Novel factors interfering with human immunodeficiency virus-type 1 replication in vivo and in vitro.

The strategy of all retroviral infections is based on establishing an equilibrium between virus replication and proviral latency in the infected host. The human immunodeficiency virus-type 1 (HIV-1), belonging to the subfamily of lentiviridae, adds an additional level of sophistication to this general rule by encoding two regulatory genes (tat and rev) and four accessory genes (nef, vif, vpr and vpu); HIV-2, structurally similar to HIV-1 but characterized by lower pathogenicity in vivo, encodes another accessory gene, vpx. The function of these accessory genes has become clear in recent years: they serve as countermeasures to host-cell restriction factors that prevent or curtail the capacity of HIV to productively infect its target cells (typically, CD4+ T lymphocytes, macrophages and dendritic cells). Some of the best characterized restriction factors for HIV-1 are Tripartite Motif-5α (TRIM5α), preventing infection of nonhuman primates, although not being effective in humans, and apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (APOBEC 3G), counteracted by the viral accessory protein Vif. In addition, several other molecules are under scrutiny for their mechanism of action and potential exploitation as novel anti-HIV agents. This review will summarize the recently emerging knowledge on these novel factors and their potential relevance for the discovery of new anti-HIV agents targeting not only the replicative, but also the latent state of HIV infection.

Iron-magnesium silicate bioweathering on Earth (and Mars?).

We examined the common, iron-magnesium silicate minerals olivine and pyroxene in basalt and in mantle rocks to determine if they exhibit textures similar to bioweathering textures found in glass. Our results show that weathering in olivine may occur as long, narrow tunnels (1-3 microm in diameter and up to 100 microm long) and as larger irregular galleries, both of which have distinctive characteristics consistent with biological activity. These weathering textures are associated with clay mineral by-products and nucleic acids. We also examined olivine and pyroxene in martian meteorites, some of which experienced preterrestrial aqueous alteration. Some olivines and pyroxenes in the martian meteorite Nakhla were found to contain tunnels that are similar in size and shape to tunnels in terrestrial iron-magnesium silicates that contain nucleic acids. Though the tunnels found in Nakhla are similar to the biosignatures found in terrestrial minerals, their presence cannot be used to prove that the martian alteration features had a biogenic origin. The abundance and wide distribution of olivine and pyroxene on Earth and in the Solar System make bioweathering features in these minerals potentially important new biosignatures that may play a significant role in evaluating whether life ever existed on Mars.

Interleukin-6 and glucocorticoids synergistically induce human immunodeficiency virus type-1 expression in chronically infected U1 cells by a long terminal repeat independent post-transcriptional mechanism.

BACKGROUND: Glucocorticoids (GC) such as dexamethasone (Dex) can directly upregulate human immunodeficiency virus type-1 (HIV-1) replication in acutely infected cells and potentiate HIV expression from chronically infected promonocytic U1 cells stimulated with tumor necrosis factor-alpha (TNF-alpha). We have here investigated the potential effect of Dex in U1 cells stimulated with interleukin-6 (IL-6), a cytokine inducing virus expression by acting mostly at a post-transcriptional level on the virus life cycle. MATERIALS AND METHODS: Virus production in culture supernatants was evaluated by reverse transcriptase (RT) activity. GC receptor expression was tested by both binding of [3H]-Dexamethasone 21-mesylate and Northern blotting. Cell-associated HIV protein expression was analyzed by Western blotting, whereas both HIV and monocyte chemoattractant protein-1 (MCP-1) RNA accumulation were evaluated by Northern blotting. HIV transcription was tested by long terminal repeat (LTR) chloramphenicol acetyl transferase (CAT) assay after transient transfection of U1 or U937 cells. Formation of activating protein-1 (AP-1) DNA binding complex in nuclear cell extracts was visualized by electrophoretic mobility shift assay (EMSA), whereas ERK1/2 mitogen-activated protein kinase (MAPK) phosphorylation was studied by Western blotting. RESULTS: IL-6 and Dex synergistically induced HIV expression in U1 cells, and this effect was blocked by RU 486. No substantial HIV RNA accumulation was demonstrated in U1 cells co-stimulated with IL-6 and Dex, whereas IL-6 upregulated the expression of MCP-1 RNA, and this effect was inhibited by Dex. In contrast, Dex potentiated IL-6 induced activation of AP-1 and ERK1/2 MAPK phosphorylation, as revealed by EMSA. HIV-1 LTR driven transcription was observed in U1 cells stimulated with TNF-alpha and this effect was potentiated by Dex. In sharp contrast, no induction of LTR-directed CAT activity was observed in transfected U1 cells (or in their parental uninfected U937 cells) stimulated with IL-6 and Dex either alone or in combination. CONCLUSIONS: High levels of virion production can be induced in latently infected cells by stimulation with IL-6 and Dex in the absence of activation of the HIV LTR or viral transcription in spite of activation of both ERK1/2 MAPK and AP-1. These findings suggest the existence of LTR-independent pathways influenced by cytokine and GC through which HIV can maintain substantial levels of protein expression and virion production.

