Coreceptor Usage by HIV-1 and HIV-2 Primary Isolates: The Relevance of CCR8 Chemokine Receptor as an Alternative Coreceptor

The human immunodeficiency virus replication cycle begins by sequential interactions between viral envelope glycoproteins with CD4 molecule and a member of the seven-transmembrane, G-protein-coupled, receptors' family (coreceptor). In this report we focused on the contribution of CCR8 as alternative coreceptor for HIV-1 and HIV-2 isolates. We found that this coreceptor was efficiently used not only by HIV-2 but particularly by HIV-1 isolates. We demonstrate that CXCR4 usage, either alone or together with CCR5 and/or CCR8, was more frequently observed in HIV-1 than in HIV-2 isolates. Directly related to this is the finding that the non-usage of CXCR4 is significantly more common in HIV-2 isolates; both features could be associated with the slower disease progression generally observed in HIV-2 infected patients. The ability of some viral isolates to use alternative coreceptors besides CCR5 and CXCR4 could further impact on the efficacy of entry inhibitor therapy and possibly also in HIV pathogenesis.

Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors.

Viruses have evolved many distinct strategies to avoid the host's apoptotic response. Here we describe a new family of viral inhibitors (v-FLIPs) which interfere with apoptosis signalled through death receptors and which are present in several gamma-herpesviruses (including Kaposi's-sarcoma-associated human herpesvirus-8), as well as in the tumorigenic human molluscipoxvirus. v-FLIPs contain two death-effector domains which interact with the adaptor protein FADD, and this inhibits the recruitment and activation of the protease FLICE by the CD95 death receptor. Cells expressing v-FLIPs are protected against apoptosis induced by CD95 or by the related death receptors TRAMP and TRAIL-R. The herpesvirus saimiri FLIP is detected late during the lytic viral replication cycle, at a time when host cells are partially protected from CD95-ligand-mediated apoptosis. Protection of virus-infected cells against death-receptor-induced apoptosis may lead to higher virus production and contribute to the persistence and oncogenicity of several FLIP-encoding viruses.

Regulation of the DNA-binding and transcriptional activities of Xenopus laevis NFI-X by a novel C-terminal domain.

The nuclear factor I (NFI) family consists of sequence-specific DNA-binding proteins that activate both transcription and adenovirus DNA replication. We have characterized three new members of the NFI family that belong to the Xenopus laevis NFI-X subtype and differ in their C-termini. We show that these polypeptides can activate transcription in HeLa and Drosophila Schneider line 2 cells, using an activation domain that is subdivided into adjacent variable and subtype-specific domains each having independent activation properties in chimeric proteins. Together, these two domains constitute the full NFI-X transactivation potential. In addition, we find that the X. laevis NFI-X proteins are capable of activating adenovirus DNA replication through their conserved N-terminal DNA-binding domains. Surprisingly, their in vitro DNA-binding activities are specifically inhibited by a novel repressor domain contained within the C-terminal part, while the dimerization and replication functions per se are not affected. However, inhibition of DNA-binding activity in vitro is relieved within the cell, as transcriptional activation occurs irrespective of the presence of the repressor domain. Moreover, the region comprising the repressor domain participates in transactivation. Mechanisms that may allow the relief of DNA-binding inhibition in vivo and trigger transcriptional activation are discussed.

TRADD protein is an essential component of the RIG-like helicase antiviral pathway

Upon detection of viral RNA, the helicases RIG-I and/or MDA5 trigger, via their adaptor Cardif (also known as IPS-1, MAVS, or VISA), the activation of the transcription factors NF-kappaB and IRF3, which collaborate to induce an antiviral type I interferon (IFN) response. FADD and RIP1, known as mediators of death-receptor signaling, are implicated in this antiviral pathway; however, the link between death-receptor and antiviral signaling is not known. Here we showed that TRADD, a crucial adaptor of tumor necrosis factor receptor (TNFRI), was important in RIG-like helicase (RLH)-mediated signal transduction. TRADD is recruited to Cardif and orchestrated complex formation with the E3 ubiquitin ligase TRAF3 and TANK and with FADD and RIP1, leading to the activation of IRF3 and NF-kappaB. Loss of TRADD prevented Cardif-dependent activation of IFN-beta, reduced the production of IFN-beta in response to RNA viruses, and enhanced vesicular stomatitis virus replication. Thus, TRADD is not only an essential component of proinflammatory TNFRI signaling, but is also required for RLH-Cardif-dependent antiviral immune responses

The hope for an HIV vaccine based on induction of CD8+ T lymphocytes: a review

The only long-term and cost-effective solution to the human immunodeficiency virus (HIV) epidemic in the developing world is a vaccine that prevents individuals from becoming infected or, once infected, from passing the virus on to others. There is currently little hope for an AIDS vaccine. Conventional attempts to induce protective antibody and CD8+ lymphocyte responses against HIV and simian immunodeficiency virus (SIV) have failed. The enormous diversity of the virus has only recently been appreciated by vaccinologists, and our assays to determine CD8+ lymphocyte antiviral efficacy are inadequate. The central hypothesis of a CTL-based vaccine is that particularly effective CD8+ lymphocytes directed against at least five epitopes that are derived from regions under functional and structural constraints will control replication of pathogenic SIV. This would be somewhat analogous to control of virus replication by triple drug therapy or neutralizing antibodies.

