Tubeimosides are pan-coronavirus and filovirus inhibitors that can block their fusion protein binding to Niemann-Pick C1.

SARS-CoV-2 and filovirus enter cells via the cell surface angiotensin-converting enzyme 2 (ACE2) or the late-endosome Niemann-Pick C1 (NPC1) as a receptor. Here, we screened 974 natural compounds and identified Tubeimosides I, II, and III as pan-coronavirus and filovirus entry inhibitors that target NPC1. Using in-silico, biochemical, and genomic approaches, we provide evidence that NPC1 also binds SARS-CoV-2 spike (S) protein on the receptor-binding domain (RBD), which is blocked by Tubeimosides. Importantly, NPC1 strongly promotes productive SARS-CoV-2 entry, which we propose is due to its influence on fusion in late endosomes. The Tubeimosides' antiviral activity and NPC1 function are further confirmed by infection with SARS-CoV-2 variants of concern (VOC), SARS-CoV, and MERS-CoV. Thus, NPC1 is a critical entry co-factor for highly pathogenic human coronaviruses (HCoVs) in the late endosomes, and Tubeimosides hold promise as a new countermeasure for these HCoVs and filoviruses.

The MGF300-2R protein of African swine fever virus is associated with viral pathogenicity by promoting the autophagic degradation of IKKα and IKKß through the recruitment of TOLLIP.

The multigene family genes (MGFs) in the left variable region (LVR) of the African swine fever virus (ASFV) genome have been reported to be involved in viral replication in primary porcine alveolar macrophages (PAMs) and virulence in pigs. However, the exact functions of key MGFs in the LVR that regulate the replication and virulence of ASFV remain unclear. In this study, we identified the MGF300-2R gene to be critical for viral replication in PAMs by deleting different sets of MGFs in the LVR from the highly virulent strain ASFV HLJ/18 (ASFV-WT). The ASFV mutant lacking the MGF300-2R gene (Del2R) showed a 1-log reduction in viral titer, and induced higher IL-1ß and TNF-α production in PAMs than did ASFV-WT. Mechanistically, the MGF300-2R protein was found to interact with and degrade IKKα and IKKß via the selective autophagy pathway. Furthermore, we showed that MGF300-2R promoted the K27-linked polyubiquitination of IKKα and IKKß, which subsequently served as a recognition signal for the cargo receptor TOLLIP-mediated selective autophagic degradation. Importantly, Del2R exhibited a significant reduction in both replication and virulence compared with ASFV-WT in pigs, likely due to the increased IL-1ß and TNF-α, indicating that MGF300-2R is a virulence determinant. These findings reveal that MGF300-2R suppresses host innate immune responses by mediating the degradation of IKKα and IKKß, which provides clues to paving the way for the rational design of live attenuated vaccines to control ASF.

RNF185 regulates proteostasis in Ebolavirus infection by crosstalk between the calnexin cycle, ERAD, and reticulophagy.

Virus infection affects cellular proteostasis and provides an opportunity to study this cellular process under perturbation. The proteostasis network in the endoplasmic reticulum (ER) is composed of the calnexin cycle, and the two protein degradation pathways ER-associated protein degradation (ERAD) and ER-to-lysosome-associated degradation (ERLAD/ER-phagy/reticulophagy). Here we show that calnexin and calreticulin trigger Zaire Ebolavirus (EBOV) glycoprotein GP1,2 misfolding. Misfolded EBOV-GP1,2 is targeted by ERAD machinery, but this results in lysosomal instead of proteasomal degradation. Moreover, the ER Ub ligase RNF185, usually associated with ERAD, polyubiquitinates EBOV-GP1,2 on lysine 673 via ubiquitin K27-linkage. Polyubiquinated GP1,2 is subsequently recruited into autophagosomes by the soluble autophagy receptor sequestosome 1 (SQSTM1/p62), in an ATG3- and ATG5-dependent manner. We conclude that EBOV hijacks all three proteostasis mechanisms in the ER to downregulate GP1,2 via polyubiquitination and show that this increases viral fitness. This study identifies linkages among proteostasis network components previously thought to function independently.