Spreading of HIV-specific CD8+ T-cell repertoire in long-term nonprogressors and its role in the control of viral load and disease activity.

Long-term non-progressors (LTNP) represent a minority of human immunodeficiency virus (HIV) infected individuals characterized by stable or even increasing CD4+ T-cell count and by stronger immune responses against HIV than progressors. In this study, HIV-specific effector CD8+ T cells, as detected by both a sensitive ex vivo enzyme-linked immunospot (ELISPOT) assay and specific major histocompatibility complex (MHC) peptide tetramers, were at a low frequency in the peripheral blood of LTNP, and recognized a lower number of HIV peptides than their memory resting cell counterparts. Both factors may account for the lack of complete HIV clearance by LTNP, who could control the viral spread, and displayed a higher magnitude of cytotoxic T lymphocyte (CTL) responses than progressors. By combining cell purification and ELISPOT assays this study demonstrates that both effector and memory resting cells were confined to a CD8+ population with memory CD45RO+ phenotype, with the former being CD28- and the latter CD28+. Longitudinal studies highlighted a relatively stable HIV-specific effector repertoire, viremia, and CD4+ T-cell counts, which were all correlated with maintenance of nonprogressor status. In conclusion, the analysis of HIV-specific cellular responses in these individuals may help define clear correlates of protective immunity in HIV infection.

The binding subunit of pertussis toxin inhibits HIV replication in human macrophages and virus expression in chronically infected promonocytic U1 cells.

We have recently shown that the binding subunit of pertussis toxin (PTX-B) inhibits the entry and replication of macrophage-tropic (R5) HIV-1 strains in activated primary T lymphocytes. Furthermore, PTX-B suppressed the replication of T cell-tropic (X4) viruses at a postentry level in the same cells. In this study we demonstrate that PTX-B profoundly impairs entry and replication of the HIV-1(ADA) (R5), as well as of HIV pseudotyped with either murine leukemia virus or vesicular stomatitis virus envelopes, in primary monocyte-derived macrophages. In addition, PTX-B strongly inhibited X4 HIV-1 replication in U937 promonocytic cells and virus expression in the U937-derived chronically infected U1 cell line stimulated with cytokines such as TNF-alpha and IL-6. Of interest, TNF-alpha-mediated activation of the cellular transcription factor NF-kappaB was unaffected by PTX-B. Therefore, PTX-B may represent a novel and potent inhibitor of HIV-1 replication to be tested for efficacy in infected individuals. In support of this proposition, a genetically modified mutant of PTX (PT-9K/129G), which is safely administered for prevention of Bordetella pertussis infection, showed an in vitro anti-HIV profile superimposable to that of PTX-B.

Tumor necrosis factor-alpha drives HIV-1 replication in U937 cell clones and upregulates CXCR4.

