EcoHIV Infection of Primary Murine Brain Cell Cultures to Model HIV Replication and Neuropathogenesis.

BACKGROUND: EcoHIV is a chimeric HIV that replicates in mice in CD4+ T cells, macrophages, and microglia (but not in neurons), causing lasting neurocognitive impairment resembling neurocognitive disease in people living with HIV. The present study was designed to develop EcoHIV-susceptible primary mouse brain cultures to investigate the indirect effects of HIV infection on neuronal integrity. RESULTS: We used two EcoHIV clones encoding EGFP and mouse bone marrow-derived macrophages (BMM), mixed mouse brain cells, or enriched mouse glial cells from two wild-type mouse strains to test EcoHIV replication efficiency, the identity of productively infected cells, and neuronal apoptosis and integrity. EcoHIV replicated efficiently in BMM. In mixed brain cell cultures, EcoHIV targeted microglia but did not cause neuronal apoptosis. Instead, the productive infection of the microglia activated them and impaired synaptophysin expression, dendritic density, and axonal structure in the neurons. EcoHIV replication in the microglia and neuronal structural changes during infection were prevented by culture with an antiretroviral. CONCLUSIONS: In murine brain cell cultures, EcoHIV replication in the microglia is largely responsible for the aspects of neuronal dysfunction relevant to cognitive disease in infected mice and people living with HIV. These cultures provide a tool for further study of HIV neuropathogenesis and its control.

CCL2 is required for initiation but not persistence of HIV infection mediated neurocognitive disease in mice.

HIV enters the brain within days of infection causing neurocognitive impairment (NCI) in up to half of infected people despite suppressive antiretroviral therapy. The virus is believed to enter the brain in infected monocytes through chemotaxis to the major monocyte chemokine, CCL2, but the roles of CCL2 in established NCI are not fully defined. We addressed this question during infection of conventional and CCL2 knockout mice with EcoHIV in which NCI can be verified in behavioral tests. EcoHIV enters mouse brain within 5 days of infection, but NCI develops gradually with established cognitive disease starting 25 days after infection. CCL2 knockout mice infected by intraperitoneal injection of virus failed to develop brain infection and NCI. However, when EcoHIV was directly injected into the brain, CCL2 knockout mice developed NCI. Knockout of CCL2 or its principal receptor, CCR2, slightly reduced macrophage infection in culture. Treatment of mice prior to and during EcoHIV infection with the CCL2 transcriptional inhibitor, bindarit, prevented brain infection and NCI and reduced macrophage infection. In contrast, bindarit treatment of mice 4 weeks after infection affected neither brain virus burden nor NCI. Based on these findings we propose that HIV enters the brain mainly through infected monocytes but that resident brain cells are sufficient to maintain NCI. These findings suggest that NCI therapy must act within the brain.

Efficient Expression of HIV in Immunocompetent Mouse Brain Reveals a Novel Nonneurotoxic Viral Function in Hippocampal Synaptodendritic Injury and Memory Impairment.

HIV causes neurodegeneration and dementia in AIDS patients, but its function in milder cognitive impairments in virologically suppressed patients on antiretroviral therapy is unknown. Such patients are immunocompetent, have low peripheral and brain HIV burdens, and show minimal brain neuropathology. Using the model of HIV-related memory impairment in EcoHIV-infected conventional mice, we investigated the neurobiological and cognitive consequences of efficient EcoHIV expression in the mouse brain after intracerebral infection. HIV integrated and persisted in an expressed state in brain tissue, was detectable in brain monocytic cells, and caused neuroinflammatory responses and lasting spatial, working, and associative memory impairment. Systemic antiretroviral treatment prevented direct brain infection and memory dysfunction indicating the requirement for HIV expression in the brain for disease. Similarly inoculated murine leukemia virus used as a control replicated in mouse brain but not in monocytic cells and was cognitively benign, linking the disease to HIV-specific functions. Memory impairment correlated in real time with hippocampal dysfunction shown by defective long-term potentiation in hippocampal slices ex vivo and with diffuse synaptodendritic injury in the hippocampus reflected in significant reduction in microtubule-associated protein 2 and synapsin II staining. In contrast, there was no evidence of overt neuronal loss in this region as determined by neuron-specific nuclear protein quantification, TUNEL assay, and histological observations. Our results reveal a novel capacity of HIV to induce neuronal dysfunction and memory impairment independent of neurotoxicity, distinct from the neurotoxicity of HIV infection in dementia.IMPORTANCE HIV neuropathogenesis has been attributed in large measure to neurotoxicity of viral proteins and inflammatory factors produced by infected monocytic cells in the brain. We show here that HIV expression in mouse brain causes lasting memory impairment by a mechanism involving injury to hippocampal synaptodendritic arbors and neuronal function but not overt neuronal loss in the region. Our results mirror the observation of minimal neurodegeneration in cognitively impaired HIV patients on antiretroviral therapy and demonstrate that HIV is nonneurotoxic in certain brain abnormalities that it causes. If neurons comprising the cognition-related networks survive HIV insult, at least for some time, there is a window of opportunity for disease treatment.