Cul3-KLHL20 E3 ubiquitin ligase plays a key role in the arms race between HIV-1 Nef and host SERINC5 restriction.

HIV-1 must counteract various host restrictions to establish productive infection. SERINC5 is a potent restriction factor that blocks HIV-1 entry from virions, but its activity is counteracted by Nef. The SERINC5 and Nef activities are both initiated from the plasma membrane, where SERINC5 is packaged into virions for viral inhibition or downregulated by Nef via lysosomal degradation. However, it is still unclear how SERINC5 is localized to and how its expression is regulated on the plasma membrane. We now report that Cullin 3-KLHL20, a trans-Golgi network (TGN)-localized E3 ubiquitin ligase, polyubiquitinates SERINC5 at lysine 130 via K33/K48-linked ubiquitination. The K33-linked polyubiquitination determines SERINC5 expression on the plasma membrane, and the K48-linked polyubiquitination contributes to SERINC5 downregulation from the cell surface. Our study reveals an important role of K130 polyubiquitination and K33/K48-linked ubiquitin chains in HIV-1 infection by regulating SERINC5 post-Golgi trafficking and degradation.

Protein disulfide isomerases (PDIs) negatively regulate ebolavirus structural glycoprotein expression in the endoplasmic reticulum (ER) via the autophagy-lysosomal pathway.

Zaire ebolavirus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates with high morbidity and mortality. EBOV infection is dependent on its structural glycoprotein (GP), but high levels of GP expression also trigger cell rounding, detachment, and downregulation of many surface molecules that is thought to contribute to its high pathogenicity. Thus, EBOV has evolved an RNA editing mechanism to reduce its GP expression and increase its fitness. We now report that the GP expression is also suppressed at the protein level in cells by protein disulfide isomerases (PDIs). Although PDIs promote oxidative protein folding by catalyzing correct disulfide formation in the endoplasmic reticulum (ER), PDIA3/ERp57 adversely triggered the GP misfolding by targeting GP cysteine residues and activated the unfolded protein response (UPR). Abnormally folded GP was targeted by ER-associated protein degradation (ERAD) machinery and, unexpectedly, was degraded via the macroautophagy/autophagy-lysosomal pathway, but not the proteasomal pathway. PDIA3 also decreased the GP expression from other ebolavirus species but increased the GP expression from Marburg virus (MARV), which is consistent with the observation that MARV-GP does not cause cell rounding and detachment, and MARV does not regulate its GP expression via RNA editing during infection. Furthermore, five other PDIs also had a similar inhibitory activity to EBOV-GP. Thus, PDIs negatively regulate ebolavirus glycoprotein expression, which balances the viral life cycle by maximizing their infection but minimizing their cellular effect. We suggest that ebolaviruses hijack the host protein folding and ERAD machinery to increase their fitness via reticulophagy during infection.Abbreviations: 3-MA: 3-methyladenine; 4-PBA: 4-phenylbutyrate; ACTB: ß-actin; ATF: activating transcription factor; ATG: autophagy-related; BafA1: bafilomycin A1; BDBV: Bundibugyo ebolavirus; CALR: calreticulin; CANX: calnexin; CHX: cycloheximide; CMA: chaperone-mediated autophagy; ConA: concanamycin A; CRISPR: clusters of regularly interspaced short palindromic repeats; Cas9: CRISPR-associated protein 9; dsRNA: double-stranded RNA; EBOV: Zaire ebolavirus; EDEM: ER degradation enhancing alpha-mannosidase like protein; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; Env: envelope glycoprotein; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; GP: glycoprotein; HA: hemagglutinin; HDAC6: histone deacetylase 6; HMM: high-molecular-mass; HIV-1: human immunodeficiency virus type 1; HSPA5/BiP: heat shock protein family A (Hsp70) member 5; IAV: influenza A virus; IP: immunoprecipitation; KIF: kifenesine; Lac: lactacystin; LAMP: lysosomal associated membrane protein; MAN1B1/ERManI: mannosidase alpha class 1B member 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MARV: Marburg virus; MLD: mucin-like domain; NHK/SERPINA1: alpha1-antitrypsin variant null (Hong Kong); NTZ: nitazoxanide; PDI: protein disulfide isomerase; RAVV: Ravn virus; RESTV: Reston ebolavirus; SARS-CoV: severe acute respiratory syndrome coronavirus; SBOV: Sudan ebolavirus; sGP: soluble GP; SQSTM1/p62: sequestosome 1; ssGP: small soluble GP; TAFV: Taï Forest ebolavirus; TIZ: tizoxanide; TGN: thapsigargin; TLD: TXN (thioredoxin)-like domain; Ub: ubiquitin; UPR: unfolded protein response; VLP: virus-like particle; VSV: vesicular stomatitis virus; WB: Western blotting; WT: wild-type; XBP1: X-box binding protein 1.

