Serine incorporator 5 (SERINC5) is a recently identified restriction factor that strongly blocks HIV-1 entry but is counteracted by Nef. Notably, tier 1 HIV-1 Env proteins are sensitive to SERINC5, whereas the majority of tier 2/3 Env proteins are resistant to SERINC5, when viruses are produced from CD4-negative cells and tested by a single-round replication assay. Here, we investigated the Env-dependent SERINC5 antiviral mechanism by comparing tier 1 NL Env with tier 3 AD8 Env proteins. We found that when NL and AD8 viruses were inoculated into CD4+ T cells and human peripheral blood mononuclear cells (PBMCs), the propagation of the two viruses was restricted to a similar level when Nef was not expressed. Using a bimolecular fluorescence complementation (BiFC) assay, we detected Env-Env association and Env-SERINC5 interactions. A much greater level of NL Env-SERINC5 interactions was detected than was AD8 Env-SERINC5 interactions, which was further validated by immunoprecipitation assays. In addition, SERINC5 dissociated the NL Env trimeric complex more effectively than the AD8 Env trimeric complex when CD4 was not expressed. However, when CD4 was expressed, SERINC5 became more capable of interacting with AD8 Env and dissociating its trimeric complex. Moreover, AD8 and several other tier 2/3 viruses produced in the presence of CD4 became sensitive to SERINC5 when measured by the single-round replication assay. Because tier 1 and tier 2/3 Env trimers have open and closed conformations, respectively, and CD4 opens the closed conformation, we conclude that SERINC5 selectively dissociates Env trimers with an open conformation to restrict HIV-1 replication. IMPORTANCE Restriction factors provide the first line of defense against retrovirus infection by posing several blocks to the viral replication cycle. SERINC5 is a novel restriction factor that strongly blocks HIV-1 entry, although it is counteracted by Nef. Currently, it is still unclear how HIV-1 entry is blocked by SERINC5. Notably, this entry block is dependent on viral Env proteins. Laboratory-adapted HIV-1 strains are sensitive, whereas primary isolates are highly resistant to SERINC5. Env proteins mediate virus entry via extensive conformational rearrangements from a closed ground state to a CD4-bound open state. We detected Env-Env associations and Env-SERINC5 interactions in live cells by a novel bimolecular fluorescence assay. We demonstrate that CD4 expression increases the Env sensitivity to SERINC5 and allows SERINC5 to dissociate the Env complex, suggesting that SERINC5 restriction is dependent on Env conformation. Our results provide new insights into the poorly defined Env-dependent SERINC5 antiviral mechanism.

Among the five serine incorporator (SERINC) family members, SERINC5 (Ser5) was reported to strongly inhibit HIV-1 replication, which is counteracted by Nef. Ser5 produces 5 alternatively spliced isoforms: Ser5-001 has 10 putative transmembrane domains, whereas Ser5-004, -005, -008a, and -008b do not have the last one. Here, we confirmed the strong Ser5 anti-HIV-1 activity and investigated its isoforms' expression and antiviral activities. It was found that Ser5-001 transcripts were detected at least 10-fold more than the other isoforms by real-time quantitative PCR. When Ser5-001 and its two isoforms Ser5-005 and Ser5-008a were expressed from the same mammalian expression vector, only Ser5-001 was stably expressed, whereas the others were poorly expressed due to rapid degradation. In addition, unlike the other isoforms, which are located mainly in the cytoplasm, Ser5-001 is localized primarily to the plasma membrane. To map the critical determinant, Ser5 mutants bearing C-terminal deletions were created. It was found that the 10th transmembrane domain is required for Ser5 stable expression and plasma membrane localization. As expected, only Ser5-001 strongly inhibits HIV-1 infectivity, whereas the other Ser5 isoforms and mutants that do not have the 10th transmembrane domain show very poor activity. It was also observed that the Nef counteractive activity could be easily saturated by Ser5 overexpression. Thus, we conclude that Ser5-001 is the predominant antiviral isoform that restricts HIV-1, and the 10th transmembrane domain plays a critical role in this process by regulating its protein stability and plasma membrane targeting. IMPORTANCE Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express a small protein, Nef, to enhance viral pathogenesis in vivo.Nef has an important in vitro function, which is to make virus particles more infectious, but the mechanism has been unclear. Recently, Nef was reported to counteract a novel anti-HIV host protein, SERINC5 (Ser5). Ser5 has five alternatively spliced isoforms, Ser5-001, -004, -005, -008a, and -008b, and only Ser5-001 has an extra C-terminal transmembrane domain. We now show that the Ser5-001 transcripts are produced at least 10-fold more than the others, and only Ser5-001 produces stable proteins that are targeted to the plasma membrane. Importantly, only Ser5-001 shows strong anti-HIV-1 activity. We further demonstrate that the extra transmembrane domain is required for Ser5 stable expression and plasma membrane localization. These results suggest that plasma membrane localization is required for Ser5 antiviral activity, and Ser5-001 is the predominant isoform that contributes to the activity.KEYWORDS HIV-1, Nef, SERINC3, SERINC5, infectivity, restriction factor