Intranasal insulin therapy reverses hippocampal dendritic injury and cognitive impairment in a model of HIV-associated neurocognitive disorders in EcoHIV-infected mice.

OBJECTIVE: Almost half of HIV-positive people on antiretroviral therapy have demonstrable mild neurocognitive impairment (HIV-NCI), even when virologically suppressed. Intranasal insulin therapy improves cognition in Alzheimer's disease and diabetes. Here we tested intranasal insulin therapy in a model of HIV-NCI in EcoHIV-infected conventional mice. DESIGN AND METHODS: Insulin pharmacokinetics following intranasal administration to mice was determined by ELISA. Mice were inoculated with EcoHIV to cause NCI; 23 days or 3 months after infection they were treated daily for 9 days with intranasal insulin (2.4 IU/mouse) and examined for NCI in behavioral tests and HIV burdens by quantitative PCR. Some animals were tested for hippocampal neuronal integrity by immunostaining and expression of neuronal function-related genes by real time-quantitative PCR. The effect of insulin treatment discontinuation on cognition and neuropathology was also examined. RESULTS: Intranasal insulin administration to mice resulted in µIU/ml levels of insulin in cerebrospinal fluid with a half-life of about 2 h, resembling pharmacokinetic parameters of patients receiving 40 IU. Intranasal insulin treatment starting 23 days or 3 months after infection completely reversed NCI in mice. Murine NCI correlated with reductions in hippocampal dendritic arbors and downregulation of neuronal function genes; intranasal insulin reversed these changes coincident with restoration of cognitive acuity, but they returned within 24 h of treatment cessation. Intranasal insulin treatment reduced brain HIV DNA when started 23 but not 90 days after infection. CONCLUSION: Our preclinical studies support the use of intranasal insulin administration for treatment of HIV-NCI and suggest that some dendritic injury in this condition is reversible.

EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment.

Suppression of HIV replication by antiretroviral therapy (ART) or host immunity can prevent AIDS but not other HIV-associated conditions including neurocognitive impairment (HIV-NCI). Pathogenesis in HIV-suppressed individuals has been attributed to reservoirs of latent-inducible virus in resting CD4+ T cells. Macrophages are persistently infected with HIV but their role as HIV reservoirs in vivo has not been fully explored. Here we show that infection of conventional mice with chimeric HIV, EcoHIV, reproduces physiological conditions for development of disease in people on ART including immunocompetence, stable suppression of HIV replication, persistence of integrated, replication-competent HIV in T cells and macrophages, and manifestation of learning and memory deficits in behavioral tests, termed here murine HIV-NCI. EcoHIV established latent reservoirs in CD4+ T lymphocytes in chronically-infected mice but could be induced by epigenetic modulators ex vivo and in mice. In contrast, macrophages expressed EcoHIV constitutively in mice for up to 16 months; murine leukemia virus (MLV), the donor of gp80 envelope in EcoHIV, did not infect macrophages. Both EcoHIV and MLV were found in brain tissue of infected mice but only EcoHIV induced NCI. Murine HIV-NCI was prevented by antiretroviral prophylaxis but once established neither persistent EcoHIV infection in mice nor NCI could be reversed by long-acting antiretroviral therapy. EcoHIV-infected, athymic mice were more permissive to virus replication in macrophages than were wild-type mice, suffered cognitive dysfunction, as well as increased numbers of monocytes and macrophages infiltrating the brain. Our results suggest an important role of HIV expressing macrophages in HIV neuropathogenesis in hosts with suppressed HIV replication.

