Simian immunodeficiency virus SIVagm from African green monkeys does not antagonize endogenous levels of African green monkey tetherin/BST-2

Abstract

The Vpu accessory gene that originated in the primate lentiviral lineage leading to human immunodeficiency virus type 1 is an antagonist of human tetherin/BST-2 restriction. Most other primate lentivirus lineages, including the lineage represented by simian immunodeficiency virus SIVagm from African green monkeys (AGMs), do not encode Vpu. While some primate lineages encode gene products other than Vpu that overcome tetherin/BST-2, we find that SIVagm does not antagonize physiologically relevant levels of AGM tetherin/BST-2. AGM tetherin/BST-2 can be induced by low levels of type I interferon and can potently restrict two independent strains of SIVagm. Although SIVagm Nef had an effect at low levels of AGM tetherin/BST-2, simian immunodeficiency virus SIVmus Vpu, from a virus that infects the related monkey Cercopithecus cephus, is able to antagonize even at high levels of AGM tetherin/BST-2 restriction. We propose that since the replication of SIVagm does not induce interferon production in vivo, tetherin/BST-2 is not induced, and therefore, SIVagm does not need Vpu. This suggests that primate lentiviruses evolve tetherin antagonists such as Vpu or Nef only if they encounter tetherin during the typical course of natural infection.

FIG. 1.

AGM and cephus tetherins restrict HIV1VpuFS and are counteracted by SIV_mus Vpu. (A) 293T cells were cotransfected with HIV1VpuFS (100 ng), human (200 ng), C. cephus (Cephus) (400 ng), or AGM tetherin (400 ng); HIV-1 Vpu (125 ng); and SIV_mus Vpu (100 ng) for 48 h. The respective empty vectors were used, indicated by no tetherin or no Vpu. Equal amounts of virus-containing supernatant were used to infect TZM-bl reporter cells for 48 h. Relative infectivity was determined by the rate of β-galactosidase activity. Bars represent the average data for eight infections, normalized to the relative infectivity of the respective Vpu proteins in the absence of tetherin. Error bars indicate standard deviations. (B) 293T cell lysates (A) were collected 48 h after transfection and analyzed by Western blot analysis for HA-tagged tetherin and β-actin expression levels.

FIG. 2.

SIV_agm does not encode Vpu-like activity. (A) 293T cells were cotransfected with HIV1VpuFS, SIV_agmTan Env⁻ Nef⁻ (and VSV-G for pseudotyping), SIV_agmTan Env⁺ Nef⁺ (left), or SIV_agmSab (right) and no tetherin (empty vector) or human or AGM tetherin for 48 h. Virus was used to infect TZM-bl reporter cells for 48 h to determine the relative infectivity. (B) 293T cells were transfected with SIV_agmTan (left) or SIV_agmSab (right) with or without AGM tetherin. Virions from the supernatant were collected and pelleted, while cells were lysed to yield intracellular virions. Western blot analysis was performed to compare the amounts of cell-free virions.

FIG. 3.

AGM tetherin retains SIV_agmSab on the plasma membrane. Shown is thin-section electron microscopy of 293T cells transfected for 48 h with SIV_agmSab in the absence of AGM tetherin (A and B) or in the presence of AGM tetherin (C and D). (A) SIV_agmSab budding from the plasma membrane in a single layer in the absence of tetherin. The arrow indicates the location of the magnified image. (B) Magnified image from A. (C) Accumulation of budding SIV_agmSab cells on the plasma membrane in the presence of AGM tetherin. Note that mature virions form multiple layers. The arrow indicates the location of the magnified image. (D) Magnified image of C. Scale bars, 2 μm (A and C) and 200 nm (B and D).

FIG. 4.

AGM tetherin is not antagonized by SIV_agm Nef but retains interface with SIV_mus Vpu. (A) 293T cells were cotransfected with HIV1VpuFSLuc2 (100 ng), SIV_agmTan Nef (250 ng), and human (100 ng), chimpanzee (100 ng), or AGM (100 ng) tetherin. Virus was assayed on SupT1 cells, and relative infectivity was normalized in the absence of tetherin. Bars represent average data for four infections, and error bars indicate standard deviations. (B) 293T cells were cotransfected with VSV-G and SIV_agmTan (solid circles) or SIV_agmTan Env⁻ Nef⁻ (open circles) with increasing amounts of untagged AGM tetherin (0, 3.12, 6.25, 12.5, 25, 50, and 100 ng). Virus infectivity was assayed on TZM-bl reporter cells, and relative infectivity was normalized in the absence of tetherin. Points represent average data for four infections, and error bars represent standard deviations. (C) 293T cells were cotransfected with HIV1VpuFSLuc2 (100 ng), AGM tetherin (100 ng), SIV_mus Vpu (100 ng), SIV_agmTan Nef (250 ng), or SIV_agmSab Nef (250 ng). Virus was assayed on SupT1 cells, and relative infectivity was normalized in the absence of tetherin. Bars represent average data for four infections, and error bars indicate standard deviations. (D) 293T cells were cotransfected with HIV1VpuFS or SIV_agmTan, AGM tetherin, and SIV_mus Vpu where indicated for 48 h. Virus was added to TZM.b1 reporter cells for 48 h, and relative infectivity was determined as described in the legend of Fig. 1A, normalized to the respective virus in the absence of tetherin. Bars represent average data for eight readings, and error bars indicate standard deviations.

FIG. 5.

Type I IFNs induce AGM tetherin and the tetherin virion retention phenotype. (A) 293T (human), COS-7 (AGM), and C. sabaeus primary fibroblast (AGM) cells were incubated with 0, 100, or 1,000 IU/ml IFN-α2a or IFN-β1b for 24 h. Cells were harvested and lysed for RT-PCR analysis. (B and C) Thin-section electron microscopy of accumulated SIV_agmTan budding from COS-7 cells exposed to no IFN (B) or 1,000 IU/ml IFN-β1b (C). COS-7 cells were infected with VSV-G-pseudotyped SIV_agmTan. Six hours after infection, cells were exposed to 1,000 IU/ml IFN-β1b and fixed for thin-section electron microscopy after an additional 42 h. Scale bars, 0.2 nm.

FIG. 6.

Type I IFNs induce AGM tetherin restriction of virus release. COS-7 (AGM) cells were infected with VSV-G-pseudotyped SIV_agmTan Env⁻ Nef⁻ (top) or VSV-G-pseudotyped SIV_agmTan (bottom) and exposed to 0, 10, or 100 IU/ml IFN-β1b 6 h postinfection. Virions from the supernatant were collected and pelleted, while cells were lysed to yield intracellular virions. Western blot analysis was performed to compare the amounts of cell-free virions.