Expression of simian immunodeficiency virus (SIV) nef in astrocytes during acute and terminal infection and requirement of nef for optimal replication of neurovirulent SIV in vitro

Abstract

As the most numerous cells in the brain, astrocytes play a critical role in maintaining central nervous system homeostasis, and therefore, infection of astrocytes by human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) in vivo could have important consequences for the development of HIV encephalitis. In this study, we establish that astrocytes are infected in macaques during acute SIV infection (10 days postinoculation) and during terminal infection when there is evidence of SIV-induced encephalitis. Additionally, with primary adult rhesus macaque astrocytes in vitro, we demonstrate that the macrophage-tropic, neurovirulent viruses SIV/17E-Br and SIV/17E-Fr replicate efficiently in astrocytes, while the lymphocyte-tropic, nonneurovirulent virus SIV(mac)239 open-nef does not establish productive infection. Furthermore, aminoxypentane-RANTES abolishes virus replication, suggesting that these SIV strains utilize the chemokine receptor CCR5 for entry into astrocytes. Importantly, we show that SIV Nef is required for optimal replication in primary rhesus macaque astrocytes and that normalizing input virus by particle number rather than by infectivity reveals a disparity between the ability of a Nef-deficient virus and a virus encoding a nonmyristoylated form of Nef to replicate in these central nervous system cells. Since the myristoylated form of Nef has been implicated in functions such as CD4 and major histocompatibility complex I downregulation, kinase association, and enhancement of virion infectivity, these data suggest that an as yet unidentified function of Nef may exist to facilitate SIV replication in astrocytes that may have important implications for in vivo pathogenesis.

FIG. 1.

(A) Immunohistochemical staining of brain from an SIV-infected macaque euthanatized at 10 days postinoculation, demonstrating colocalization of GFAP [black, SG substrate (Vector Laboratories, Burlingame, Calif.)] and SIV Nef [brown, 3,3,-diaminobenzidine; Biogenics, San Ramon, Calif.)] (magnification, 200×). Arrow indicates an astrocyte expressing SIV Nef. (B) Immunohistochemical staining of brain tissue from a macaque with severe encephalitis (3 months postinfection; magnification, 200×). Arrow indicates an SIV Nef-expressing astrocyte. (C) Immunohistochemical staining of brain tissue from a macaque with severe encephalitis (3 months postinfection; magnification, 400×) dually labeled for SIV Nef (3,3,-diaminobenzidine) and SIV gp41 (Texas Red; Vector Labs). Shown is a multinucleated giant cell expressing both SIV Nef and gp41, while the arrow indicates an astrocyte expressing only Nef. (D) Phase contrast of primary rhesus macaque astrocytes plated on glass coverslips and displaying stellate morphology typical of astrocytes (magnification, 400×). (E) GFAP expression in the astrocyte cultures shown in D and stained with a monoclonal antibody to human GFAP (indocarbocyanine-conjugated; Sigma; magnification, 400×).

FIG. 2.

SIV infection of primary rhesus astrocytes. Astrocyte cultures were inoculated with SIV/17E-Br, SIV/17E-Fr, and SIV_mac239 normalized by TCID₅₀ (multiplicity of infection = 0.1) and allowed to incubate for 6 h before the cells were washed extensively, and replication was monitored over time postinfection (PI) for the presence of SIV p27. Results shown are representative of several independent experiments.

FIG. 3.

Colabeling of GFAP-positive cells with SIV p27. (A) Phase contrast of infected astrocytes plated on glass coverslips, infected with 50,000 cpm of SIV/17E-Br reverse transcriptase units, washed, and allowed to incubate for 1 week. (B) Immunofluorescence of astrocytes in A stained for viral p27 (fluorescein isothiocyanate; Dako) and GFAP (indocarbocyanine) (magnification, 400×). (C) Phase contrast of infected astrocytes (magnification, 400×). (D) Immunofluorescence of the astrocytes shown in C stained with isotype controls for both p27 and GFAP.

FIG. 4.

Transmission electron microscopy of SIV-infected primary macaque astrocytes. (A) Low magnification of astrocyte cultures infected with SIV/17E-Fr and allowed to incubate for 14 days before samples were prepared as described in Materials and Methods and sent to Electron Microscopy Bioservices for analysis. Bar, 1 μm. (B) Higher magnification of the cell shown in panel A, showing SIV budding from the plasma membrane. Bar, 0.2 μm. B, budding virions; M, mature particle; I, intermediate filaments.

FIG. 5.

Infectivity of astrocyte-derived SIV. Supernatants derived from astrocytes and macrophages infected with SIV/17E-Br were normalized for p27, and the infectivities were quantitated by the LuSIV assay as described in Materials and Methods.

FIG. 6.

AOP-RANTES blocks SIV infection of primary rhesus astrocytes. Astrocytes were incubated with or without 500 ng of AOP-RANTES per ml for 1 h prior to infection with SIV/17E-Br or SIV/17E-Fr [input virus normalized by TCID₅₀ (multiplicity of infection = 0.1)], and supernatants were sampled at various times postinfection (PI) and assayed for the presence of SIV p27. Data are representative of several independent experiments.

FIG. 7.

Nef is required for optimal SIV replication in rhesus astrocytes. (A) Astrocytes were infected with SIV/17E-Fr or SIV/Fr-2 [input normalized by TCID₅₀ (multiplicity of infection = 0.1)]. (B) Astrocytes were infected with SIV/17E-Fr, SIV/17E-Fr(−myr), SIV/17E-Fr(−nef), or SIV/17E-FrΔnef [input normalized by TCID₅₀ (multiplicity of infection = 0.1)]. (C) Astrocytes were infected with SIV/17E-Fr, SIV/Fr-2, SIV/17E-Fr(−myr), or SIV/17E-FrΔnef [input normalized by particle number (50 ng of p27)]. Supernatants in all experiments were sampled at various times postinfection (PI) and assayed for viral p27. Data are representative of several independent experiments.