Analysis of HIV-1 expression level and sense of transcription by high-throughput sequencing of the infected cell.

Next-generation sequencing offers an unprecedented opportunity to jointly analyze cellular and viral transcriptional activity without prerequisite knowledge of the nature of the transcripts. SupT1 cells were infected with a vesicular stomatitis virus G envelope protein (VSV-G)-pseudotyped HIV vector. At 24 h postinfection, both cellular and viral transcriptomes were analyzed by serial analysis of gene expression followed by high-throughput sequencing (SAGE-Seq). Read mapping resulted in 33 to 44 million tags aligning with the human transcriptome and 0.23 to 0.25 million tags aligning with the genome of the HIV-1 vector. Thus, at peak infection, 1 transcript in 143 is of viral origin (0.7%), including a small component of antisense viral transcription. Of the detected cellular transcripts, 826 (2.3%) were differentially expressed between mock- and HIV-infected samples. The approach also assessed whether HIV-1 infection modulates the expression of repetitive elements or endogenous retroviruses. We observed very active transcription of these elements, with 1 transcript in 237 being of such origin, corresponding on average to 123,123 reads in mock-infected samples (0.40%) and 129,149 reads in HIV-1-infected samples (0.45%) mapping to the genomic Repbase repository. This analysis highlights key details in the generation and interpretation of high-throughput data in the setting of HIV-1 cellular infection.

Role of the host cell's unfolded protein response in arenavirus infection.

Arenaviruses are enveloped RNA viruses with a nonlytic life cycle that cause acute and persistent infections. Here, we investigated the role of the host cell's unfolded protein response (UPR) in infection of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). In mammalian cells, the endoplasmic reticulum (ER) chaperone protein GRP78/BiP functions as the principal sensor for the induction of the UPR and interacts with three mediators: kinase/endonuclease inositol-requiring protein 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Acute infection with LCMV resulted in a selective induction of the ATF6-regulated branch of the UPR, whereas pathways controlled by PERK and IRE1 were neither activated nor blocked. Expression of individual LCMV proteins revealed that the viral glycoprotein precursor (GPC), but not that of other viral proteins, was responsible for the induction of ATF6. Rapid downregulation of the viral GPC during transition from acute to persistent LCMV infection restored basal levels of UPR signaling. To address a possible role of ATF6 signaling in LCMV infection, we used cells deficient in site 2 protease (S2P), a metalloprotease required for the activation of ATF6. Cells deficient in S2P showed significantly lower levels of production of infectious virus during acute but not persistent infection, indicating a requirement for ATF6-mediated signaling for optimal virus multiplication. In summary, acute LCMV infection seems to selectively induce the ATF6-regulated branch of the UPR that is likely beneficial for virus replication and cell viability, but it avoids induction of PERK and IRE1, whose activation may be detrimental for virus and the host cell.

Antiviral Immunity in HIV-1 infected long term non-progressors (LTNPs).

Now that the acquired immunodeficiency syndrome (AIDS) epidemic is well into its second decade, it has become evident that a small percentage (approximately 5%) of HIV-infected individuals do not experience progression of HIV disease even after several years of being infected with HIV. These individuals have been designated as 'long term non-progressors' (LTNPs). From a virologic standpoint, these LTNPs have low viral burden in mononuclear cells, but persistent virus replication as manifested by chronic and generally low levels of plasma viremia. From an immunologic standpoint, immune functions including CD8(+) T-cell- and CD4(+) T-cell-mediated functions are preserved. In addition, they show a vigorous humoral immune response. More importantly, lymphoid tissue structure and function are preserved in LTNPs. Despite persistent low-level virus replication and chronic stimulation of the immune system, immune activation is qualitatively and quantitatively different in LTNPs compared to that observed in HIV-infected individuals whose HIV disease has progressed.

Effects of immunosuppressive drugs on HIV infection: implications for solid-organ transplantation.

With the advent of highly active antiretroviral therapy (HAART), HIV infection has become a chronic disease. Various end-stage organ failures have now become common co-morbidities and are primary causes of mortality in HIV-infected patients. Solid-organ transplantation therefore has been proposed to these patients, as HIV infection is not anymore considered an absolute contraindication. The initial results of organ transplantation in HIV-infected patients are encouraging with no differences in patient and graft survival compared with non-HIV-infected patients. The use of immunosuppressive drug therapy in HIV-infected patients has so far not shown major detrimental effects, and some drugs in combination with HAART have even demonstrated possible beneficial effects for specific HIV settings. Nevertheless, organ transplantation in HIV-infected patients remains a complex intervention, and more studies will be required to clarify open questions such as long-term effects of drug interactions between antiretroviral and immunosuppressive drugs, outcome of recurrent HCV infection in HIV-infected patients, incidence of graft rejection, or long-term graft and patient survival. In this article, we first review the immunological pathogenesis of HIV infection and the rationale for using immunosuppression combined with HAART. We then discuss the most recent results of solid-organ transplantation in HIV-infected patients.

