Human cyclin T1 expression ameliorates a T-cell-specific transcriptional limitation for HIV in transgenic rats, but is not sufficient for a spreading infection of prototypic R5 HIV-1 strains ex vivo.

BACKGROUND: Cells derived from native rodents have limits at distinct steps of HIV replication. Rat primary CD4 T-cells, but not macrophages, display a profound transcriptional deficit that is ameliorated by transient trans-complementation with the human Tat-interacting protein Cyclin T1 (hCycT1). RESULTS: Here, we generated transgenic rats that selectively express hCycT1 in CD4 T-cells and macrophages. hCycT1 expression in rat T-cells boosted early HIV gene expression to levels approaching those in infected primary human T-cells. hCycT1 expression was necessary, but not sufficient, to enhance HIV transcription in T-cells from individual transgenic animals, indicating that endogenous cellular factors are critical co-regulators of HIV gene expression in rats. T-cells from hCD4/hCCR5/hCycT1-transgenic rats did not support productive infection of prototypic wild-type R5 HIV-1 strains ex vivo, suggesting one or more significant limitation in the late phase of the replication cycle in this primary rodent cell type. Remarkably, we identify a replication-competent HIV-1 GFP reporter strain (R7/3 YU-2 Env) that displays characteristics of a spreading, primarily cell-to-cell-mediated infection in primary T-cells from hCD4/hCCR5-transgenic rats. Moreover, the replication of this recombinant HIV-1 strain was significantly enhanced by hCycT1 transgenesis. The viral determinants of this so far unique replicative ability are currently unknown. CONCLUSION: Thus, hCycT1 expression is beneficial to de novo HIV infection in a transgenic rat model, but additional genetic manipulations of the host or virus are required to achieve full permissivity.

Primary T-cells from human CD4/CCR5-transgenic rats support all early steps of HIV-1 replication including integration, but display impaired viral gene expression.

BACKGROUND: In vivo studies on HIV-1 pathogenesis and testing of antiviral strategies have been hampered by the lack of an immunocompetent small animal model that is highly susceptible to HIV-1 infection. Since native rodents are non-permissive, we developed transgenic rats that selectively express the HIV-1 receptor complex, hCD4 and hCCR5, on relevant target cells. These animals display a transient low-level plasma viremia after HIV-1YU-2 infection, demonstrating HIV-1 susceptibility in vivo. However, unlike macrophages, primary CD4 T-cells from double-transgenic animals fail to support viral spread ex vivo. To identify quantitative limitations or absolute blocks in this rodent species, we quantitatively assessed the efficiency of key steps in the early phase of the viral replication cycle in a side-by-side comparison in infected cell lines and primary T-cells from hCD4/hCCR5-transgenic rats and human donors. RESULTS: Levels of virus entry, HIV-1 cDNA synthesis, nuclear import, and integration into the host genome were shown to be remarkably similar in cell lines and, where technically accessible, in primary T-cells from both species. In contrast, a profound impairment at the level of early HIV gene expression was disclosed at the single-cell level in primary rat T-cells and most other rat-derived cells. Macrophages were a notable exception, possibly reflecting the unique transcriptional milieu in this evolutionarily conserved target cell of all lentiviruses. Importantly, transient trans-complementation by ex vivo nucleofection with the Tat-interacting protein Cyclin T1 of human origin markedly elevated HIV gene expression in primary rat T-cells. CONCLUSION: This is the first study that has quantitatively determined the efficiency of consecutive steps in the HIV-1 replication cycle in infected primary HIV target cells from a candidate transgenic small animal and compared it to human cells. Unlike cells derived from mice or rabbits, rat cells complete all of the early steps in the HIV-1 replication cycle, including provirus integration in vivo, with high efficiency. A deficiency in gene expression was disclosed at the single cell level and could be counteracted by the human pTEFb transcription complex factor Cyclin T1. Collectively, these results provide the basis for the advancement of this transgenic rat model through strategies aimed at boosting HIV-1 gene expression in primary rat CD4 T-cells, including human Cyclin T1 transgenesis.

Expression of Nef downregulates CXCR4, the major coreceptor of human immunodeficiency virus, from the surfaces of target cells and thereby enhances resistance to superinfection.