HIV-1 Nef interacts with the cyclin K/CDK13 complex to antagonize SERINC5 for optimal viral infectivity.

HIV-1-negative factor (Nef) protein antagonizes serine incorporator 5 (SERINC5) by redirecting this potent restriction factor to the endosomes and lysosomes for degradation. However, the precise mechanism remains unclear. Using affinity purification/mass spectrometry, we identify cyclin K (CycK) and cyclin-dependent kinase 13 (CDK13) as a Nef-associated kinase complex. CycK/CDK13 phosphorylates the serine at position 360 (S360) in SERINC5, which is required for Nef downregulation of SERINC5 from the cell surface and its counteractivity of the SERINC5 antiviral activity. To understand the role of S360 phosphorylation, we generate chimeric proteins between CD8 and SERINC5 to study their response to Nef. Nef not only downregulates but, importantly, also binds to this chimera in an S360-dependent manner. Thus, S360 phosphorylation increases interactions between Nef and SERINC5 and initiates the destruction of SERINC5 by the endocytic machinery.

Host Immune Response Driving SARS-CoV-2 Evolution.

The transmission and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of paramount importance in controlling and combating the coronavirus disease 2019 (COVID-19) pandemic. Currently, over 15,000 SARS-CoV-2 single mutations have been recorded, which have a great impact on the development of diagnostics, vaccines, antibody therapies, and drugs. However, little is known about SARS-CoV-2's evolutionary characteristics and general trend. In this work, we present a comprehensive genotyping analysis of existing SARS-CoV-2 mutations. We reveal that host immune response via APOBEC and ADAR gene editing gives rise to near 65% of recorded mutations. Additionally, we show that children under age five and the elderly may be at high risk from COVID-19 because of their overreaction to the viral infection. Moreover, we uncover that populations of Oceania and Africa react significantly more intensively to SARS-CoV-2 infection than those of Europe and Asia, which may explain why African Americans were shown to be at increased risk of dying from COVID-19, in addition to their high risk of COVID-19 infection caused by systemic health and social inequities. Finally, our study indicates that for two viral genome sequences of the same origin, their evolution order may be determined from the ratio of mutation type, C > T over T > C.

MARCH8 Inhibits Ebola Virus Glycoprotein, Human Immunodeficiency Virus Type 1 Envelope Glycoprotein, and Avian Influenza Virus H5N1 Hemagglutinin Maturation.

