Paola D'Aloja

Massive Secretion by T Cells Is Caused by HIV Nef in Infected Cells and by Nef Transfer to Bystander Cells

The HIV Nef protein mediates endocytosis of surface receptors that correlates with disease progression, but the link between this Nef function and HIV pathogenesis is not clear. Here, we report that Nef-mediated activation of membrane trafficking is bidirectional, connecting endocytosis with exocytosis as occurs in activated T cells. Nef expression induced an extensive secretory activity in infected and, surprisingly, also in noninfected T cells, leading to the massive release of microvesicle clusters, a phenotype observed in vitro and in 36%-87% of primary CD4 T cells from HIV-infected individuals. Consistent with exocytosis in noninfected cells, Nef is transferred to bystander cells upon cell-to-cell contact and subsequently induces secretion in an Erk1/2-dependent manner. Thus, HIV Nef alters membrane dynamics, mimicking those of activated T cells and causing a transfer of infected cell signaling (TOS) to bystander cells. This mechanism may help explain the detrimental effect on bystander cells seen in HIV infection.

T-tropic human immunodeficiency virus (HIV) type 1 Nef protein enters human monocyte-macrophages and induces resistance to HIV replication: a possible mechanism of HIV T-tropic emergence in AIDS.

Increasing interest has been devoted to the role that monocyte-macrophages play in the pathogenesis of AIDS. The hypothesis of an involvement in AIDS pathogenesis of human/simian immunodeficiency virus (HIV/SIV) Nef also is currently under evaluation by many investigators. The original basis of this hypothesis came from evidence that monkeys infected with a nef-deleted SIV strain failed to develop simian AIDS. Here, we show that treatment of human monocyte-derived macrophages (MDM) with recombinant HIV-1 Nef protein (rNef) induces a strong inhibition of the replication of either macrophage (M-) or dual-tropic HIV-1 strains. Through cytofluorimetric analyses, we detected internalization of FITC-conjugated rNef in MDM as early as 6 h after treatment. Confocal microscope observations demonstrated that the intracellular distribution of internalized rNef was identical to that of endogenously produced Nef. Down-regulation of the CD4 HIV receptor detected upon rNef treatment of MDM suggested that the rNef-induced HIV inhibition occurred at the virus entry step. This deduction was strengthened by the observation that CD4-independent infection was totally insensitive to rNef treatment. The specificity of all observed effects was demonstrated by immunodepletion of rNef. Finally, we showed that the resistance to HIV replication induced by rNef treatment in MDM favours the spread of T-tropic over M-tropic HIV strains in doubly infected CD4(+) lymphocyte-MDM co-cultures. We propose that extracellular Nef contributes to AIDS pathogenesis by inducing resistance to M-tropic HIV replication in MDM, thereby facilitating the switching from M- to T-tropic HIV prevalence that correlates frequently with AIDS progression.

cis Expression of the F12 Human Immunodeficiency Virus (HIV) Nef Allele Transforms the Highly Productive NL4-3 HIV Type 1 to a Replication-Defective Strain: Involvement of both Env gp41 and CD4 Intracytoplasmic Tails

ABSTRACT F12 human immunodeficiency virus type 1 (HIV-1) nef is a naturally occurring nef mutant cloned from the provirus of a nonproductive, nondefective, and interfering HIV-1 variant (F12-HIV). We have already shown that cells stably transfected with a vector expressing the F12-HIV nef allele do not downregulate CD4 receptors and, more peculiarly, become resistant to the replication of wild type (wt) HIV. In order to investigate the mechanism of action of such an HIV inhibition, the F12-HIVnef gene was expressed in the context of the NL4-3 HIV-1 infectious molecular clone by replacing the wt nef gene (NL4-3/chi). Through this experimental approach we established the following. First, NL4-3/chi and nef-defective (Δnef) NL4-3 viral particles behave very similarly in terms of viral entry and HIV protein production during the first replicative cycle. Second, no viral particles were produced from cells infected with NL4-3/chi virions, whatever the multiplicity of infection used. The viral inhibition apparently occurs at level of viral assembling and/or release. Third, this block could not be relieved by in-trans expression of wt nef. Finally, NL4-3/chi reverts to a producer HIV strain when F12-HIV Nef is deprived of its myristoyl residue. Through a CD4 downregulation competition assay, we demonstrated that F12-HIV Nef protein potently inhibits the CD4 downregulation induced by wt Nef. Moreover, we observed a redistribution of CD4 receptors at the cell margin induced by F12-HIV Nef. These observations strongly suggest that F12-HIV Nef maintains the ability to interact with the intracytoplasmic tail of the CD4 receptor molecule. Remarkably, we distinguished the intracytoplasmic tails of Env gp41 and CD4 as, respectively, viral and cellular targets of the F12-HIV Nef-induced viral retention. For the first time, the inhibition of the viral life cycle by means of in-cis expression of a Nef mutant is here reported. Delineation of the F12-HIV Nef mechanism of action may offer additional approaches to interference with the propagation of HIV infection.