Lentiviral Nef proteins are key factors for pathogenesis and are known to downregulate functionally important molecules, including CD4 and major histocompatibility complex class I (MHC-I), from the surfaces of infected cells. Recently, we demonstrated that Nef reduces cell surface levels of the human immunodeficiency virus type 1 (HIV-1) entry coreceptor CCR5 (N. Michel, I. Allespach, S. Venzke, O. T. Fackler, and O. T. Keppler, Curr. Biol. 15:714-723, 2005). Here, we report that Nef downregulates the second major HIV-1 coreceptor, CXCR4, from the surfaces of HIV-infected primary CD4 T lymphocytes with efficiencies comparable to those of the natural CXCR4 ligand, stromal cell-derived factor-1 alpha. Analysis of a panel of mutants of HIV-1(SF2) Nef revealed that the viral protein utilized the same signature motifs for downmodulation of CXCR4 and MHC-I, including the proline-rich motif P(73)P(76)P(79)P(82) and the acidic cluster motif E(66)E(67)E(68)E(69.) Expression of wild-type Nef, but not of specific Nef mutants, resulted in a perinuclear accumulation of the coreceptor. Remarkably, the carboxy terminus of CXCR4, which harbors the classical motifs critical for basal and ligand-induced receptor endocytosis, was dispensable for the Nef-mediated reduction of surface exposure. Functionally, the ability of Nef to simultaneously downmodulate CXCR4 and CD4 correlated with maximum-level protection of Nef-expressing target cells from fusion with cells exposing X4 HIV-1 envelopes. Furthermore, the Nef-mediated downregulation of CXCR4 alone on target T lymphocytes was sufficient to diminish cells' susceptibility to X4 HIV-1 virions at the entry step. The downregulation of chemokine coreceptors is a conserved activity of Nef to modulate infected cells, an important functional consequence of which is an enhanced resistance to HIV superinfection.

The Nef protein of human immunodeficiency virus is a broad-spectrum modulator of chemokine receptor cell surface levels that acts independently of classical motifs for receptor endocytosis and Galphai signaling.

Chemokine receptors (CKRs) are important physiological mediators of immune defense, inflammatory responses, and angiogenesis, and they have also been implicated in a number of viral disease processes. Here, we report that the Nef protein of human immunodeficiency virus (HIV) reduces cell surface levels of eight different members of the CC- and CXC-family of CKRs by up to 92%. This broad-range activity required specific elements in HIV(SF2) Nef, including the proline-rich motif P73P76P79P82 as well as the acidic cluster motif E66E67E68E69, and Nef expression induced a marked perinuclear accumulation of CKRs. Surprisingly, receptor mutagenesis demonstrated that the cytoplasmic tail of CCR5 and CXCR4, which is critical for basal and ligand-mediated endocytosis, was completely dispensable for this Nef activity. In contrast, triple-mutation of the highly conserved DRY motif in the second intracellular CKR loop abolished the Nef-mediated down-regulation of CXCR4 independently of this motif's role in CKR binding to heterotrimeric G proteins and signaling via the Galphai subunit. Thus, we identify the lentiviral pathogenicity factor Nef as a unique and broad-range modulator of CKR cell surface levels. Nef uses a mechanism that is distinct from well-established pathways orchestrating CKR metabolism and offers an interesting tool to study the multifaceted biology of CKRs.

The HIV-1 pathogenicity factor Nef interferes with maturation of stimulatory T-lymphocyte contacts by modulation of N-Wasp activity.