Membrane-associated RING-CH-type 8 (MARCH8) strongly blocks human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) incorporation into virions by downregulating its cell surface expression, but the mechanism is still unclear. We now report that MARCH8 also blocks the Ebola virus (EBOV) glycoprotein (GP) incorporation via surface downregulation. To understand how these viral fusion proteins are downregulated, we investigated the effects of MARCH8 on EBOV GP maturation and externalization via the conventional secretion pathway. MARCH8 interacted with EBOV GP and furin when detected by immunoprecipitation and retained the GP/furin complex in the Golgi when their location was tracked by a bimolecular fluorescence complementation (BiFC) assay. MARCH8 did not reduce the GP expression or affect the GP modification by high-mannose N-glycans in the endoplasmic reticulum (ER), but it inhibited the formation of complex N-glycans on the GP in the Golgi. Additionally, the GP O-glycosylation and furin-mediated proteolytic cleavage were also inhibited. Moreover, we identified a novel furin cleavage site on EBOV GP and found that only those fully glycosylated GPs were processed by furin and incorporated into virions. Furthermore, the GP shedding and secretion were all blocked by MARCH8. MARCH8 also blocked the furin-mediated cleavage of HIV-1 Env (gp160) and the highly pathogenic avian influenza virus H5N1 hemagglutinin (HA). We conclude that MARCH8 has a very broad antiviral activity by prohibiting different viral fusion proteins from glycosylation and proteolytic cleavage in the Golgi, which inhibits their transport from the Golgi to the plasma membrane and incorporation into virions.IMPORTANCE Enveloped viruses express three classes of fusion proteins that are required for their entry into host cells via mediating virus and cell membrane fusion. Class I fusion proteins are produced from influenza viruses, retroviruses, Ebola viruses, and coronaviruses. They are first synthesized as a type I transmembrane polypeptide precursor that is subsequently glycosylated and oligomerized. Most of these precursors are cleaved en route to the plasma membrane by a cellular protease furin in the late secretory pathway, generating the trimeric N-terminal receptor-binding and C-terminal fusion subunits. Here, we show that a cellular protein, MARCH8, specifically inhibits the furin-mediated cleavage of EBOV GP, HIV-1 Env, and H5N1 HA. Further analyses uncovered that MARCH8 blocked the EBOV GP glycosylation in the Golgi and inhibited its transport from the Golgi to the plasma membrane. Thus, MARCH8 has a very broad antiviral activity by specifically inactivating different viral fusion proteins.

Host immune response driving SARS-CoV-2 evolution.

The transmission and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of paramount importance to the controlling and combating of coronavirus disease 2019 (COVID-19) pandemic. Currently, near 15,000 SARS-CoV-2 single mutations have been recorded, having a great ramification to the development of diagnostics, vaccines, antibody therapies, and drugs. However, little is known about SARS-CoV-2 evolutionary characteristics and general trend. In this work, we present a comprehensive genotyping analysis of existing SARS-CoV-2 mutations. We reveal that host immune response via APOBEC and ADAR gene editing gives rise to near 65\% of recorded mutations. Additionally, we show that children under age five and the elderly may be at high risk from COVID-19 because of their overreacting to the viral infection. Moreover, we uncover that populations of Oceania and Africa react significantly more intensively to SARS-CoV-2 infection than those of Europe and Asia, which may explain why African Americans were shown to be at increased risk of dying from COVID-19, in addition to their high risk of getting sick from COVID-19 caused by systemic health and social inequities. Finally, our study indicates that for two viral genome sequences of the same origin, their evolution order may be determined from the ratio of mutation type C$>$T over T$>$C.

Wild-type p53-induced phosphatase 1 down-regulation promotes apoptosis by activating the DNA damage-response pathway in amyotrophic lateral sclerosis.

Accumulation of DNA damage has been detected in the spinal cord of patients as well as in the G93A mouse model of amyotrophic lateral sclerosis (ALS). Wild-type p53-induced phosphatase 1 (Wip1) is a p53-inducible serine/threonine phosphatase that terminates DNA-damage responses via dephosphorylation of DNA-damage response proteins, namely ataxia-telangiectasia mutated (ATM) kinase, checkpoint kinase 2, and p53, thus enhancing cell proliferation. However, the role of Wip1, DNA-damage responses, and their interaction in ALS development remains to be elucidated. Here, we showed that Wip1 expression levels were substantially decreased in ALS motor neurons compared with wild-type controls both in vivo and in vitro. The DNA-damage response was activated in superoxide dismutase 1 (SOD1) G93A-transfected cells. However, increased expression of Wip1 improved cell viability and inhibited the DNA-damage response in mutated SOD1G93A cells. Further studies demonstrated that decreased Wip1 expression reduced cell viability and further activated the DNA-damage response in chronic H2O2-treated NSC34 cells. In contrast, Wip1 promoted cell survival and suppressed DNA damage-induced apoptosis during persistent DNA damage conditions. Over-expression of Wip1 in the central nervous system (CNS) can delay the onset of disease symptoms, extended the survival, decreased MN loss improved motor function and inhibit the DNA-damage response in SOD1 G93A mice. Furthermore, homeodomain-interacting protein kinase 2 (HIPK2) promoted the degradation of Wip1 via the ubiquitin-proteasome system during chronic stress. These findings indicate that persistent accumulation of DNA damage and subsequent chronic activation of the downstream DNA damage-response ATM and p53 pro-apoptotic signaling pathways may trigger neuronal dysfunction and neuronal death in ALS. Wip1 may play a protective role by targeting the DNA-damage response in ALS motor neurons. Importantly, these findings provide a novel direction for therapeutic options for patients with ALS.