Nef from Human Immunodeficiency Virus Type 1F12 Inhibits Viral Production and Infectivity

ABSTRACT Human immunodeficiency virus type 1F12(HIV-1F12) interferes with the replication of other strains of HIV. Its accessory protein, Nef, is sufficient for this phenotype, where the production and infectivity of HIV are impaired significantly. The analysis of three rare mutations in this Nef protein revealed that these effects could be separated genetically. Moreover, the defect in virus production correlated with the lack of processing of the p55Gag precursor in the presence of Nef from HIV-1F12. Importantly, the introduction of one of these mutations (E177G) into Nef from HIV-1NL4-3 also created a dominant-negative Nef protein. Effects of Nef from HIV-1F12on virus production and Gag processing correlated with its altered subcellular distribution. Moreover, the association with two new cellular proteins with molecular masses of 74 and 75 kDa, which do not interact with other Nef proteins, correlated with the decreased virion infectivity. The identification of a dominant-negative protein for the production and infectivity of HIV suggests that Nef plays an active role at this stage of the viral replicative cycle.

Genetic and functional analysis of the human immunodeficiency virus (HIV) type 1-inhibiting F12-HIVnef allele

The primary human immunodeficiency virus type 1 (HIV-1) Nef mutant F12-HIVNef is characterized by three rare amino acid substitutions, G(140)E, V(153)L and E(177)G. It was reported previously that the expression of F12-HIVNef in the context of the highly productive NL4-3 HIV-1 strain blocks virus replication at the level of virus assembly and/or release by a mechanism depending on the presence of the CD4 intracytoplasmic tail. Here, it is reported that NL4-3 HIV-1 strains expressing F12-HIVnef alleles that were back-mutated in each amino acid substitution readily replicated in CD4(+) cells. Attempting to correlate possible functional alterations with antiviral effects, both F12-HIVNef and its back mutants were tested in terms of well-characterized markers of Nef expression. Both F12-HIVNef and its G(177)E back mutant did not down-regulate CD4 as the consequence of a greatly reduced rate of CD4 internalization. On the other hand, F12-HIVNef as well as the E(140)G and L(153)V back mutants failed to activate the p62 Nef-associated kinase (p62NAK). Thus, only F12-HIVNef was defective in both accelerated rates of CD4 internalization and p62NAK activation, whereas at least one Nef function was restored in all of the back mutants. Infection of cells expressing Nef-resistant CD4 molecules with HIV-1 strains encoding F12-HIVNef back mutants showed that both the lack of accelerated CD4 endocytosis and an, as yet, still unidentified function are required for the F12-HIVNef inhibitory phenotype. These results provide a detailed functional analysis of the F12-HIVnef allele and support the idea that both CD4 accelerated internalization and p62NAK activation are part of the essential steps in the virus replication cycle.

Generation and characterization of a stable cell population releasing fluorescent HIV-1-based Virus Like Particles in an inducible way

BackgroundThe availability of cell lines releasing fluorescent viral particles can significantly support a variety of investigations, including the study of virus-cell interaction and the screening of antiviral compounds. Regarding HIV-1, the recovery of such biologic reagents represents a very hard challenge due to the intrinsic cytotoxicity of many HIV-1 products. We sought to overcome such a limitation by using a cell line releasing HIV-1 particles in an inducible way, and by exploiting the ability of a HIV-1 Nef mutant to be incorporated in virions at quite high levels.ResultsHere, we report the isolation and characterization of a HIV-1 packaging cell line, termed 18-4s, able to release valuable amounts of fluorescent HIV-1 based Virus-Like Particles (VLPs) in an inducible way. 18-4s cells were recovered by constitutively expressing the HIV-1 NefG3C mutant fused with the enhanced-green fluorescent protein (NefG3C-GFP) in a previously isolated inducible HIV-1 packaging cell line. The G3C mutation creates a palmitoylation site which results in NefG3C-GFP incorporation into virions greatly exceeding that of the wild type counterpart. Upon induction of 18-4s cells with ponasterone A and sodium butyrate, up to 4 μg/ml of VLPs, which had incorporated about 150 molecules of NefG3C-GFP per viral particle, were released into the culture supernatant. Due to their intrinsic strong fluorescence, the 18-4s VLPs were easily detectable by a novel cytofluorometric-based assay developed here. The treatment of target cells with fluorescent 18-4 VLPs pseudotyped with different glycoprotein receptors resulted in these becoming fluorescent as early as two hours post-challenge.ConclusionWe created a stable cell line releasing fluorescent HIV-1 based VLPs upon induction useful for several applications including the study of virus-cell interactions and the screening of antiviral compounds.