The Nef protein is a key determinant of human immunodeficiency virus (HIV) pathogenicity that, among other activities, sensitizes T-lymphocytes for optimal virus production. The initial events by which Nef modulates the T-cell receptor (TCR) cascade are poorly understood. TCR engagement triggers actin rearrangements that control receptor clustering for signal initiation and dynamic organization of signaling protein complexes to form an immunological synapse. Here we report that Nef potently interferes with cell spreading and formation of actin-rich circumferential rings in T-lymphocytes upon surface-supported TCR stimulation. These effects were conserved among Nef proteins from different lentiviruses and occurred in HIV-1-infected primary human T-lymphocytes. This novel Nef activity critically depended on its Src homology 3 domain binding motif and required efficient association with Pak2 activity. Notably, whereas overall signaling microcluster formation immediately following TCR engagement occurred normally in Nef-expressing cells, the viral protein inhibited the concomitant activation of the actin organizer N-Wasp. During the subsequent maturation phase of the stimulatory contact, Nef interfered with the translocation of N-Wasp to the cell periphery, the overall induction of tyrosine phosphorylation, and the selective recruitment of phosphorylated LAT to stimulatory contacts. Consistent with such a critical role of N-Wasp in this process, Nef also blocked morphological changes induced by the known N-Wasp regulators Rac1 and Cdc42. Together, our results demonstrate that Nef alters both the amount and composition of signaling microclusters. We propose modulation of actin dynamics as an important mechanism for Nef-induced alterations of TCR signaling.

The Nef protein of human immunodeficiency virus establishes superinfection immunity by a dual strategy to downregulate cell-surface CCR5 and CD4.

BACKGROUND: Viruses frequently render cells refractory to subsequent infection with the same virus. This state of superinfection immunity counteracts potentially detrimental consequences for the infected cell and facilitates high-level replication and viral spread in the host. RESULTS: Here, we show that human immunodeficiency virus (HIV) employs its early gene product Nef to efficiently interfere with superinfection at the viral-entry step. In this context, we identify the downregulation of cell-surface CCR5, the major HIV coreceptor, as a novel and highly conserved activity of Nef. Nef targets the CCR5 coreceptor and the HIV binding receptor CD4 via distinct cellular machineries to enhance the endocytosis rate of both HIV receptor components and to accelerate their degradation. Functionally, these genetically separable actions by Nef synergized to efficiently protect cells from HIV superinfection at the level of fusion of the viral envelope with the plasma membrane. CONCLUSIONS: HIV has evolved two independent activities for Nef to downregulate the receptor complex and to facilitate its efficient replication and spread. This evasion strategy likely represents a mechanism by which the pathogenicity factor Nef elevates viral replication in vivo and thus promotes AIDS pathogenesis.

Enhanced immunogenicity of HPV 16 E7 fusion proteins in DNA vaccination.

DNA vaccination is a promising approach for inducing both humoral and cellular immune responses. For immunotherapy of HPV-16-associated diseases the E7 protein is considered a prime candidate, as it is expressed in all HPV-16-positive tumors. Unfortunately, the E7 protein is a very poor inducer of a cytotoxic T-cell response, when being used as antigen in DNA vaccination. Here we demonstrate that after fusion to protein export/import signals such as the herpes simplex virus ferry protein VP22, E7 can translocate in vitro from VP22-E7-expressing cells to neighboring cells that do not carry the VP22-E7 gene. In vivo, the VP22-E7 fusion shows significantly increased efficiency in inducing a cytotoxic T-cell response. Our data suggest that the export function of VP22 plays a major role in this phenomenon, since VP22 can be replaced by classical protein export signals, without impairing the induction of the E7-specific cellular immune response. However, all E7 fusion constructs showed significantly elevated protein steady-state levels, which might also account for the observed boost in immunogenicity.

T cell response to human papillomavirus 16 E7 in mice: comparison of Cr release assay, intracellular IFN-gamma production, ELISPOT and tetramer staining.

Successful vaccination against infections by high-risk papillomaviruses aiming at the prevention of cervical cancer most likely requires the induction of neutralizing antibodies and human papillomavirus (HPV)-specific T cells directed against early viral proteins such as E7. Whereas the technology for detection of antibodies is well established, measurement of T cells is more cumbersome and standardization of assays is difficult. By using chromium release assay, ELISPOT, tetramer staining and intracellular IFN-gamma assay, we compared the levels of HPV 16 E7-specific T cells obtained after immunization of C57BL/6 mice with different DNA expression vectors. We found that all four assays gave highly comparable results. ELISPOT can be recommended for future studies as it indicates the presence of activated (i.e. IFN-gamma-secreting) T cells in a quantitative manner and combines high sensitivity with relatively low T cell demand.