cells are recognized by plasmacytoid dendritic cells (pDCs) and by other cells. We show that infected lymphocytes are more potent inducers of IFN than virions. There are target cell-type differences in the recognition of infected lymphocytes. In primary pDCs and pDC-like cells, recognition occurs in large part through TLR7, as demonstrated by the use of inhibitors and by TLR7 silencing. Donor cells expressing replication-defective viruses, carrying mutated reverse transcriptase, integrase or nucleocapsid proteins induced IFN production by target cells as potently as wild-type virus. In contrast, Env-deleted or fusion defective HIV-1 mutants were less efficient, suggesting that in addition to TLR7, cytoplasmic cellular sensors may also mediate sensing of infected cells. Furthermore, in a model of TLR7-negative cells, we demonstrate that the IRF3 pathway, through a process requiring access of incoming viral material to the cytoplasm, allows sensing of HIV-infected lymphocytes. Therefore, detection of HIV-infected lymphocytes occurs through both endosomal and cytoplasmic pathways. Characterization of the mechanisms of innate recognition of HIV-infected cells allows a better understanding of the pathogenic and exacerbated immunologic events associated with HIV infection

APOBEC3A Is a Specific Inhibitor of the Early Phases of HIV-1 Infection in Myeloid Cells

Myeloid cells play numerous roles in HIV-1 pathogenesis serving as a vehicle for viral spread and as a viral reservoir. Yet, cells of this lineage generally resist HIV-1 infection when compared to cells of other lineages, a phenomenon particularly acute during the early phases of infection. Here, we explore the role of APOBEC3A on these steps. APOBEC3A is a member of the APOBEC3 family that is highly expressed in myeloid cells, but so far lacks a known antiviral effect against retroviruses. Using ectopic expression of APOBEC3A in established cell lines and specific silencing in primary macrophages and dendritic cells, we demonstrate that the pool of APOBEC3A in target cells inhibits the early phases of HIV-1 infection and the spread of replication-competent R5-tropic HIV-1, specifically in cells of myeloid origins. In these cells, APOBEC3A affects the amount of vDNA synthesized over the course of infection. The susceptibility to the antiviral effect of APOBEC3A is conserved among primate lentiviruses, although the viral protein Vpx coded by members of the SIVSM/HIV-2 lineage provides partial protection from APOBEC3A during infection. Our results indicate that APOBEC3A is a previously unrecognized antiviral factor that targets primate lentiviruses specifically in myeloid cells and that acts during the early phases of infection directly in target cells. The findings presented here open up new venues on the role of APOBEC3A during HIV infection and pathogenesis, on the role of the cellular context in the regulation of the antiviral activities of members of the APOBEC3 family and more generally on the natural functions of APOBEC3A