THE NON-PRODUCER PHENOTYPE OF THE HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 PROVIRUS F12/HIV-1 IS THE RESULT OF MULTIPLE GENETIC VARIATIONS

A cell clone (Hut-78/F12) chronically infected with a non-producer human immunodeficiency virus type 1 (HIV-1) variant showed an abnormal pattern of virus structural proteins and released no detectable virus particles. Exchanges of homologous parts of the F12/HIV provirus and a replication-competent HIV (strain NL4-3) were undertaken to define the genetic determinants of the F12/HIV phenotype. The non-infectious phenotype was reproduced by replacing an NL4-3 genomic fragment encoding the C terminus of gp 120 and the N terminus of gp41 with the corresponding parts of the F12/HIV provirus. Conversely, a much more extended genomic fragment (encompassing the vif, pol and env genes) was necessary to convert the F12/HIV phenotype. These results demonstrate that the F12/HIV non-producer phenotype is the result of mutations scattered along most of the genome, rendering the conversion to an infectious phenotype a very unlikely event. The F12/HIV genome is thus a reliable model for preclinical studies of anti-HIV gene therapy.

FULL EXPRESSION OF TRANSFECTED NONPRODUCER INTERFERING HIV-1 PROVIRAL DNA ABROGATES SUSCEPTIBILITY OF HUMAN HE-LA CD4+ CELLS TO HIV

An Hut-78 cell clone (F12) harboring a nonproducer human immunodeficiency virus-1 (HIV) variant, and showing a complete resistance to HIV-1 or HIV-2 superinfection, was previously characterized. We demonstrated that the replication of the superinfecting HIVs is blocked at the retrotranscription step, despite the CD4 down-regulation, since HIVs are able to cross the Hut-78/F12 cell membrane. In order to establish if the expression of the HIV-1 variant (F12/HIV) could be per se sufficient to induce the homologous viral interference shown in the F12 cells, the whole F12/HIV provirus was cloned and transfected in He-La CD4+ cells. In F12/HIV expressing He-La CD4+ clones, both the viral proteins expressed and the HIV nonproducer phenotype remain unmodified compared to F12 cells. Furthermore, despite the full expression of CD4 HIV receptors, the life cycle of the superinfecting HIV could be either strongly inhibited or totally abolished, depending on the cell clone considered. The inhibition of the superinfecting HIV was also reproduced when an HIV infectious molecular clone was transfected in F12/HIV He-La CD4+ clones, thus indicating that a post-cDNA synthesis block may operate against the superinfecting HIV. These data demonstrate that HIV susceptibility could be abrogated in cells expressing the F12/HIV genome, even in absence of any CD4 down-regulation.

Aberrant, noninfectious HIV-1 particles are released by chronically infected human T cells transduced with a retroviral vector expressing an interfering HIV-1 Variant

The expression of the nonproducer F12-HIV-1 genome has been previously shown to protect the host cell from HIV superinfection. In order to estimate the efficacy of the F12-HIV genome as an anti-HIV reagent also in cells already infected, an HIV-1 chronically infected Hut-78 cell clone (D10) was superinfected with an amphotropic mouse/human pseudotype retrovirus whose genome expresses both the F12-HIV genome and the selection marker gene (ie the c-DNA of a truncated form of the nerve growth factor receptor, NGFr) under the control of F12-HIV 5′LTR. D10 cells homogenously expressing the F12-HIV genome (T-D10) released unaltered amounts of retrovirions whose infectivity was, however, dramatically impaired (from 9 × 103 in D10 to <100.5 TCID50/ml in T-D10 super- natants). Electron microscopy showed that the morphology of retrovirions released by T-D10 cells was heavily altered, both in size and shape. Furthermore, no retrotranscription products were detectable in CD4+ cells challenged with T-D10 retrovirions. For the first time, the block in the infectivity of HIV released from already infected cells through the expression of an anti-HIV retroviral vector was demonstrated. These data could have important implications both from a perspective of F12-HIV-based anti-HIV gene therapy and, in general, on the role that nonproducer and/or defective HIV could play ‘in vivo’ in HIV infection and AIDS pathogenesis.

Maternal mortality in Italy: Results and perspectives of record‐linkage analysis

Accurate estimates and reliable classification of maternal deaths are imperative steps in the chain of actions targeted at reducing avoidable maternal mortality. The aims of this study were to estimate the maternal mortality ratio (MMR) in 10 Italian regions covering 77% of total national births and to identify the most suitable approach to classify the causes of death.