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

Li, Sunan; Ahmad, Iqbal; Shi, Jing; Wang, Bin; Yu, Changqing; Zhang, Lixin; Zheng, Yong Hui

doi:10.1128/jvi.01651-18

Cited by 35 publications

(59 citation statements)

References 34 publications

(67 reference statements)

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“…In order to elucidate the Ser5 antiviral mechanism, we adopted a bimolecular fluorescence complementation (BiFC) assay to detect Env-Env association in live cells (24). We previously detected the specific Nef-Ser5, glycoGag-Ser5, and S2-Ser5 interactions using this assay (8)(9)(10). A hemagglutinin (HA)-tagged VN or FLAG-tagged VC gene fragment was linked to the last codon of NL and AD8 gp160, and these new gene fragments were inserted into a proviral vector, pH22, by replacing the entire gp160 and 5= portion of nef ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Detection of Env-Ser5 interaction by BiFC. We created vectors expressing Ser5-VN and Ser5-VC fusion proteins in our previous studies (9,10). Because Ser2 does not have any antiviral activity (4), we additionally created vectors expressing Ser2-VN and Ser2-VC fusion proteins as controls.…”

Section: Resultsmentioning

confidence: 99%

“…In addition to Nef, Ser5 is antagonized by murine leukemia virus (MLV) glycosylated Gag (glycoGag) (2,3) and equine infectious anemia virus (EIAV) S2 proteins (6,7). We recently reported that Nef, glycoGag, and S2 proteins all target Ser5 to the endosome/lysosome pathways for degradation (8)(9)(10). Thus, Ser5 is an important restriction factor for a wide range of retroviruses.…”

mentioning

confidence: 99%

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

Zhang

Shi

Qiu

et al. 2019

J Virol

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Zhang

Shi

Qiu

et al. 2019

J Virol

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“…To construct BiFC expression vectors, the furin, MARCH8, and GP-ΔMLD cDNAs were inserted into pcDNA3.1-VN, pcDNA3.1-VN-HA, or pcDNA3.1-FLAG-VC (C-terminal VN/VC) vectors by EcoRI/AgeI digestion, which generated pcDNA3.1-Furin-VN, pcDNA3.1-Furin-VN-HA, pcDNA3.1-MARCH8-VN-HA, or pcDNA3.1-GPΔMLD-FLAG-VC, respectively. pcDNA3.1-SERINC5-FLAG-VC was reported previously ( 32 ). mCherry-TGNP-N-10 and mCherry-Calnexin-N-14 expression vectors were obtained from Michael Davidson via Addgene.…”

Section: Methodsmentioning

confidence: 99%

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

Zhang

et al. 2020

mBio

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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.

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“…It plays multiple roles in CME, such as promoting the assembly of clathrin-coated pits and coordinating interactions between clathrin and other CME proteins (59,60). To date, the AP-2 complex has been reported to be involved in the endocytosis processes of different viruses, including HIV (61,62), murine cytomegalovirus (63), and white spot syndrome virus (64). As for IAV, a previous study by Chen and Zhuang reported that siRNA knockdown of the AP2M1 subunit of the AP-2 complex did not inhibit the clathrin-mediated uptake of IAV (11).…”

Section: Discussionmentioning

confidence: 99%

The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry

Wang

et al. 2020

J Virol

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Influenza A virus (IAV) coopts numerous host factors to complete its replication cycle. Here, we identify free fatty acid receptor 2 (FFAR2) as a cofactor for IAV entry into host cells. We found that downregulation of FFAR2 or Ffar2 expression significantly reduced the replication of IAV in A549 or RAW 264.7 cells. The treatment of A549 cells with small interfering RNA (siRNA) targeting FFAR2 or the FFAR2 pathway agonists 2-(4-chlorophenyl)-3-methyl-N-(thiazol-2-yl)butanamide (4-CMTB) and compound 58 (Cmp58) [(S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide] dramatically inhibited the nuclear accumulation of viral nucleoprotein (NP) at early time points postinfection, indicating that FFAR2 functions in the early stage of the IAV replication cycle. FFAR2 downregulation had no effect on the expression of sialic acid (SA) receptors on the cell membrane, the attachment of IAV to the SA receptors, or the activity of the viral ribonucleoprotein (vRNP) complex. Rather, the amount of internalized IAVs was significantly reduced in FFAR2-knocked-down or 4-CMTB- or Cmp58-treated A549 cells. Further studies showed that FFAR2 associated with β-arrestin1 and that β-arrestin1 interacted with the β2-subunit of the AP-2 complex (AP2B1), the essential adaptor of the clathrin-mediated endocytosis pathway. Notably, siRNA knockdown of either β-arrestin1 or AP2B1 dramatically impaired IAV replication, and AP2B1 knockdown or treatment with Barbadin, an inhibitor targeting the β-arrestin1/AP2B1 complex, remarkably decreased the amount of internalized IAVs. Moreover, we found that FFAR2 interacted with three G protein-coupled receptor (GPCR) kinases (i.e., GRK2, GRK5, and GRK6) whose downregulation inhibited IAV replication. Together, our findings demonstrate that the FFAR2 signaling cascade is important for the efficient endocytosis of IAV into host cells. IMPORTANCE To complete its replication cycle, IAV hijacks the host endocytosis machinery to invade cells. However, the underlying mechanisms of how IAV is internalized into host cells remain poorly understood, emphasizing the need to elucidate the role of host factors in IAV entry into cells. In this study, we identified FFAR2 as an important host factor for the efficient replication of both low-pathogenic and highly pathogenic IAV. We revealed that FFAR2 facilitates the internalization of IAV into target cells during the early stage of infection. Upon further characterization of the role of FFAR2-associated proteins in virus replication, we found that the FFAR2–β-arrestin1–AP2B1 signaling cascade is important for the efficient endocytosis of IAV. Our findings thus further our understanding of the biological details of IAV entry into host cells and establish FFAR2 as a potential target for antiviral drug development.

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

Cited by 35 publications

References 34 publications

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

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

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

The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry

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