The Antagonism of HIV-1 Nef to SERINC5 Particle Infectivity Restriction Involves the Counteraction of Virion-Associated Pools of the Restriction Factor

Abstract

SERINC3 (serine incorporator 3) and SERINC5 are recently identified host cell inhibitors of HIV-1 particle infectivity that are counteracted by the viral pathogenesis factor Nef. Here we confirm that HIV-1 Nef, but not HIV-1 Vpu, antagonizes the particle infectivity restriction of SERINC5. SERINC5 antagonism occurred in parallel with other Nef activities, including cell surface receptor downregulation, trans-Golgi network targeting of Lck, and inhibition of host cell actin dynamics. Interaction motifs with host cell endocytic machinery and the Nef-associated kinase complex, as well as CD4 cytoplasmic tail/HIV-1 protease, were identified as essential Nef determinants for SERINC5 antagonism. Characterization of antagonism-deficient Nef mutants revealed that counteraction of SERINC5 occurs in the absence of retargeting of the restriction factor to intracellular compartments and reduction of SERINC5 cell surface density is insufficient for antagonism. Consistent with virion incorporation of SERINC5 being a prerequisite for its antiviral activity, the infectivity of HIV-1 particles produced in the absence of a SERINC5 antagonist decreased with increasing amounts of virion SERINC5. At low levels of SERINC5 expression, enhancement of virion infectivity by Nef was associated with reduced virion incorporation of SERINC5 and antagonism-defective Nef mutants failed to exclude SERINC5 from virions. However, at elevated levels of SERINC5 expression, Nef maintained infectious HIV particles, despite significant virion incorporation of the restriction factor. These results suggest that in addition to virion exclusion, Nef employs a cryptic mechanism to antagonize virion-associated SERINC5. The involvement of common determinants suggests that the antagonism of Nef to SERINC5 and the downregulation of cell surface CD4 by Nef involve related molecular mechanisms. IMPORTANCEHIV-1 Nef critically determines virus spread and disease progression in infected individuals by incompletely defined mechanisms. SERINC3 and SERINC5 were recently identified as potent inhibitors of HIV particle infectivity whose antiviral activity is antagonized by HIV-1 Nef. To address the mechanism of SERINC5 antagonism, we identified four molecular determinants of Nef antagonism that are all linked to the mechanism by which Nef downregulates cell surface CD4. Functional characterization of these mutants revealed that endosomal targeting and cell surface downregulation of SERINC5 are dispensable and insufficient for antagonism, respectively. In contrast, virion exclusion and antagonism of SERINC5 were correlated; however, Nef was also able to enhance the infectivity of virions that incorporated robust levels of SERINC5. These results suggest that the antagonism of HIV-1 Nef to SERINC5 restriction of virion infectivity is mediated by a dual mechanism that is related to CD4 downregulation. Nef is a myristoylated 25-to 34-kDa protein that, in addition to human immunodeficiency virus type 1 (HIV-1), is encoded by HIV-2 and simian immunodeficiency virus (S...

A Long Cytoplasmic Loop Governs the Sensitivity of the Anti-viral Host Protein SERINC5 to HIV-1 Nef

“…More recent work confirmed that Nef excludes SERINC5 from virions but suggested that Nef additionally inactivates the antiviral activity of any SERINC5 that becomes virion-associated even in the presence of Nef as a consequence of overexpression (Trautz et al, 2016). In the present study, the ability of Nef proteins to reduce the incorporation of native or chimeric versions of frog or human SERINC5 correlated well with their ability to enhance HIV-1 infectivity, which supports the notion that virion exclusion is the primary mechanism by which Nef counteracts SERINC5.…”

Alterations of redox and iron metabolism accompany development of HIV latency

“…To assess how single viral proteins modulate the redox status of the cell in the context of infection, we employed three different viral clones bearing mutations in the env (Pizzato et al 2007) , nef (Trautz et al 2016) or tat (Bejarano et al 2019) genes . Compared to wild type HIV-1 NL4-3 , all mutant viruses exhibit the expected decrease in viral transcription ( Figure S2B ) and integration ( Figure S2C ), with the Δ tat mutant displaying the lowest fitness ( Figure S2B and S2C ).…”

SERINC5 protein inhibits HIV-1 fusion pore formation by promoting functional inactivation of envelope glycoproteins

“…Accumulating evidence implies that Nef is incapable of blocking SER5 incorporation into HIV-1 particles upon overexpression of this restriction factor (16,17) but that Env itself is a major determinant of SER5 sensitivity (16). Specifically, the Nef/glycoGag dependence of HIV-1 infectivity (and thus its sensitivity to SER3/SER5) has been mapped to the gp120 V1/V2 loops (18), while a recent study revealed a critical role of the V3 loop in modulating the Env sensitivity to SER5 (16).…”

“…Specifically, the Nef/glycoGag dependence of HIV-1 infectivity (and thus its sensitivity to SER3/SER5) has been mapped to the gp120 V1/V2 loops (18), while a recent study revealed a critical role of the V3 loop in modulating the Env sensitivity to SER5 (16). New evidence also suggests that, in addition to its role in SER5 internalization from the plasma membrane, Nef antagonizes the activity of virus-incorporated SER5 by a cryptic mechanism (17).…”