Proteasomal degradation of human SERINC4: A potent host anti-HIV-1 factor that is antagonized by nef.

The serine incorporator (SERINC) protein family has five paralogous members with 9-11 transmembrane domains. SERINC5 is a potent host restriction factor and antagonized by HIV-1 Nef and two other retroviral accessory proteins via the lysosomal degradation pathway. Here, we investigated human SERINC4 expression and antiviral mechanisms. Unlike its four paralogs, human SERINC4 is subjected to proteasome-mediated turnover, resulting in ~250-fold lower expression than SERINC5. However, when expression was normalized, human SERINC4 restricted HIV-1 replication as effectively as SERINC5, and SERINC4 was also antagonized by Nef via the lysosomal pathway. Although SERINC4 proteins are conserved within primates or rodents, their N-terminal regions are highly variable across species. Interestingly, unlike human SERINC4, murine SERINC4 was stably expressed but had a very poor antiviral activity. We created stable SERINC4 chimeras by replacing the N-terminal region and found that the 1-34 and 35-92 amino acids determine SERINC4 antiviral activity or protein expression, respectively. Using these chimeras, we demonstrate that SERINC4 is incorporated into HIV-1 virions and restricts Tier 1 HIV-1 more effectively than Tier 3 HIV-1. Importantly, SERINC4 increases HIV-1 sensitivity to broadly neutralizing antibodies. Thus, human SERINC4 strongly restricts HIV-1 replication when it is overexpressed, which reflects a potential antiviral activity of this gene product under physiological conditions.

ANP32A and ANP32B are key factors in the Rev-dependent CRM1 pathway for nuclear export of HIV-1 unspliced mRNA.

The nuclear export receptor CRM1 is an important regulator involved in the shuttling of various cellular and viral RNAs between the nucleus and the cytoplasm. HIV-1 Rev interacts with CRM1 in the late phase of HIV-1 replication to promote nuclear export of unspliced and single spliced HIV-1 transcripts. However, other cellular factors involved in the CRM1-dependent viral RNA nuclear export remain largely unknown. Here, we demonstrate that ANP32A and ANP32B mediate the export of unspliced or partially spliced viral mRNA via interactions with Rev and CRM1. We found that a double, but not single, knockout of ANP32A and ANP32B significantly decreased the expression of gag protein. Reconstitution of either ANP32A or ANP32B restored the viral production equally. Disruption of both ANP32A and ANP32B expression led to a dramatic accumulation of unspliced viral mRNA in the nucleus. We further identified that ANP32A and ANP32B interact with both Rev and CRM1 to promote RNA transport. Our data strongly suggest that ANP32A and ANP32B play an important role in the Rev-CRM1 pathway, which is essential for HIV-1 replication, and our findings provide a candidate therapeutic target for host defense against retroviral infection.

CD4 Expression and Env Conformation Are Critical for HIV-1 Restriction by SERINC5.

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.

The retroviral accessory proteins S2, Nef, and glycoMA use similar mechanisms for antagonizing the host restriction factor SERINC5.

