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. 2019 Jun 11;10(3):e01071-19.
doi: 10.1128/mBio.01071-19.

Potent Enhancement of HIV-1 Replication by Nef in the Absence of SERINC3 and SERINC5

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Free PMC article

Potent Enhancement of HIV-1 Replication by Nef in the Absence of SERINC3 and SERINC5

Yuanfei Wu et al. mBio. .
Free PMC article

Abstract

It has recently emerged that HIV-1 Nef counteracts the antiviral host proteins SERINC3 and SERINC5. In particular, SERINC5 inhibits the infectivity of progeny virions when incorporated. SERINC3 and SERINC5 are also counteracted by the unrelated murine leukemia virus glycosylated Gag (glycoGag) protein, which possesses a potent Nef-like activity on HIV-1 infectivity. We now report that a minimal glycoGag termed glycoMA can fully substitute for Nef in promoting HIV-1 replication in Jurkat T lymphoid cells, indicating that Nef enhances replication in these cells mainly by counteracting SERINCs. In contrast, the SERINC antagonist glycoMA was unable to substitute for Nef in MOLT-3 T lymphoid cells, in which HIV-1 replication was highly dependent on Nef, and remained so even in the absence of SERINC3 and SERINC5. As in MOLT-3 cells, glycoMA was unable to substitute for Nef in stimulating HIV-1 replication in primary human cells. Although the ability of Nef mutants to promote HIV-1 replication in MOLT-3 cells correlated with the ability to engage endocytic machinery and to downregulate CD4, Nef nevertheless rescued virus replication under conditions where CD4 downregulation did not occur. Taken together, our observations raise the possibility that Nef triggers the endocytosis of a novel antiviral factor that is active against both laboratory-adapted and primary HIV-1 strains.IMPORTANCE The Nef protein of HIV-1 and the unrelated glycoGag protein of a murine leukemia virus similarly prevent the uptake of antiviral host proteins called SERINC3 and SERINC5 into HIV-1 particles, which enhances their infectiousness. We now show that although both SERINC antagonists can in principle similarly enhance HIV-1 replication, glycoGag is unable to substitute for Nef in primary human cells and in a T cell line called MOLT-3. In MOLT-3 cells, Nef remained crucial for HIV-1 replication even in the absence of SERINC3 and SERINC5. The pronounced effect of Nef on HIV-1 spreading in MOLT-3 cells correlated with the ability of Nef to engage cellular endocytic machinery and to downregulate the HIV-1 receptor CD4 but nevertheless persisted in the absence of CD4 downregulation. Collectively, our results provide evidence for a potent novel restriction activity that affects even relatively SERINC-resistant HIV-1 isolates and is counteracted by Nef.

Keywords: Nef; SERINC5; human immunodeficiency virus; infectivity; virus replication.