Replicative phenotyping adds value to genotypic resistance testing in heavily pre-treated HIV-infected individuals--the Swiss HIV Cohort Study.

BACKGROUND: Replicative phenotypic HIV resistance testing (rPRT) uses recombinant infectious virus to measure viral replication in the presence of antiretroviral drugs. Due to its high sensitivity of detection of viral minorities and its dissecting power for complex viral resistance patterns and mixed virus populations rPRT might help to improve HIV resistance diagnostics, particularly for patients with multiple drug failures. The aim was to investigate whether the addition of rPRT to genotypic resistance testing (GRT) compared to GRT alone is beneficial for obtaining a virological response in heavily pre-treated HIV-infected patients. METHODS: Patients with resistance tests between 2002 and 2006 were followed within the Swiss HIV Cohort Study (SHCS). We assessed patients' virological success after their antiretroviral therapy was switched following resistance testing. Multilevel logistic regression models with SHCS centre as a random effect were used to investigate the association between the type of resistance test and virological response (HIV-1 RNA <50 copies/mL or ≥1.5 log reduction). RESULTS: Of 1158 individuals with resistance tests 221 with GRT+rPRT and 937 with GRT were eligible for analysis. Overall virological response rates were 85.1% for GRT+rPRT and 81.4% for GRT. In the subgroup of patients with >2 previous failures, the odds ratio (OR) for virological response of GRT+rPRT compared to GRT was 1.45 (95% CI 1.00-2.09). Multivariate analyses indicate a significant improvement with GRT+rPRT compared to GRT alone (OR 1.68, 95% CI 1.31-2.15). CONCLUSIONS: In heavily pre-treated patients rPRT-based resistance information adds benefit, contributing to a higher rate of treatment success.

Type I interferons protect T cells against NK cell attack mediated by the activating receptor NCR1.

Direct type I interferon (IFN) signaling on T cells is necessary for the proper expansion, differentiation, and survival of responding T cells following infection with viruses prominently inducing type I IFN. The reasons for the abortive response of T cells lacking the type I IFN receptor (Ifnar1(-/-)) remain unclear. We report here that Ifnar1(-/-) T cells were highly susceptible to natural killer (NK) cell-mediated killing in a perforin-dependent manner. Depletion of NK cells prior to lymphocytic choriomeningitis virus (LCMV) infection completely restored the early expansion of Ifnar1(-/-) T cells. Ifnar1(-/-) T cells had elevated expression of natural cytotoxicity triggering receptor 1 (NCR1) ligands upon infection, rendering them targets for NCR1 mediated NK cell attack. Thus, direct sensing of type I IFNs by T cells protects them from NK cell killing by regulating the expression of NCR1 ligands, thereby revealing a mechanism by which T cells can evade the potent cytotoxic activity of NK cells.

An adenovirus-based fluorescent reporter vector to identify and isolate HIV-infected cells.

A procedure is described that allows the simple identification and sorting of live human cells that transcribe actively the HIV virus, based on the detection of GFP fluorescence in cells. Using adenoviral vectors for gene transfer, an expression cassette including the HIV-1 LTR driving the reporter gene GFP was introduced into cells that expressed stably either the Tat transcriptional activator, or an inactive mutant of Tat. Both northern and fluorescence-activated cell sorting (FACS) analysis indicate that cells containing the functional Tat protein presented levels of GFP mRNA and GFP fluorescence several orders of magnitude higher than control cells. Correspondingly, cells infected with HIV-1 showed similar enhanced reporter gene activation. HIV-1-infected cells of the lymphocytic line Jurkat were easily identified by fluorescence-activated cell sorting (FACS) as they displayed a much higher green fluorescence after transduction with the reporter adenoviral vector. This procedure could also be applied on primary human cells as blood monocyte-derived macrophages exposed to the adenoviral LTR-GFP reporter presented a much higher fluorescence when infected with HIV-1 compared with HIV-uninfected cells. The vector described has the advantages of labelling cells independently of their proliferation status and that analysis can be carried on intact cells which can be isolated subsequently by fluorescence-activated cell sorting (FACS) for further culture. This work suggests that adenoviral vectors carrying a virus-specific transcriptional control element controlling the expressions of a fluorescent protein will be useful in the identification and isolation of cells transcribing actively the viral template, and to be of use for drug screening and susceptibility assays.

Cytomegalovirus (CMV)-specific cellular immune responses.

A large percentage of healthy individuals (50-90%) is chronically infected with Cytomegalovirus (CMV). Over the past few years, several techniques were developed in order to monitor CMV-specific T-cell responses. In addition to the identification of antigen-specific T cells with peptide-loaded MHC complexes, most of the current strategies to identify CMV-specific T cells are centered on the assessment of the functions of memory T cells including their ability to mediate effector function, to proliferate or to secrete cytokines following antigen-specific stimulation. The investigation of these functions has allowed the characterization of the CMV-specific T-cell responses that are present during different phases of the infection. Furthermore, it has also been shown that the combination of virus-specific CD4 and CD8 T-cell responses are critical components of the immune response in the control of virus replication.