U937 cell clones in which efficient (plus) vs poor (minus) replication of HIV-1 occurs have been described. We evaluated the role of host factors in their differential ability to support HIV-1 replication. Plus clones constitutively produced TNF-alpha and viral replication was inhibited by neutralization of endogenous TNF-alpha. However, HIV-1 replication was strongly upregulated in minus clones by exogenous TNF-alpha, which also further accelerated the kinetics of infection in plus clones. We observed an increased accumulation of proviral DNA within one round of HIV-1 replication following TNF-a treatment of plus cells. This effect was associated with increased surface density of CXCR4 in both plus and minus clones. Our results identify TNF-alpha as one correlate that contributes to the higher ability of U937-plus clones to sustain HIV-1 replication. Furthermore, we suggest that TNF-alpha may affect steps of the viral life cycle that occur earlier than transcription and also enhance HIV-1 replication by increasing the surface density of CXCR4.

Selective inhibition of HIV replication in primary macrophages but not T lymphocytes by macrophage-derived chemokine.

Macrophage-derived chemokine (MDC) has been reported to inhibit different HIV-1 strains in activated peripheral blood mononuclear cells (T cell blasts), although other investigators have not confirmed these findings. Here we demonstrate that MDC inhibits the replication of CCR5-dependent (R5) HIV-1(BaL) in monocyte-derived macrophages (MDM), but not in T cell blasts, although with variable potency depending on donor variability. Analysis of HIV-1(BaL) proviral DNA synthesis in MDM indicated that the suppressive effect of MDC did not involve inhibition of early events such as entry or reverse transcription. Finally, an inverse correlation was observed between the levels of endogenous MDC secreted by uninfected MDM of different donors and the efficiency of different HIV strains, including two primary isolates with different coreceptor usage, to replicate in these cells. Thus, MDC represents an example of a chemokine inhibiting HIV replication in macrophages acting at one or more postentry levels in the virus life cycle.

HTLV-II down-regulates HIV-1 replication in IL-2-stimulated primary PBMC of coinfected individuals through expression of MIP-1alpha.

The influence of human T-cell leukemia/lymphoma virus type II (HTLV-II) in individuals also infected with HIV-1 is poorly understood. To evaluate the reciprocal influence of HTLV-II and HIV-1 infection, primary peripheral blood mononuclear cell (PBMC) cultures from coinfected individuals were established in the presence of interleukin 2 (IL-2). In these cultures, the kinetics of HTLV-II replication always preceded those of HIV-1. Noteworthy, the kinetics of HIV-1 production were inversely correlated to the HTLV-II proviral load in vivo and its replication ex vivo. These observations suggested a potential interaction between the 2 retroviruses. In this regard, the levels of IL-2, IL-6, and tumor necrosis factor-alpha (TNF-alpha) were measured in the same coinfected PBMC cultures. Endogenous IL-2 was not produced, whereas IL-6 and TNF-alpha were secreted at levels compatible with their known ability to up-regulate HIV-1 expression. The HIV-suppressive CC-chemokines RANTES, macrophage inflammatory protein-1alpha (MIP-1alpha), and MIP-1beta were also determined in IL-2-stimulated PBMC cultures. Of interest, their kinetics and concentrations were inversely related to those of HIV-1 replication. Experiments were performed in which CD8(+) T cells or PBMCs from HTLV-II monoinfected individuals were cocultivated with CD4(+) T cells from HIV-1 monoinfected individuals separated by a semipermeable membrane in the presence or absence of antichemokine neutralizing antibodies. The results indicate that HTLV-II can interfere with the replicative potential of HIV-1 by up-regulating viral suppressive CC-chemokines and, in particular, MIP-1alpha. This study is the first report indicating that HTLV-II can influence HIV replication, at least in vitro, via up-regulation of HIV-suppressive chemokines. (Blood. 2000;95:2760-2769)

Envelope-dependent restriction of human immunodeficiency virus type 1 spreading in CD4(+) T lymphocytes: R5 but not X4 viruses replicate in the absence of T-cell receptor restimulation.