HIV induces expression of complement component C3 in astrocytes by NF-κB-dependent activation of interleukin-6 synthesis.

BACKGROUND: Abnormal activation of the complement system contributes to some central nervous system diseases but the role of complement in HIV-associated neurocognitive disorder (HAND) is unclear. METHODS: We used real-time PCR and immunohistochemistry to detect complement expression in postmortem brain tissue from HAND patients and controls. To further investigate the basis for viral induction of gene expression in the brain, we studied the effect of HIV on C3 expression by astrocytes, innate immune effector cells, and targets of HIV. Human fetal astrocytes (HFA) were infected with HIV in culture and cellular pathways and factors involved in signaling to C3 expression were elucidated using pharmacological pathway inhibitors, antisense RNA, promoter mutational analysis, and fluorescence microscopy. RESULTS: We found significantly increased expression of complement components including C3 in brain tissues from patients with HAND and C3 was identified by immunocytochemistry in astrocytes and neurons. Exposure of HFA to HIV in culture-induced C3 promoter activity, mRNA expression, and protein production. Use of pharmacological inhibitors indicated that induction of C3 expression by HIV requires NF-κB and protein kinase signaling. The relevance of NF-κB regulation to C3 induction was confirmed through detection of NF-κB translocation into nuclei and inhibition through overexpression of the physiological NF-κB inhibitor, I-κBα. C3 promoter mutation analysis revealed that the NF-κB and SP binding sites are dispensable for the induction by HIV, while the proximal IL-1ß/IL-6 responsive element is essential. HIV-treated HFA secreted IL-6, exogenous IL-6 activated the C3 promoter, and anti-IL-6 antibodies blocked HIV activation of the C3 promoter. The activation of IL-6 transcription by HIV was dependent upon an NF-κB element within the IL-6 promoter. CONCLUSIONS: These results suggest that HIV activates C3 expression in primary astrocytes indirectly, through NF-κB-dependent induction of IL-6, which in turn activates the C3 promoter. HIV induction of C3 and IL-6 in astrocytes may contribute to HIV-mediated inflammation in the brain and cognitive dysfunction.

Chronic, highly productive HIV infection in monocytes during supportive culture.

Peripheral blood monocytes of HIV-infected individuals carry virus, constituting one potential reservoir. However, most studies of infection in tissue culture find monocytes refractory to HIV replication, suggesting that culture conditions limit the relative susceptibility of this target cell. We employed a tissue culture system optimized for maintenance of human monocytes without differentiation and compared HIV infection efficiency of monocytes and fully differentiated monocyte derived macrophages (MDM). We tested direct virus-cell fusion, expression of cell lineage markers, and productive HIV infection in fresh monocytes, monocytes after varying periods of supportive culture, and fully differentiated MDM comparing cells from individual donors. Fresh, uncultured monocytes allowed modest HIV fusion, however one week culture was sufficient to allow efficient fusion and an increase in expression of CD14, CD16, CD33, and CD105. Compared to freshly isolated monocytes, monocytes infected after a few days in culture produced p24 more quickly, but the peaks of production were similar. Fresh monocytes were highly susceptible to productive HIV infection in supportive culture, roughly equal to MDM from the same donor in expression of extracellular p24 up to five weeks after infection. Taken together our findings indicate that monocytes are biologically capable of supporting chronic, highly productive HIV infection, a capacity that may reflect their status in HIV-infected persons.

Transmission of chimeric HIV by mating in conventional mice: prevention by pre-exposure antiretroviral therapy and reduced susceptibility during estrus.