Serine incorporator 5 (SERINC5) is a recently identified restriction factor that blocks virus entry but is antagonized by three unrelated retroviral accessory proteins. The S2 protein from equine infectious anemia virus (EIAV) has been reported to reduce SERINC5 expression at steady-state levels likely via the endocytic pathway; however, the precise mechanism is still unclear. Here, we investigated how EIAV S2 protein down-regulates SERINC5 compared with down-regulation induced by Nef from HIV-1 and glycoMA proteins from murine leukemia virus (MLV). Using bimolecular fluorescence complementation (BiFC) assay and immunoprecipitation (IP), we detected an interaction between S2 and SERINC5. We found that this interaction relies on the S2 myristoylation site, indicating that it may occur on the plasma membrane. S2 internalized SERINC5 via receptor-mediated endocytosis and targeted it to endosomes and lysosomes, resulting in a ubiquitination-dependent decrease in SERINC5 expression at steady-state levels. Both BiFC and IP detected a glycoMA-SERINC5 interaction, but a Nef-SERINC5 interaction was detected only by BiFC. Moreover, S2 and glycoMA down-regulated SERINC5 more effectively than did Nef. We further show that unlike Nef, both S2 and glycoMA effectively down-regulate SERINC2 and also SERINC5 from Xenopus tropicalis (xSERINC5). Moreover, we detected expression of the equine SERINC5 (eSERINC5) protein and observed that its expression is much weaker than expression levels of SERINC5 from other species. Nonetheless, eSERINC5 had a strong antiviral activity that was effectively counteracted by S2. We conclude that HIV-1, EIAV, and MLV share a similar mechanism to antagonize viral restriction by host SERINC5.

In vitro Infection of Primary Human Monocytes with HIV-1.

Monocyte infection by HIV-1 is an important component of chronic HIV pathogenesis. Following infection by HIV-1, monocytes are able to cross the blood brain barrier and set up a viral reservoir in the central nervous system. Additionally, in the setting of chronic HIV-1 infection, monocytes can become activated either directly through HIV-1 infection or indirectly via HIV-1-mediated systemic immune activation. Currently, there are few studies looking at HIV-1 infection of primary human monocytes in vitro. Furthermore, detection of successful HIV-1 infection of monocytes can be laborious requiring an ELISA for p24 or assessing levels of HIV-1 mRNA or DNA. This protocol utilizes an HIV-1 strain expressing GFP to allow for easy quantification of HIV-1 infection by fluorescence-assisted cell sorting (FACS). By determining HIV-1 infection by FACS one can take advantage of its multiparametric nature allowing for the use of less cells and the ability to assess the expression of other markers on HIV-1+ and HIV-1- cells in the same experiment. Additionally, this protocol could be modified to study HIV-1 infection of other cells including CD4+ T cells.

Murine Leukemia Virus Glycosylated Gag Reduces Murine SERINC5 Protein Expression at Steady-State Levels via the Endosome/Lysosome Pathway to Counteract SERINC5 Antiretroviral Activity.

Glycosylated Gag (glycoGag) is an accessory protein expressed by most gammaretroviruses, including murine leukemia virus (MLV). MLV glycoGag not only enhances MLV replication and disease progression but also increases human immunodeficiency virus type 1 (HIV-1) infectivity as Nef does. Recently, SERINC5 (Ser5) was identified as the target for Nef, and the glycoGag Nef-like activity has been attributed to the Ser5 antagonism. Here, we investigated how glycoGag antagonizes Ser5 using MLV glycoMA and murine Ser5 proteins. We confirm previous observations that glycoMA relocalizes Ser5 from plasma membrane to perinuclear punctated compartments and the important role of its Y36XXL39 motif in this process. We find that glycoMA decreases Ser5 expression at steady-state levels and identify two other glycoGag crucial residues, P31 and R63, for the Ser5 downregulation. The glycoMA and Ser5 interaction is detected in live cells using a bimolecular fluorescence complementation assay. Ser5 is internalized via receptor-mediated endocytosis and relocalized to Rab5+ early, Rab7+ late, and Rab11+ recycling endosomes by glycoMA. Although glycoMA is not polyubiquitinated, the Ser5 downregulation requires Ser5 polyubiquitination via the K48- and K63-linkage, resulting in Ser5 destruction in lysosomes. Although P31, Y36, L39, and R63 are not required for glycoMA interaction with Ser5, they are required for Ser5 relocalization to lysosomes for destruction. In addition, although murine Ser1, Ser2, and Ser3 exhibit very poor antiviral activity, they are also targeted by glycoMA for lysosomal destruction. We conclude that glycoGag has a broad activity to downregulate SERINC proteins via the cellular endosome/lysosome pathway, which promotes viral replication.IMPORTANCE MLV glycoGag not only enhances MLV replication but also increases HIV-1 infectivity similarly as Nef. Recent studies have discovered that both glycoGag and Nef antagonize a novel host restriction factor Ser5 and promote viral replication. Compared to Nef, the glycoGag antagonism of Ser5 is still poorly understood. MLV glycoGag is a transmembrane version of the structural Gag protein with an extra 88-amino-acid leader region that determines its activity. We now show that glycoGag interacts with Ser5 in live cells and internalizes Ser5 via receptor-mediated endocytosis. Ser5 is polyubiquitinated and relocalized to endosomes and lysosomes for massive destruction. In addition to the previously identified tyrosine-based sorting signal, we find two more important residues for Ser5 relocalization and downregulation. We also find that the Ser5 sensitivity to glycoGag is conserved in the SERINC family. Together, our findings highlight the important role of endosome/lysosome pathway in the enhancement of viral replication by viral proteins.

HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System.

The primate lentiviral accessory protein Nef downregulates CD4 and major histocompatibility complex class I (MHC-I) from the cell surface via independent endosomal trafficking pathways to promote viral pathogenesis. In addition, Nef antagonizes a novel restriction factor, SERINC5 (Ser5), to increase viral infectivity. To explore the molecular mechanism of Ser5 antagonism by Nef, we determined how Nef affects Ser5 expression and intracellular trafficking in comparison to CD4 and MHC-I. We confirm that Nef excludes Ser5 from human immunodeficiency virus type 1 (HIV-1) virions by downregulating its cell surface expression via similar functional motifs required for CD4 downregulation. We find that Nef decreases both Ser5 and CD4 expression at steady-state levels, which are rescued by NH4Cl or bafilomycin A1 treatment. Nef binding to Ser5 was detected in living cells using a bimolecular fluorescence complementation assay, where Nef membrane association is required for interaction. In addition, Nef triggers rapid Ser5 internalization via receptor-mediated endocytosis and relocalizes Ser5 to Rab5+ early, Rab7+ late, and Rab11+ recycling endosomes. Manipulation of AP-2, Rab5, Rab7, and Rab11 expression levels affects the Nef-dependent Ser5 and CD4 downregulation. Moreover, although Nef does not promote Ser5 polyubiquitination, Ser5 downregulation relies on the ubiquitination pathway, and both K48- and K63-specific ubiquitin linkages are required for the downregulation. Finally, Nef promotes Ser5 colocalization with LAMP1, which is enhanced by bafilomycin A1 treatment, suggesting that Ser5 is targeted to lysosomes for destruction. We conclude that Nef uses a similar mechanism to downregulate Ser5 and CD4, which sorts Ser5 into a point-of-no-return degradative pathway to counteract its restriction.IMPORTANCE Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express an accessory protein called Nef to promote viral pathogenesis. Nef drives immune escape in vivo through downregulation of CD4 and MHC-I from the host cell surface. Recently, Nef was reported to counteract a novel host restriction factor, Ser5, to increase viral infectivity. Nef downregulates cell surface Ser5, thus preventing its incorporation into virus particles, resulting in disruption of its antiviral activity. Here, we report mechanistic studies of Nef-mediated Ser5 downregulation in comparison to CD4 and MHC-I. We demonstrate that Nef binds directly to Ser5 in living cells and that Nef-Ser5 interaction requires Nef association with the plasma membrane. Subsequently, Nef internalizes Ser5 from the plasma membrane via receptor-mediated endocytosis, and targets ubiquitinated Ser5 to endosomes and lysosomes for destruction. Collectively, these results provide new insights into our ongoing understanding of the Nef-Ser5 arms race in HIV-1 infection.

Innate Sensing of Influenza A Virus Hemagglutinin Glycoproteins by the Host Endoplasmic Reticulum (ER) Stress Pathway Triggers a Potent Antiviral Response via ER-Associated Protein 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.

Identification of SERINC5-001 as the Predominant Spliced Isoform for HIV-1 Restriction.

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.