The human immunodeficiency virus (HIV) replicates in activated CD4(+) T lymphocytes. However, only CD4(+) Th2 and Th0, but not Th1, CD4(+) T-cell clones have been reported to efficiently support HIV-1 replication. This dichotomous pattern was further investigated in the present study in Th1, Th2, or Th0 cell lines derived from umbilical human cord blood and in T-cell clones obtained from the peripheral blood mononuclear cells (PBMC) of healthy adults. Both primary and laboratory-adapted HIV-1 strains with CCR5 as the exclusive entry coreceptor (R5 viruses) efficiently replicated in Th1, Th2, and Th0 cells. In sharp contrast, CXCR4-dependent (X4) viruses poorly replicated in both polarized and unpolarized CD4(+) T cells, including adults' PBMC infected several days after mitogenic stimulation. Unlike the X4 HIV-1(NL4-3), a chimera in which the env gene had been replaced with that of the R5 HIV-1(NL(AD8)), efficiently replicated in both Th1 and Th2 cells. This X4-dependent restriction of HIV replication was not explained by either the absence of functional CXCR4 on the cell surface or by the inefficient viral entry and reverse transcription. T-cell receptor stimulation by anti-CD3 monoclonal antibodies fully rescued X4 HIV-1 replication in both Th1 and Th2 cells, whereas it did not alter the extent and kinetics of R5 HIV-1 spreading. Thus, R5 HIVs show a replicative advantage in comparison to X4 viruses in their ability to efficiently propagate among suboptimally activated T lymphocytes, regardless of their polarized or unpolarized functional profiles. This observation may help to explain the absolute predominance of R5 HIVs over X4 viruses observed after viral transmission and during early-stage disease.

Human CD34(+) cells express CXCR4 and its ligand stromal cell-derived factor-1. Implications for infection by T-cell tropic human immunodeficiency virus.

Human CD34(+) hematopoietic progenitor cells obtained from bone marrow (BM), umbilical cord blood (UCB), and mobilized peripheral blood (MPB) were purified and investigated for the expression of the chemokine receptor CXCR4 and its ligand, stromal cell-derived factor-1 (SDF-1). CXCR4 was found present on the cell surface of all CD34(+) cells, although it was expressed at lower density on MPB with respect to BM CD34(+) cells. Freshly isolated and in vitro-cultured CD34(+) cells also coexpressed SDF-1 mRNA, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Of interest, CD34(+)/CD38(+) committed progenitor cells, unlike primitive CD34(+)/CD38(-) cells, expressed SDF-1 mRNA. Supernatants from in vitro-cultured CD34(+) cells contained substantial (3 to 8 ng/mL) amounts of SDF-1 by enzyme-linked immunosorbent assay and induced migration of CD34(+) cells. Because CD34(+) cells express low levels of CD4, the primary receptor of the human immunodeficiency virus (HIV), and CXCR4 is a coreceptor for T-cell tropic (X4) HIV strains, we investigated the susceptibility of CD34(+) cells to infection by this subset of viruses. Lack of productive infection was almost invariably observed as determined by a conventional RT activity in culture supernatants and by real-time PCR for HIV DNA in CD34(+) cells exposed to both laboratory adapted (LAI) and primary (BON) X4 T-cell tropic HIV-1 strain. Soluble gp120 Env (sgp120) from X4 HIV-1 efficiently blocked binding of the anti-CD4 Leu3a monoclonal antibody (MoAb) to either human CD4(+) T cells or CD34(+) cells. In contrast, sgp120 interfered with an anti-CXCR4 MoAb binding to human T lymphocytes, but not to CD34(+) cells. However, CXCR4 on CD34(+) cells was downregulated by SDF-1. These results suggest that CXCR4 and its ligand SDF-1 expressed in CD34(+) progenitors may play an important role in regulating the local and systemic trafficking of these cells. Moreover, these findings suggest multiple and potentially synergistic mechanisms at the basis of the resistance of CD34(+) cells to X4 HIV infection, including their ability to produce SDF-1, and the lack of CXCR4 internalization following gp120 binding to CD4.

Human immunodeficiency virus replication induces monocyte chemotactic protein-1 in human macrophages and U937 promonocytic cells.