Figures

FIG 1
FIG 1
MLV glycoMA can substitute for Nef in promoting HIV-1 replication in Jurkat cells. (A) Western blots showing the effects of Nef and glycoMA on HIV-1 spreading in parental CD4high JTAg cells, double knockout cells lacking SERINC3 and SERINC5, and SERINC3- and SERINC5-reconstituted double-knockout cells. The cells were infected with equal amounts (2 ng/ml p24) of Nef+, Nef, or glycoMA+ HIV-1NL4-3, and cell lysates were examined with anti-CA and anti-actin 12 days after infection. A duplicate experiment gave similar results. (B and C) Nef and glycoMA similarly enhance HIV-1NL4-3 replication in Jurkat E6.1 cells, as examined by Western blotting of cell lysates 11 days after infection (B) and by monitoring p24 accumulation in the supernatants (C). The cells were infected with 0.2 ng p24/ml. The data in panels B and C are from independent experiments.
FIG 2
FIG 2
Potent enhancement of HIV-1 replication in MOLT-3 cells and PBMC by Nef but not glycoMA. (A) SERINC mRNA expression in Jurkat E6.1 and MOLT-3 cells quantified by transcriptome sequencing (RNA-seq) as fragments per kilobase of transcript per million mapped reads (FPKM) (n = 2). (B) Nef but not glycoMA (gMA) potently enhances HIV-1NL4-3 replication in MOLT-3 cells. Gag expression at day 11 after infection with equal amounts (0.2 ng/ml p24) of Nef+, Nef, or glycoMA+ HIV-1NL4-3 was examined by Western blotting. (C) Virus replication in MOLT-3 cells infected with the same viruses (at 2 ng/ml p24) monitored by a p24 ELISA. (D) Virus replication in MOLT-3 cells and in PBMC examined in parallel by Western blotting of cell lysates with anti-CA. MOLT-3 cells stimulated or not with 100 ng/ml TNF-α were infected with Nef+, Nef, or glycoMA+ HIV-1NL4-3 (0.2 ng/ml p24 each). Unstimulated PBMC were infected with the same viruses (at 0.25 ng/ml p24) and stimulated with PHA on day 4 after infection. (E and F) Virus replication in the same cultures monitored by a p24 ELISA. (G) Virus replication in prestimulated PBMC monitored by a p24 ELISA after infection with Nef+, Nef, or glycoMA+ HIV-1NL4-3 (0.25 ng/ml p24 each). This experiment was performed twice. The data in panels B to D are from independent experiments. The results in panels E and F were confirmed in independent experiments.
FIG 3
FIG 3
Replication of SERINC5-resistant HIV-1 in MOLT-3 cells depends on Nef. (A and B) Virus replication in MOLT-3/CCR5 cells (A) or CD4high MOLT-3/CCR5 cells (B) infected with equal amounts (1 ng p24/ml) of NL-JRFL or NL-JRFL/nef. HIV-1 replication was monitored by a p24 ELISA. (C) Virus replication in CD4high MOLT-3/CCR5 cells infected with equal amounts (10 ng p24/ml) of NL-ADA or NL-ADA/nef. The results in panels B and C were confirmed in experiments performed with different amounts of input virus.
FIG 4
FIG 4
Nef is required for efficient HIV-1 replication in MOLT-3 cells lacking SERINC3 and SERINC5. (A) Mutant SERINC alleles identified in SERINC5 knockout and SERINC3/5 double-knockout clones. The sgRNA target sites are highlighted, and the predicted Cas9 target sites are indicated by arrowheads. Inserted nucleotides are in red. One of the mutated SERINC3 alleles in MOLT-3 S3/5 KO cells has a large deletion that removes the splice site at the 3′ end of the targeted exon. All other mutations cause frameshifts. No WT alleles were detected in any of the KO clones. (B) Western blots showing the effects of Nef on HIV-1 spreading in MOLT-3 cells lacking SERINC5 (S5 KO) or SERINC3 and SERINC5 (S3/5 KO). The cells were infected with equal amounts (0.2 ng/ml p24) of Nef+ or Nef HIV-1NL4-3. This experiment was performed twice.
FIG 5
FIG 5
Replication of Nef mutants in MOLT-3 cells. (A) Effects of mutations that abrogate AP-2 binding and/or CD4 downregulation by Nef. (B) Effects of mutations that disrupt AP-1 binding (E160A), PAK2 binding (F191I), or Nef myristylation (G2A). (C) Effects of mutations that disrupt interactions with Src kinases (P69/72A) or dynamin 2 (L112A, F121A, and D123A). MOLT-3 cells were infected with 5 ng (A) or 2 ng (B and C) p24/ml, and virus replication was monitored by a p24 ELISA.
FIG 6
FIG 6
Nef enhances HIV-1 replication in MOLT-3 cells expressing high levels of a Nef-resistant CD4. (A) CD4 surface levels on parental MOLT-3 cells and on MOLT-3 cells stably transduced with a retroviral vector expressing a Nef-resistant CD4 (M3/CD4ΔCT cells). (B) Virus replication in M3/CD4ΔCT cells monitored by a p24 ELISA after infection with equal amounts (2 ng p24/ml) of Nef+ or Nef HIV-1NL4-3.
FIG 7
FIG 7
Nef enhances HIV-1 replication in MOLT-3 cells in the absence of CD4 downregulation. (A) CD4 surface levels on M3/CD4ΔCT cells stably transduced with the empty pCX4pur retroviral vector (M3/CD4ΔCT/vector cells) or a version expressing NefLAI (M3/CD4ΔCT/NefLAI cells). (B) Virus replication in M3/CD4ΔCT/vector and M3/CD4ΔCT/NefLAI cells monitored by a p24 ELISA after infection with equal amounts (2 ng p24/ml) of Nef+ or Nef HIV-1NL4-3. The effect of Nef in trans on Nef HIV-1NL4-3 was confirmed in an independent experiment. (C and D) Replication of Nef HIV-1NL4-3 in M3/CD4ΔCT cells stably expressing various group M Nef proteins (C) or a group N Nef protein (D).
FIG 8
FIG 8
Nef but not glycoMA enhances the infectivity of HIV-1 produced in MOLT-3 cells. (A) Dot blots showing ZsGreen expression in MOLT-3/ZsGreen reporter cells after infection with equal amounts of Nef+, Nef, or glycoMA+ HIV-1NL4-3 produced in MOLT-3 cells. FSC, forward scatter. (B) HIV-1 virions produced in MOLT-3 cells are relatively poorly infectious. Relative infectivities of Nef+ and Nef HIV-1NL4-3 produced in 293T or MOLT-3 cells were measured using TZM-bl indicator cells.

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