The Ebola virus (EBOV) trimeric envelope glycoprotein (GP) precursors are cleaved into the receptor-binding GP and the fusion-mediating GP subunits and incorporated into virions to initiate infection. GP and GP form heterodimers that have 15 or two -glycosylation sites (NGSs), respectively. Here we investigated the mechanism of how-glycosylation contributes to GP expression, maturation, and function. As reported before, we found that, although GP NGSs are not critical, the two GP NGSs, Asn and Asn, are essential for GP function. Further analysis uncovered that Asn and Asn regulate GP processing, demannosylation, oligomerization, and conformation. Consequently, these two NGSs are required for GP incorporation into EBOV-like particles and HIV type 1 (HIV-1) pseudovirions and determine viral transduction efficiency. Using CRISPR/Cas9 technology, we knocked out the two classical endoplasmic reticulum chaperones calnexin (CNX) and/or calreticulin (CRT) and found that both CNX and CRT increase GP expression. Nevertheless, NGSs are not required for the GP interaction with CNX or CRT. Together, we conclude that, although Asn and Asn are not required for EBOV GP expression, they synergistically regulate its maturation, which determines its functionality.

Background: HIV-1 envelope (Env) glycoprotein is targeted to endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway for degradation after infecting cells. Results: ER class I ␣-mannosidase (ERManI) interacts with Env and initiates this degradation process. Conclusion: ERManI is essential for the Env degradation. Significance: These findings define a novel endogenous and potential therapeutically applicable antiretroviral mechanism by targeting Env for degradation.

Innate immunity provides an immediate defense against infection after host cells sense danger signals from microbes. Endoplasmic reticulum (ER) stress arises from accumulation of misfolded/unfolded proteins when protein load overwhelms the ER folding capacity, which activates the unfolded protein response (UPR) to restore ER homeostasis. Here, we show that a mechanism for antiviral innate immunity is triggered after the ER stress pathway senses viral glycoproteins. When hemagglutinin (HA) glycoproteins from influenza A virus (IAV) are expressed in cells, ER stress is induced, resulting in rapid HA degradation via proteasomes. The ER-associated protein degradation (ERAD) pathway, an important UPR function for destruction of aberrant proteins, mediates HA degradation. Three class I α-mannosidases were identified to play a critical role in the degradation process, including EDEM1, EDEM2, and ERManI. HA degradation requires either ERManI enzymatic activity or EDEM1/EDEM2 enzymatic activity when ERManI is not expressed, indicating that demannosylation is a critical step for HA degradation. Silencing of EDEM1, EDEM2, and ERManI strongly increases HA expression and promotes IAV replication. Thus, the ER stress pathway senses influenza HA as “nonself” or misfolded protein and sorts HA to ERAD for degradation, resulting in inhibition of IAV replication. IMPORTANCE Viral nucleic acids are recognized as important inducers of innate antiviral immune responses that are sensed by multiple classes of sensors, but other inducers and sensors of viral innate immunity need to be identified and characterized. Here, we used IAV to investigate how host innate immunity is activated. We found that IAV HA glycoproteins induce ER stress, resulting in HA degradation via ERAD and consequent inhibition of IAV replication. In addition, we have identified three class I α-mannosidases, EDEM1, EDEM2, and ERManI, which play a critical role in initiating HA degradation. Knockdown of these proteins substantially increases HA expression and IAV replication. The enzymatic activities and joint actions of these mannosidases are required for this antiviral activity. Our results suggest that viral glycoproteins induce a strong innate antiviral response through activating the ER stress pathway during viral infection.