We have recently described a significant correlation between human immunodeficiency virus-1 (HIV-1) RNA replication and monocyte chemotactic protein-1 (MCP-1) levels in the cerebrospinal fluid (CSF) of individuals with the acquired immunodeficiency syndrome (AIDS) with HIV encephalitis (E). Because local macrophages (microglia) are the cells predominantly infected in the brain, we investigated whether in vitro HIV infection affects MCP-1 production in mononuclear phagocytes (MP). MCP-1 secretion and expression were consinstently upregulated over constitutive levels in human monocyte-derived macrophages (MDM) infected with the M-tropic R5 BaL strain of HIV-1. HIV replication was required for this effect, as demonstrated by the absence of chemokine upregulation after infection in the presence of 3'-azido-3'-deoxythimidine (AZT) or cell-exposure to heat-inactivated (triangle up degrees ) virus. MCP-1 induction was not restricted to HIV-1 BaL, but was also observed during productive infection of MDM with two primary isolates differing for entry coreceptor usage and of U937 cells with the X4 HIV-1 MN strain. Based on the observation that exogenous HIV-1 Tat induced MCP-1 expression in astrocytes, we also investigated its role in MDM and U937 cells. Exogenous Tat induced MCP-1 production from MDM in a concentration-dependent manner, however, it was not effective on uninfected U937 cells or on the chronically infected U937-derived cell line U1. Transfection of Tat-expressing plasmids moderately activated HIV expression in U1 cells, but failed to induce MCP-1 expression in this cell line or in uninfected U937 cells. HIV replication-dependent expression of MCP-1 in MP may be of particular relevance for the pathogenesis of HIV infection in nonlymphoid organs such as the brain.

Role of pro-inflammatory cytokines and beta-chemokines in controlling HIV replication.

Several members of the cytokine network play an important role in controlling the replication of the human immunodeficiency virus (HIV) in several experimental systems. Their effects can be categorized in the following three functional groups: (1) HIV-inductive cytokines; (2) HIV-suppressive cytokines; (3) cytokines with both activating and inhibiting capacities. Studies on the mechanism of action of these molecules have highlighted the fact that several steps of the retrovirus life cycle, from binding to budding of progeny virions from the infected cell, are affected by cytokines. This general concept has been recently substantiated by the discovery that certain beta-chemokines can act as blockers of viral entry by interfering with HIV co-receptors. Finally, it is important to recognize that cytokines have gone beyond their role as potential pathogenetic or protective endogenous cofactors in HIV replication and disease progression, and are becoming experimental therapeutic agents for HIV disease, best illustrated thus far by the case of interleukin-2.

Experiences in immune reconstitution. The rationale for interleukin-2 administration to HIV-infected individuals.

Since the clinical earliest descriptions of patients with acquired immune deficiency syndrome (AIDS) it has been very clear that a profound state of immunologic dysfunction was the underlying cause of the emergence of life-threatening opportunistic infections and tumors. In addition to the progressive loss of CD4 "helper" T lymphocytes, a profound defect in interleukin-2 (IL-2) production was recognized as a major pathogenic component of the new disease. For these reasons, attempts to administer IL-2 to individuals infected with the human immunodeficiency virus (HIV), the causative agent of AIDS, have been made since the mid eighties, however with little success. On the other hand, the propensity of HIV to replicate in activated lymphocytes and macrophages, under the influence of the cytokine network, has represented, and in part still does, a major hurdle for the rationale of administering IL-2 or other cytokines to HIV-infected individuals. Major steps forward towards an understanding of the role of multiple components of the immune system, coupled with a potentially successful protocol of IL-2 administration in vivo, resulting in the stable uprising of circulating CD4+ T cells, shed an optimistic light on the possibility to achieve a substantial immune reconstitution in HIV-infected individuals, thus preventing the onset of AIDS.

Selective elevation of monocyte chemotactic protein-1 in the cerebrospinal fluid of AIDS patients with cytomegalovirus encephalitis.

The CC chemokine monocyte chemotactic protein-1 (MCP-1) was markedly elevated in the cerebrospinal fluid (CSF) of human immunodeficiency virus (HIV)-infected patients with cytomegalovirus (CMV) encephalitis. The MCP-1 CSF levels in CMV encephalitis were markedly higher than those in the CSF of HIV-infected patients with or without unrelated neurologic diseases, including progressive multifocal leukoencephalopathy, cryptococcal meningitis, toxoplasmic encephalitis, and primary lymphoma. Interleukin-8, RANTES, macrophage inflammatory protein (MIP)-1 alpha, and MIP-1 beta were not substantially increased in the CSF of CMV encephalitis patients. High levels of MCP-1 may underlie monocyte recruitment and tissue damage in CMV encephalitis and may represent a rapid and useful tool in the diagnostic armamentarium for neurologic disorders associated with HIV infection.

