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Viral envelope glycoproteins are an important structural component on the surfaces of enveloped viruses that direct virus binding and entry and also serve as targets for the host adaptive immune response. In this study, we investigate the mechanism of action of the MARCH family of cellular proteins that disrupt the trafficking and virion incorporation of viral glycoproteins across several virus families.
Cheng Man Lun; Abdul A. Waheed; Ahlam Majadly; Nicole Powell; Eric O. Freed. Mechanism of Viral Glycoprotein Targeting by Membrane-Associated RING-CH Proteins. mBio 2021, 12, 1 .
AMA StyleCheng Man Lun, Abdul A. Waheed, Ahlam Majadly, Nicole Powell, Eric O. Freed. Mechanism of Viral Glycoprotein Targeting by Membrane-Associated RING-CH Proteins. mBio. 2021; 12 (2):1.
Chicago/Turabian StyleCheng Man Lun; Abdul A. Waheed; Ahlam Majadly; Nicole Powell; Eric O. Freed. 2021. "Mechanism of Viral Glycoprotein Targeting by Membrane-Associated RING-CH Proteins." mBio 12, no. 2: 1.
HIV virion assembly begins with the construction of an immature lattice consisting of Gag hexamers. Upon virion release, protease-mediated Gag cleavage leads to a maturation event in which the immature lattice disassembles and the mature capsid assembles. The cellular metabolite inositiol hexakisphosphate (IP6) and maturation inhibitors (MIs) both bind and stabilize immature Gag hexamers, but whereas IP6 promotes virus maturation, MIs inhibit it. Here we show that HIV is evolutionarily constrained to maintain an immature lattice stability that ensures IP6 packaging without preventing maturation. Replication-deficient mutant viruses with reduced IP6 recruitment display increased infectivity upon treatment with the MI PF46396 (PF96) or the acquisition of second-site compensatory mutations. Both PF96 and second-site mutations stabilise the immature lattice and restore IP6 incorporation, suggesting that immature lattice stability and IP6 binding are interdependent. This IP6 dependence suggests that modifying MIs to compete with IP6 for Gag hexamer binding could substantially improve MI antiviral potency.
Donna L. Mallery; Alex B. Kleinpeter; Nadine Renner; K. M. Rifat Faysal; Mariia Novikova; Leo Kiss; Miranda S. C. Wilson; Bilal Ahsan; Zunlong Ke; John A. G. Briggs; Adolfo Saiardi; Till Böcking; Eric O. Freed; Leo C. James. A stable immature lattice packages IP6 for HIV capsid maturation. Science Advances 2021, 7, eabe4716 .
AMA StyleDonna L. Mallery, Alex B. Kleinpeter, Nadine Renner, K. M. Rifat Faysal, Mariia Novikova, Leo Kiss, Miranda S. C. Wilson, Bilal Ahsan, Zunlong Ke, John A. G. Briggs, Adolfo Saiardi, Till Böcking, Eric O. Freed, Leo C. James. A stable immature lattice packages IP6 for HIV capsid maturation. Science Advances. 2021; 7 (11):eabe4716.
Chicago/Turabian StyleDonna L. Mallery; Alex B. Kleinpeter; Nadine Renner; K. M. Rifat Faysal; Mariia Novikova; Leo Kiss; Miranda S. C. Wilson; Bilal Ahsan; Zunlong Ke; John A. G. Briggs; Adolfo Saiardi; Till Böcking; Eric O. Freed; Leo C. James. 2021. "A stable immature lattice packages IP6 for HIV capsid maturation." Science Advances 7, no. 11: eabe4716.
An emerging class of cellular inhibitory proteins has been identified that targets viral glycoproteins. These include the membrane-associated RING-CH (MARCH) family of E3 ubiquitin ligases that, among other functions, downregulate cell-surface proteins involved in adaptive immunity. The RING-CH domain of MARCH proteins is thought to function by catalyzing the ubiquitination of the cytoplasmic tails (CTs) of target proteins, leading to their degradation. MARCH proteins have recently been reported to target retroviral envelope glycoproteins (Env) and vesicular stomatitis virus G glycoprotein (VSV-G). However, the mechanism of antiviral activity remains poorly defined. Here we show that MARCH8 antagonizes the full-length forms of HIV-1 Env, VSV-G, Ebola virus glycoprotein (EboV-GP), and the spike (S) protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) thereby impairing the infectivity of virions pseudotyped with these viral glycoproteins. This MARCH8-mediated targeting of viral glycoproteins requires the E3 ubiquitin ligase activity of the RING-CH domain. We observe that MARCH8 protein antagonism of VSV-G is CT dependent. In contrast, MARCH8-mediated targeting of HIV-1 Env, EboV-GP and SARS-CoV-2 S protein by MARCH8 does not require the CT, suggesting a novel mechanism of MARCH-mediated antagonism of these viral glycoproteins. Confocal microscopy data demonstrate that MARCH8 traps the viral glycoproteins in an intracellular compartment. We observe that the endogenous expression of MARCH8 in several relevant human cell types is rapidly inducible by type I interferon. These results help to inform the mechanism by which MARCH proteins exert their antiviral activity and provide insights into the role of cellular inhibitory factors in antagonizing the biogenesis, trafficking, and virion incorporation of viral glycoproteins. Importance Viral envelope glycoproteins are an important structural component on the surface of enveloped viruses that direct virus binding and entry and also serve as targets for the host adaptive immune response. In this study, we investigate the mechanism of action of the MARCH family of cellular proteins that disrupt the trafficking and virion incorporation of viral glycoproteins across several virus families. This research provides novel insights into how host cell factors antagonize viral replication, perhaps opening new avenues for therapeutic intervention in the replication of a diverse group of highly pathogenic enveloped viruses.