Heterosexual transmission accounts for the majority of new human immunodeficiency virus (HIV) cases worldwide. The current approach to investigate HIV heterosexual transmission in animals involves application of virus stock to the vaginal surface, a method that does not reproduce the physiological conditions of vaginal intercourse that influence the rate of transmission. We have previously described efficient infection of conventional mice using EcoHIV/NL4-3 and EcoHIV/NDK, chimeric HIV molecular clones constructed to express all HIV structural and regulatory genes except envelope, which is replaced by a rodent-tropic envelope gene. Here we investigated whether EcoHIV/NDK-infected male mice transmit virus to females during coitus, and the sensitivity of this transmission to HIV pre-exposure prophylaxis and the estrus state. Our general approach was to allow mating between EcoHIV/NDK-infected male mice and uninfected females for 1-7 nights. At 1-6 weeks after mating, mice were euthanized and virus burdens were measured by quantitative PCR (qPCR) amplification of HIV RNA or DNA in peritoneal macrophages, inguinal lymph node cells, spleen cells or vas deferens, or by ELISA for antibodies to HIV Gag. We found that 70-100% of female mice mated to EcoHIV/NDK-infected males acquired infection. Pericoital treatment of females with either 2',3'-dideoxcytidine (ddC) or tenofovir largely prevented their EcoHIV/NDK infection by mating (P<0.05 and P<0.003, respectively). In males, T cells were dispensable for virus transmission. The rate of EcoHIV/NDK sexual transmission to females in estrus declined sharply (P=0.003) but their infection by injection was unaffected, indicating that the local environment in the female reproductive tract influences susceptibility to HIV. We conclude that this system of EcoHIV/NDK transmission during mouse mating reproduces key features of heterosexual transmission of HIV in humans and can be used to investigate its biology and control.

T cells induced by recombinant chimpanzee adenovirus alone and in prime-boost regimens decrease chimeric EcoHIV/NDK challenge virus load.

The popularity of nonreplicating adenoviruses of chimpanzee origin (ChAdVs) as vectors for subunit vaccines is on the rise. This is mainly for their excellent safety and impressive immunogenicity observed in human studies to date. Here, we recloned the chimpanzee adenovirus sero type 68 (ChAdV-68), also designated SAdV-25 and AdC68, genome and demonstrated its straightforward genetic manipulation facilitated by the use of bacterial artificial chromosome recombineering. To generate the ChAdV68.GagB vaccine, the HIV-1 consensus clade B Gag-derived Tg was inserted into the E1 region. In part confirming previous observations, the ChAdV68.GagB vaccine alone and in heterologous prime-boost regimens with plasmid DNA- and modified vaccinia virus Ankara (MVA)-vectored vaccines induced robust polyfunctional HIV-1-specific CD8(+) and CD4(+) T-cell responses with a gut-homing phenotype. Importantly, we showed that when a single epitope is expressed as an immunodominant CD8(+) T-cell determinant, responses elicited by ChAdV68.GagB alone and in combination lowered surrogate challenge EcoHIV/NDK (where EcoHIV is chimeric ecotropic HIV) virus load in mice both at the peak T-cell frequencies 2 weeks after vaccination and 16 weeks later indicating development of protective effector memory. These results parallel the immunogenicity of similar vaccine regimens in macaques and an ongoing phase I/IIa trial in humans, and support further development of vaccines vectored by ChAdVs.

Mice chronically infected with chimeric HIV resist peripheral and brain superinfection: a model of protective immunity to HIV.

Infection by some viruses induces immunity to reinfection, providing a means to identify protective epitopes. To investigate resistance to reinfection in an animal model of HIV disease and its control, we employed infection of mice with chimeric HIV, EcoHIV. When immunocompetent mice were infected by intraperitoneal (IP) injection of EcoHIV, they resisted subsequent secondary infection by IP injection, consistent with a systemic antiviral immune response. To investigate the potential role of these responses in restricting neurotropic HIV infection, we established a protocol for efficient EcoHIV expression in the brain following intracranial (IC) inoculation of virus. When mice were inoculated by IP injection and secondarily by IC injection, they also controlled EcoHIV replication in the brain. To investigate their role in EcoHIV antiviral responses, CD8+ T lymphocytes were isolated from spleens of EcoHIV infected and uninfected mice and adoptively transferred to isogenic recipients. Recipients of EcoHIV primed CD8+ cells resisted subsequent EcoHIV infection compared to recipients of cells from uninfected donors. CD8+ spleen cells from EcoHIV-infected mice also mounted modest but significant interferon-γ responses to two HIV Gag peptide pools. These findings suggest EcoHIV-infected mice may serve as a useful system to investigate the induction of anti-HIV protective immunity for eventual translation to human beings.