Cytokines in the acquired immunodeficiency syndrome and other infectious diseases.

The pathogenesis of several infectious diseases is frequently related not only to their etiological agents of viral, bacterial, or parasitic nature, but also to the host immune response. Both inflammatory responses and specific immune responses to the invading microorganisms are controlled by complex networks of intercellular signalling molecules, namely cytokines. This rapidly growing family of mediators includes lymphokines, interleukins, and molecules such as tumor necrosis factors and interferons. Patterns of cytokine production from antigen- or allergen-specific T lymphocytic cell clones have been identified, firstly in animal models and subsequently in man, and are commonly referred to as TH0, TH1, and TH2 profiles. The predominance of one of these profiles strongly influences the type of immune response (humoral versus cellular) and, at least in some experimental models, whether the immune response is protective or harmful. This is most convincingly demonstrated in models of parasitic diseases, but has also been hypothesized to be involved in the pathogenesis of human immunodeficiency virus infection and the acquired immunodeficiency syndrome. Earlier studies on human immunodeficiency virus infection revealed that the replicative capacity of this retrovirus, like the herpes viruses, is strongly influenced by the cytokine milieu of infected cells. Transcriptional and post-transcriptional regulation of human immunodeficiency virus expression by human cytokines are examples of the complex interdigitation of viruses with the host immune system.

Ultraviolet irradiation and cytokines as regulators of HIV latency and expression.

The ability of the human immunodeficiency virus (HIV) to persist and replicate in human CD4+ T lymphocytes and mononuclear phagocytes is under the control of both virally encoded proteins and a variety of host-related factors. Ultraviolet (UV) light has been shown to induce transcription and expression of HIV. Both DNA damage and repair and DNA damage/repair-independent pathways caused by UV irradiation lead to expression of proviral HIV genomes via activation of the cellular transcription factor NF-kappa B. Transgenic mice that contain either long terminal repeat (LTR)-reporter genes or HIV genomes, either full length or deleted in the gag-pol region, express RNA and proteins at the epidermal level, particularly after UV irradiation. Furthermore, UV-triggered release of soluble factors capable of inducing expression of HIV in non-irradiated cells has been observed. Among other host factors, the functional network of pro-inflammatory and immunoregulatory cytokines has been demonstrated to act as a potent regulator of HIV replication, at least in different in vitro systems of infection.

Ultraviolet radiation increases HIV-long terminal repeat-directed expression in transgenic mice.

Previously described FVB/N mice harboring a human immunodeficiency virus (HIV) long terminal repeat (LTR)/chloramphenicol acetyl transferase (CAT) transgene were treated with varying amounts of 254 nm UV-C radiation or 312 nm UV-B radiation. At optimal exposure periods, a 20-fold increase in HIV-LTR-directed expression was observed in ear specimens collected 24 h following UV-C exposure; a fourfold increase in expression was induced by UV-B exposure. Investigation of the kinetics of UV-C induction in vivo revealed that LTR-directed gene expression began to increase 2 hours after exposure and reached a maximum on Day 3 following exposure (greater than 30-fold induction). In experiments examining the kinetics of UV-B activation, the maximum level of CAT activity in the ears of irradiated transgenic animals was fivefold above levels in unirradiated transgenic controls (Day 5). Furthermore, CAT activity was not induced in fur-bearing skin following UV exposure; however, a fourfold increase in HIV-LTR-directed expression could be elicited when hair was removed by shaving prior to UV-B treatment.

Transforming growth factor alpha: an aromatic side chain at position 38 is essential for biological activity.

Site-directed mutagenesis has been performed in the human transforming growth factor alpha gene. When tyrosine 38 is mutated into phenylalanine or tryptophane, biological activity is retained. In contrast, other alterations between cysteine 34 and cysteine 43 and disruption of disulfide bonds 8 to 21 and 34 to 43 resulted in loss of activities. The presence of an aromatic side chain at position 38 of transforming growth factor alpha seems to be essential for its activity.