Public health. A sound rationale needed for phase III HIV-1 vaccine trials.
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AIDS Vaccines↗
Biomedical subjects
Mario Stevenson
Publications and source records attributed to Mario Stevenson.
Characterization of restrictions to human immunodeficiency virus type 1 infection of monocytes.
Tissue macrophages are an important cellular reservoir for replication of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus. In vitro, the ability of macrophages to support viral replication is differentiation dependent in that precursor monocytes are refractory to infection. There is, however, no consensus as to the exact point at which infection is restricted in monocytes. We have revisited this issue and have compared the efficiencies of early HIV-1 replication events in monocytes and in differentiated macrophages. Although virus entry in monocytes was comparable to that in differentiated macrophages, synthesis of full-length viral cDNAs was very inefficient. Relative to differentiated macrophages, monocytes contained low levels of dTTP due to low thymidine phosphorylase activity. Exogenous addition of D-thymidine increased dTTP levels to that in differentiated macrophages but did not correct the reverse transcription defect. These results point to a restriction in monocytes that is independent of reverse transcription precursors and suggest that differentiation-dependent cellular cofactors of reverse transcription are rate limiting in monocytes.
Cell Differentiation↗
Inhibition of human immunodeficiency virus type 1 replication by RNA interference directed against human transcription elongation factor P-TEFb (CDK9/CyclinT1).
The human positive transcription elongation factor P-TEFb is composed of two subunits, cyclin T1 (hCycT1) and CDK9, and is involved in transcriptional regulation of cellular genes as well as human immunodeficiency virus type 1 (HIV-1) mRNA. Replication of HIV-1 requires the Tat protein, which activates elongation of RNA polymerase II at the HIV-1 promoter by interacting with hCycT1. To understand the cellular functions of P-TEFb and to test whether suppression of host proteins such as P-TEFb can modulate HIV infectivity without causing cellular toxicity or lethality, we used RNA interference (RNAi) to specifically knock down P-TEFb expression by degrading hCycT1 or CDK9 mRNA. RNAi-mediated gene silencing of P-TEFb in HeLa cells was not lethal and inhibited Tat transactivation and HIV-1 replication in host cells. We also found that CDK9 protein stability depended on hCycT1 protein levels, suggesting that the formation of P-TEFb CDK-cyclin complexes is required for CDK9 stability. Strikingly, P-TEFb knockdown cells showed normal P-TEFb kinase activity. Our studies suggest the existence of a dynamic equilibrium between active and inactive pools of P-TEFb in the cell and indicate that this equilibrium shifts towards the active kinase form to sustain cell viability when P-TEFb protein levels are reduced. The finding that a P-TEFb knockdown was not lethal and still showed normal P-TEFb kinase activity suggested that there is a critical threshold concentration of activated P-TEFb required for cell viability and HIV replication. These results provide new insights into the regulation of P-TEFb function and suggest the possibility that similar mechanisms for monitoring protein levels to modulate the activity of proteins may exist for the regulation of a variety of other enzymatic pathways.
Cell Line↗
HIV and cells of macrophage/dendritic lineage and other non-T cell reservoirs: new answers yield new questions.
Defining how human immunodeficiency virus (HIV) interacts with macrophages, dendritic cells (DC), and other non-T cell reservoirs remains a critical area of research despite widespread use in the developed world of highly active antiretroviral therapy. In fact, as highlighted at the Fifth International Workshop on HIV and Cells of Macrophage/Dendritic Lineage and Other Reservoirs, as viral suppression in T cells becomes increasingly effective, these alternative reservoirs may take on even greater relative importance as sites for viral persistence and as a target for purging. These cells may be especially important reservoirs in several critical settings of clinical relevance, and there are major differences in the molecular mechanisms that regulate HIV replication in these cells compared with T cells. Dysfunction of these cells may also play a major role in particular aspects of pathogenesis. Three broad themes emerged from the workshop regarding areas of recent progress, which also serve to identify current research challenges of (i). determining the role played by macrophages, DC, and other non-T cell viral targets in transmission and dissemination and as viral reservoirs at various stages of disease and in different compartments in vivo; (ii). identifying the molecular mechanisms by which virus-cell interactions affect the inflammatory, immune, and other functions of these cells; and (iii). defining the unique pathways that regulate infection and replication in these cellular compartments. This issue of JLB contains several reviews and original reports resulting from the workshop that address recent progress and highlight the current research questions regarding these cell types.