Novel HIV-1 clade B candidate vaccines designed for HLA-B*5101(+) patients protected mice against chimaeric ecotropic HIV-1 challenge.

Novel candidate HIV-1 vaccines have been constructed, which are tailor-designed for HLA-B*5101(+) patients infected with HIV-1 clade B. These vaccines employ novel immunogen HIVB-B*5101 derived from consensus HIV-1 clade B Gag p17 and p24 regions coupled to two Pol-derived B*5101-restricted epitopes, which are together with a third B*5101 epitope in Gag dominant in HIV-1-infected long-term non-progressing patients. Both plasmid DNA and modified vaccinia virus Ankara (MVA) vectors supported high expression levels of the HIVB-B*5101 immunogen in cultured cells. Heterologous DNA prime-recombinant MVA boost regimen induced efficiently HIV-1-specific CD8(+) T-cell responses in BALB/c mice. These vaccine-elicited T cells were multifunctional, killed efficiently target cells in vivo, and protected mice against challenge with ecotropic HIV-1/NL4-3 and ecotropic HIV-1/NDK chimaeric viruses with HIV-1 clade B or D backbones, respectively, and ecotropic murine leukemia virus gp80 envelope, and therefore did so in the absence of anti-HIV-1 gp120 antibodies. These results support further development of HIVB-B*5101 vaccines in combined heterologous-modality regimens. The use of allele-specific vaccines in humans is discussed in the context of other developments in the HIV-1 field.

Host and virus strain dependence in activation of human macrophages by human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1)-associated neuropathogenesis occurs in a large minority of infected people. Presently, there are neither viral nor cellular markers that predict the development of brain disease during HIV-1 infection. This study was conducted to determine whether there exist systematic differences among human cell donors and virus strains for the activation of macrophage gene expression by HIV-1 that may contribute to neuropathogenesis. Four HIV-1, ADA and B-aL, which were isolated from peripheral tissues of acquired immunodeficiency syndrome (AIDS) patients, and DJV and YU-2, which were isolated from brains of patients with HIV-1-associated dementia, were compared for induction of expression of cellular genes associated with antiviral activity or inflammation in monocyte-derived macrophages from several donors. Virus replication and cytokine production were scored by enzyme-linked immunosorbent assay (ELISA) and cellular transcripts were measured by real-time polymerase chain reaction (PCR). ADA and B-aL productively infected cells from all donors tested and induced all cellular transcripts tested, illustrating a common response of macrophages to HIV-1 replication. In sharp contrast, the viruses associated with neuropathogenesis, DJV and YU-2, induced intense gene expression early after infection in cells from a subset of donors but DJV did not productively infect these cells. No such heterogeneity was observed in the responses of macrophages during high-level replication of any HIV-1 tested. The susceptibility to early activation by HIV-1 may reflect susceptibility to neuropathogenesis in AIDS.

Vaccine-induced protection from infection of mice by chimeric human immunodeficiency virus type 1, EcoHIV/NL4-3.

EcoHIV/NL4-3 is a chimeric human immunodeficiency virus type 1 (HIV-1) that can productively infect mice. This study tests the utility of EcoHIV/NL4-3 infection to reveal protective immune responses to an HIV-1 vaccine. Immunocompetent mice were first immunized with VRC 4306 which encodes subtype B consensus sequences of gag, pol, and nef and then were infected by EcoHIV/NL4-3. Anti-Gag antibodies were sampled during immunization and infection. The extent of EcoHIV/NL4-3 infection in spleen cells and peritoneal macrophages was determined by quantitative real-time PCR (QPCR). Although antibody titres were not significantly different in control and vaccinated groups, VRC 4306 immunization induced protective responses that significantly reduced virus burden in both lymphocyte and macrophage compartments. These results indicate that EcoHIV/NL4-3 infection can be controlled by HIV-1 vaccine-induced responses, introducing a small animal model to test vaccine efficacy against HIV-1 infection.

