Research Article: SIVcol Nef counteracts SERINC5 by promoting its proteasomal degradation but does not efficiently enhance HIV-1 replication in human CD4+ T cells and lymphoid tissue

Date Published: August 20, 2018

Publisher: Public Library of Science

Author(s): Dorota Kmiec, Bengisu Akbil, Swetha Ananth, Dominik Hotter, Konstantin M. J. Sparrer, Christina M. Stürzel, Birthe Trautz, Ahidjo Ayouba, Martine Peeters, Zhong Yao, Igor Stagljar, Vânia Passos, Thomas Zillinger, Christine Goffinet, Daniel Sauter, Oliver T. Fackler, Frank Kirchhoff, Michael Emerman.

http://doi.org/10.1371/journal.ppat.1007269

Abstract

SERINC5 is a host restriction factor that impairs infectivity of HIV-1 and other primate lentiviruses and is counteracted by the viral accessory protein Nef. However, the importance of SERINC5 antagonism for viral replication and cytopathicity remained unclear. Here, we show that the Nef protein of the highly divergent SIVcol lineage infecting mantled guerezas (Colobus guereza) is a potent antagonist of SERINC5, although it lacks the CD4, CD3 and CD28 down-modulation activities exerted by other primate lentiviral Nefs. In addition, SIVcol Nefs decrease CXCR4 cell surface expression, suppress TCR-induced actin remodeling, and counteract Colobus but not human tetherin. Unlike HIV-1 Nef proteins, SIVcol Nef induces efficient proteasomal degradation of SERINC5 and counteracts orthologs from highly divergent vertebrate species, such as Xenopus frogs and zebrafish. A single Y86F mutation disrupts SERINC5 and tetherin antagonism but not CXCR4 down-modulation by SIVcol Nef, while mutation of a C-proximal di-leucine motif has the opposite effect. Unexpectedly, the Y86F change in SIVcol Nef had little if any effect on viral replication and CD4+ T cell depletion in preactivated human CD4+ T cells and in ex vivo infected lymphoid tissue. However, SIVcol Nef increased virion infectivity up to 10-fold and moderately increased viral replication in resting peripheral blood mononuclear cells (PBMCs) that were first infected with HIV-1 and activated three or six days later. In conclusion, SIVcol Nef lacks several activities that are conserved in other primate lentiviruses and utilizes a distinct proteasome-dependent mechanism to counteract SERINC5. Our finding that evolutionarily distinct SIVcol Nefs show potent anti-SERINC5 activity supports a relevant role of SERINC5 antagonism for viral fitness in vivo. Our results further suggest this Nef function is particularly important for virion infectivity under conditions of limited CD4+ T cell activation.

Partial Text

The accessory nef gene is present in the genomes of all primate lentiviruses that infect at least forty different African monkey species as well as great apes and humans. Nef performs a striking number of activities [1,2] and is required for efficient viral replication and pathogenicity of HIV-1 and SIVmac in humans and experimentally infected rhesus macaques, respectively [3–5]. Some Nef functions are conserved in the vast majority of primate lentiviruses. These include down-modulation of the CD4 receptor and class I major histocompatibility complex (MHC-I) from the cell surface [6], enhancement of virion infectivity [7] by counteraction of the antiviral factor SERINC5 [8–10], modulation of the actin skeleton [11,12] and T cell signaling and migration [13,14], as well as stimulation of NF-κB activity. The Nef proteins of HIV-2, which originated from several cross-species transmissions of SIVsmm found in sooty mangabeys, and most SIVs additionally down-modulate CD3 from the cell surface to suppress stimulation of virally infected CD4+ T cells and antiviral gene expression [6,15–17]. In contrast, this Nef function was lost in most primate lentiviruses encoding a vpu gene, i.e. HIV-1, its direct simian precursors SIVcpz and SIVgor from chimpanzees and gorillas, respectively, and some closely related SIVs infecting several Cercopithecus species [6,18]. These primate lentiviruses are unable to block TCR-CD3-mediated T cell activation and instead use Vpu to suppress antiviral gene expression by inhibiting activation of the transcription factor NF-κB [19,20]. Most primate lentiviruses lacking Vpu as well as SIVcpz, SIVgor and HIV-1 group O also use Nef to antagonize the restriction factor tetherin to allow efficient release of viral particles from infected cells [21–24]. Finally, many HIV-2, SIV and (to a lesser extent) HIV-1 Nef proteins down-modulate CD28 and CXCR4 from the cell surface [14,25,26]. Thus, the multifunctionality of lentiviral Nefs highlights the importance of this accessory protein but also poses a challenge for dissecting its effects on viral replication and pathogenicity.

In the present study, we show that SIVcol Nef proteins derived from naturally infected mantled guerezas lack the ability to down-modulate CD4, CD28 and CD3 but are highly potent in modulating CXCR4, suppressing TCR-induced actin remodeling, impairing T cell chemotaxis towards SDF-1α, counteracting Colobus tetherin, and enhancing viral infectivity by counteracting SERINC3 and 5. These results expand previous studies [10,20,28] suggesting that Nef proteins from SIVcol are functionally distinct from all other known primate lentiviral Nef proteins. SERINC5 counteraction by SIVcol Nef was highly efficient and involved surface down-modulation as well as effective proteasomal degradation of this restriction factor. In addition, our results show that SIVcol Nefs are broad-spectrum antagonists of vertebrate SERINC5 proteins and fully active against orthologs from humans, zebrafish and frogs. These effects were disrupted by a single point mutation of Y86F allowing us to analyze the relevance of potent anti-SERINC5 antagonism by Nef for viral replication and cytopathicity. Although the conservation and apparent independent evolution of this Nef function suggest a relevant role in vivo, efficient SERINC5 counteraction did not significantly enhance viral spread and replication in preactivated human CD4+ T cells or in tonsillar lymphoid tissue cultures in the absence of endogenous stimulation. More pronounced effects of SERINC5 antagonism on infectious virus yield and virion infectivity were observed when human PBMC cultures were first HIV-1 infected and then PHA-activated six days later probably because activation reduces expression of this restriction factor [49].

 

Source:

http://doi.org/10.1371/journal.ppat.1007269