Dendritic Cells↗
HIV-1 Nef intersects the macrophage CD40L signalling pathway to promote resting-cell infection.
All primate lentiviruses (HIV-1, HIV-2, SIV) encode Nef proteins, which are important for viral replication and pathogenicity in vivo. It is not known how Nef regulates these processes. It has been suggested that Nef protects infected cells from apoptosis and recognition by cytotoxic T lymphocytes. Other studies suggest that Nef influences the activation state of the infected cell, thereby enhancing the ability of that cell to support viral replication. Here we show that macrophages that express Nef or are stimulated through the CD40 receptor release a paracrine factor that renders T lymphocytes permissive to HIV-1 infection. This activity requires the upregulation of B-cell receptors involved in the alternative pathway of T-lymphocyte stimulation. T lymphocytes stimulated through this pathway become susceptible to viral infection without progressing through the cell cycle. We identify two proteins, soluble CD23 and soluble ICAM, that are induced from macrophages by Nef and CD40L, and which mediate their effects on lymphocyte permissivity. Our results reveal a mechanism by which Nef expands the cellular reservoir of HIV-1 by permitting the infection of resting T lymphocytes.
B-Lymphocytes↗
HIV-1 replication increases HIV-specific CD4+ T cell frequencies but limits proliferative capacity in chronically infected children.
This study investigated the relationship between HIV-1 replication and virus (HIV-1; CMV)-specific CD4(+) T cell frequency and function in HIV-1-infected children. HIV-1 gag p55-specific CD4(+) T cell IFN-gamma responses were detected in the majority of children studied. p55-specific responses were detected less commonly and at lower frequencies in children with <50 copies/ml plasma HIV-1 RNA than in children with active HIV-1 replication. In children with <50 copies/ml plasma HIV-1, p55-specific responses were detected only in children with evidence of ongoing HIV-1 replication, indicating a direct relationship between HIV-1 replication and HIV-specific CD4(+) T cell frequencies. In contrast, p55-specific proliferative responses were detected more frequently in children with <50 copies/ml plasma HIV-1. CMV-specific CD4(+) responses were more commonly detected and at higher frequencies in CMV-coinfected children with suppressed HIV-1 replication. The lack of HIV-specific CD4(+) proliferative responses, along with the preservation of CMV-specific CD4(+) responses in children with controlled HIV-1 replication, suggests that viral replication may have deleterious effects on HIV-1 and other virus-specific CD4(+) responses. Vaccination to stimulate HIV-specific CD4(+) T cell responses in these children may synergize with antiretroviral therapy to improve the long-term control of viral replication, and may perhaps allow the eventual discontinuation of antiretroviral therapy.
Adolescent↗
HIV-1 pathogenesis.
Despite considerable advances in HIV science in the past 20 years, the reason why HIV-1 infection is pathogenic is still debated and the goal of eradicating HIV-1 infection remains elusive. A deeper understanding of the interplay between HIV-1 and its host and why simian immunodeficiency virus (SIV) is nonpathogenic in some natural hosts may provide a few answers.
Animals↗
Dissecting HIV-1 through RNA interference.
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HIV Infections↗
Evidence for a cytopathogenicity determinant in HIV-1 Vpr.