Inhibition of HIV-1 or bacterial activation of macrophages by products of HIV-1-resistant human cells.

We have recently described the molecular basis of HIV-1 resistance factor (HRF)-mediated anti-viral activity in primary and transformed CD4 T cells. HRF+ cell culture supernatants or partially purified HRF were found to incapacitate the formation of the NF-kappaB/DNA complex, which is indispensable for long terminal promoter-driven transcription of virus genes. In this study, we tested whether HRF might have much broader activity against other organisms whose pathogenesis is linked to NF-kappaB activation. Specifically, we tested the effects of HRF on the NF-kappaB-mediated responses of primary macrophages to HIV-1 or several bacterial antigens. We found that exposure to HRF inhibited HIV-1 expression in macrophages and also induced the production of HRF-like activity by macrophages, which prevented replication of virus in HIV-1-infected peripheral blood lymphocytes cultured in the adjacent compartment. We investigated the mechanism of this inhibition and found that HRF impeded NF-kappaB/DNA binding in macrophages induced by either HIV-1 or lipopolysaccharide from several bacteria species, resulting in impaired tumor necrosis factor-alpha responses to these organisms.

Human immunodeficiency virus-encoded Tat activates glycogen synthase kinase-3beta to antagonize nuclear factor-kappaB survival pathway in neurons.

The pathogenesis of human immunodeficiency virus type 1 (HIV-1)-associated dementia is mediated by neuronal dysfunction and death, brought about by the action of soluble neurotoxic factors that are released by virally infected macrophages and microglia. Paradoxically, many candidate HIV-1 neurotoxins also possess the ability to activate nuclear factor-kappa B (NF-kappaB), which has a potent pro-survival effect in primary neurons. The present study explored this conundrum and investigated why NF-kappaB might fail to protect neurons that are exposed to candidate HIV-1 neurotoxins. Here, we evaluated the ability of virus-depleted conditioned medium produced by HIV-1-infected human macrophages (HIV-MCMs) to modulate NF-kappaB activity in neurons. We demonstrated that HIV-MCMs inhibit the normal signaling pathways that lead to NF-kappaB activation in neurons. This inhibitory effect of HIV-MCM is dependent upon the presence of HIV-1 Tat, which activates glycogen synthase kinase (GSK)-3beta in neurons. Activation of GSK-3beta, in turn, results in modification of the NF-kappaB subunit RelA at serine 468, thereby regulating the physical interaction of RelA with histone deacetylase-3 corepressor molecules. Furthermore, neutralization of Tat or inhibition of GSK-3beta activity prevents neuronal apoptosis induced by HIV-MCM. We conclude that HIV-1 Tat may compromise neuronal function and fate by interfering with normal survival pathways subserved by NF-kappaB. These findings may have important therapeutic implications for the management of HIV-1-associated dementia.

Novel activities of cyclophilin A and cyclosporin A during HIV-1 infection of primary lymphocytes and macrophages.

Studies conducted in cell lines indicate that cyclophilin A (CypA) is a component of HIV type 1 (HIV-1) virions, and that when CypA incorporation into virions is inhibited by treatment of infected cells with the immunosuppressive agent cyclosporin A (CsA), HIV-1 infection also is inhibited. Because HIV-1 particles assemble along a different pathway and incorporate different host proteins in macrophages than in other cell types, we investigated CypA and CsA activities in HIV-1-infected primary human macrophages, compared with primary human lymphocytes. We tested virus protein production, virion composition and infectivity, and progress through the virus life cycle under perturbation by drug treatment or mutagenesis in infected cells from multiple donors. Our findings from both primary cell types are different from that previously reported in transformed cells and show that the amount of CypA incorporated into virions is variable and that CsA inhibits HIV-1 infection at both early and late phases of virus replication, the stage affected is determined by the sequence of HIV-1 Gag. Because the cell type infected determines the identity of host proteins active in HIV-1 replication and can influence the activity of some viral inhibitors, infection of transformed cells may not recapitulate infection of the native targets of HIV-1.