HIV-1 is cytopathic for CD4(+) T lymphocytes in vitro and this property of HIV-1 is generally considered to account for some of its in vivo cytopathogenicity. Thus, the extent of lymphocyte depletion correlates with the level of viremia whereas low levels of viral replication are typically associated with stable lymphocyte levels and asymptomatic infection such as is observed in non-progressors. Here, we describe a non-progressor who did not fit this general pattern in that CD4(+) T lymphocyte homeostasis was maintained in the face of high-level viral replication. Biological viral isolates from this patient replicated in primary lymphocytes without inducing cytopathicity. Because this phenotype is reminiscent of Vpr-deleted viruses, we examined the contribution of the Vpr gene to the viral phenotype. Vpr alleles derived from this patient contained both premature stop codons and an unusual Q3R polymorphism. Insertion of patient-derived Vpr alleles or a Q3R substitution into a cytopathic HIV-1 clone resulted in a marked impairment of cytopathicity without affecting viral replication efficiency. The effect of Vpr on cytopathicity was unrelated to reported activities of Vpr including virion association, interaction with uracil DNA glycosylase, G(2) arrest, or enhancement of macrophage infection but correlated with the ability of Vpr to induce host cell apoptosis. This study suggests the presence of a determinant of in vivo cytopathogenicity within HIV-1 Vpr and further indicates that viral replication can be uncoupled from cytopathicity in vitro and in vivo.
Alleles↗
Modulation of HIV-1 replication by RNA interference.
RNA interference (RNAi) is the process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA in animal and plant cells. In mammalian cells, RNAi can be triggered by 21-nucleotide duplexes of small interfering RNA (siRNA). Here we describe inhibition of early and late steps of HIV-1 replication in human cell lines and primary lymphocytes by siRNAs targeted to various regions of the HIV-1 genome. We demonstrate that synthetic siRNA duplexes or plasmid-derived siRNAs inhibit HIV-1 infection by specifically degrading genomic HIV-1 RNA, thereby preventing formation of viral complementary-DNA intermediates. These results demonstrate the utility of RNAi for modulating the HIV replication cycle and provide evidence that genomic HIV-1 RNA, as it exists within a nucleoprotein reverse-transcription complex, is amenable to siRNA-mediated degradation.
Cell Line↗
Progress toward a human CD4/CCR5 transgenic rat model for de novo infection by human immunodeficiency virus type 1.
The development of a permissive small animal model for the study of human immunodeficiency virus type (HIV)-1 pathogenesis and the testing of antiviral strategies has been hampered by the inability of HIV-1 to infect primary rodent cells productively. In this study, we explored transgenic rats expressing the HIV-1 receptor complex as a susceptible host. Rats transgenic for human CD4 (hCD4) and the human chemokine receptor CCR5 (hCCR5) were generated that express the transgenes in CD4(+) T lymphocytes, macrophages, and microglia. In ex vivo cultures, CD4(+) T lymphocytes, macrophages, and microglia from hCD4/hCCR5 transgenic rats were highly susceptible to infection by HIV-1 R5 viruses leading to expression of abundant levels of early HIV-1 gene products comparable to those found in human reference cultures. Primary rat macrophages and microglia, but not lymphocytes, from double-transgenic rats could be productively infected by various recombinant and primary R5 strains of HIV-1. Moreover, after systemic challenge with HIV-1, lymphatic organs from hCD4/hCCR5 transgenic rats contained episomal 2-long terminal repeat (LTR) circles, integrated provirus, and early viral gene products, demonstrating susceptibility to HIV-1 in vivo. Transgenic rats also displayed a low-level plasma viremia early in infection. Thus, transgenic rats expressing the appropriate human receptor complex are promising candidates for a small animal model of HIV-1 infection.
Animals↗
Developments in basic science research.
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Animals↗
Developments in basic science research. Highlights of the 11th Conference on Retroviruses and Opportunistic Infections, February 8-11, 2004, San Francisco, California, USA.
As with last year's meeting, perhaps the greatest emphasis in the basic science categories was in the area of cellular cofactors that influence HIV-1 replication either positively or negatively. A number of presentations provided detailed insight into the mechanism by which APOBEC 3G, the cellular target of Vif, effects its antiviral activity. The surprising thing is that this antiviral activity is not restricted to primate lentiviruses but is active against retroviruses and even hepatitis viruses. In the area of positive-acting cellular cofactors, the emphasis was on those cellular proteins that facilitate egress of the virus from the infected cell. It is now apparent that viruses such as HIV-1 can bud into cytoplasmic vesicles in order to establish a unique intracellular reservoir. How viruses move between cells was also the focus of several presentations at the meeting, and there were further surprises about the mechanism by which HIV-1 may establish latent infection.
HIV Infections↗