A mouse model for study of systemic HIV-1 infection, antiviral immune responses, and neuroinvasiveness.

We created a model of HIV-1 infection of conventional mice for investigation of viral replication, control, and pathogenesis. To target HIV-1 to mice, the coding region of gp120 in HIV-1/NL4-3 was replaced with that of gp80 from ecotropic murine leukemia virus, a retrovirus that infects only rodents. The resulting chimeric virus construct, EcoHIV, productively infected murine lymphocytes, but not human lymphocytes, in culture. Adult, immunocompetent mice were readily susceptible to infection by a single inoculation of EcoHIV as shown by detection of virus in splenic lymphocytes, peritoneal macrophages, and the brain. The virus produced in animals was infectious, as shown by passage in culture, and immunogenic, as shown by induction of antibodies to HIV-1 Gag and Tat. A second chimeric virus based on clade D HIV-1/NDK was also highly infectious in mice; it was detected in both spleen and brain 3 wk after tail vein inoculation, and it induced expression of infection response genes, MCP-1, STAT1, IL-1beta, and complement component C3, in brain tissue as determined by quantitative real-time PCR. EcoHIV infection of mice forms a useful model of HIV-1 infection of human beings for convenient and safe investigation of HIV-1 therapy, vaccines, and potentially pathogenesis.

Productive infection of primary murine astrocytes, lymphocytes, and macrophages by human immunodeficiency virus type 1 in culture.

A mouse model of human immunodeficiency virus type 1 (HIV-1) infection would be extremely valuable for evaluation of therapies and vaccines; however, multiple blocks to productive infection of NIH 3T3 and other mouse cell lines have been reported. The authors investigated the replication of HIV-1 in primary mouse astrocytes, lymphocytes, and macrophages in culture by infection with intact HIV-1 pseudotyped with the vesicular stomatitis virus G envelope glycoprotein (VSV-G) or with the envelope glycoprotein of amphotropic murine leukemia virus. Astrocytes, lymphocytes, and macrophages were susceptible to productive infection as variously assayed by detection of p24 and Tat proteins, viral protease-mediated processing of Gag, appropriately spliced viral RNA, and infectious progeny virus. As expected, NIH 3T3 cells were not susceptible to productive infection by VSV/NL4. Susceptibility mapped neither to the Fv locus nor to a possible polymorphism in cyclin T1. This study indicates that there are no intrinsic intracellular barriers to HIV-1 replication in primary mouse cells when virus entry is efficient.

The macrophage response to HIV-1: Intracellular control of X4 virus replication accompanied by activation of chemokine and cytokine synthesis.

During human immunodeficiency virus (HIV)-1 infection, T lymphocytes and macrophages play dual roles. They are the primary targets for virus replication, but they are also primary effector cells in acquired and innate immunity, respectively. The authors are now investigating how these roles come together in the response of human monocyte-derived macrophages (MDM) to certain HIV-1. The authors and others have previously shown that MDM permit entry of some X4 virus strains, but control viral replication intracellularly. In the present study, viral DNA synthesis, entry into the nucleus, and transcription to RNA were all observed in X4 virus-infected MDM. MDM arrested HIV-1 replication prior to expression of mature capsid antigen p24 and production of cell-free infectious viral particles. Cell-associated transmissible HIV-1 was detected by cocultivation of infected MDM and susceptible T lymphocytes. A second protective response of MDM to specific R5 as well as X4 HIV-1 was identified in rapid and extensive secretion of tumor necrosis factor-alpha, macrophage inflammatory protein-1alpha, and RANTES. These findings support the view that MDM act aggressively to control HIV-1 replication: X4 strains by severely limiting the progeny virus production and R5 strains by producing beta-chemokines competent to block virus entry into target cells. Optimizing these innate immune responses offers another means to control HIV-1 infection in the human host.