Cheng Man Lun; Abdul A. Waheed; Alhlam Majadly; Nicole Powell; Eric O. Freed. Mechanism of Viral Glycoprotein Targeting by Membrane-associated-RING-CH Proteins. 2021, 1 .
AMA StyleCheng Man Lun, Abdul A. Waheed, Alhlam Majadly, Nicole Powell, Eric O. Freed. Mechanism of Viral Glycoprotein Targeting by Membrane-associated-RING-CH Proteins. . 2021; ():1.
Chicago/Turabian StyleCheng Man Lun; Abdul A. Waheed; Alhlam Majadly; Nicole Powell; Eric O. Freed. 2021. "Mechanism of Viral Glycoprotein Targeting by Membrane-associated-RING-CH Proteins." , no. : 1.
Despite the effectiveness of antiretroviral (ARV) therapy, virological failure can occur in some HIV-1-infected patients in the absence of mutations in drug target genes. We previously reported that, in vitro, the lab-adapted HIV-1 NL4-3 strain can acquire resistance to the integrase inhibitor dolutegravir (DTG) by acquiring mutations in the envelope glycoprotein (Env) that enhance viral cell-cell transmission. In this study, we investigated whether Env-mediated drug resistance extends to ARVs other than DTG and whether it occurs in other HIV-1 isolates. We demonstrate that Env mutations can reduce susceptibility to multiple classes of ARVs and also increase resistance to ARVs when coupled with target-gene mutations. We observe that the NL4-3 Env mutants display a more stable and closed Env conformation and lower rates of gp120 shedding than the WT virus. We also selected for Env mutations in clinically relevant HIV-1 isolates in the presence of ARVs. These Env mutants exhibit reduced susceptibility to DTG, with effects on replication and Env structure that are HIV-1 strain dependent. Finally, to examine a possible in vivo relevance of Env-mediated drug resistance, we performed single-genome sequencing of plasma-derived virus from five patients failing an integrase inhibitor-containing regimen. This analysis revealed the presence of several mutations in the highly conserved gp120-gp41 interface despite low frequency of resistance mutations in integrase. These results suggest that mutations in Env that enhance the ability of HIV-1 to spread via a cell-cell route may increase the opportunity for the virus to acquire high-level drug resistance mutations in ARV target genes. IMPORTANCE Although combination antiretroviral (ARV) therapy is highly effective in controlling the progression of HIV disease, drug resistance can be a major obstacle. Recent findings suggest that resistance can develop without ARV target gene mutations. We previously reported that mutations in the HIV-1 envelope glycoprotein (Env) confer resistance to an integrase inhibitor. Here, we investigated the mechanism of Env-mediated drug resistance and the possible contribution of Env to virological failure in vivo. We demonstrate that Env mutations can reduce sensitivity to major classes of ARVs in multiple viral isolates and define the effect of the Env mutations on Env subunit interactions. We observed that many Env mutations accumulated in individuals failing integrase inhibitor therapy despite a low frequency of resistance mutations in integrase. Our findings suggest that broad-based Env-mediated drug resistance may impact therapeutic strategies and provide clues toward understanding how ARV-treated individuals fail therapy without acquiring mutations in drug target genes.
Yuta Hikichi; Rachel Van Duyne; Phuong Pham; Jennifer L. Groebner; Ann Wiegand; John W. Mellors; Mary F. Kearney; Eric O. Freed. Mechanistic Analysis of the Broad Antiretroviral Resistance Conferred by HIV-1 Envelope Glycoprotein Mutations. mBio 2021, 12, 1 .
AMA StyleYuta Hikichi, Rachel Van Duyne, Phuong Pham, Jennifer L. Groebner, Ann Wiegand, John W. Mellors, Mary F. Kearney, Eric O. Freed. Mechanistic Analysis of the Broad Antiretroviral Resistance Conferred by HIV-1 Envelope Glycoprotein Mutations. mBio. 2021; 12 (1):1.
Chicago/Turabian StyleYuta Hikichi; Rachel Van Duyne; Phuong Pham; Jennifer L. Groebner; Ann Wiegand; John W. Mellors; Mary F. Kearney; Eric O. Freed. 2021. "Mechanistic Analysis of the Broad Antiretroviral Resistance Conferred by HIV-1 Envelope Glycoprotein Mutations." mBio 12, no. 1: 1.
P-selectin glycoprotein ligand-1 (PSGL-1) is a cell surface glycoprotein that binds to P-, E-, and L-selectins to mediate the tethering and rolling of immune cells on the surface of the endothelium for cell migration into inflamed tissues. PSGL-1 has been identified as an interferon-γ (INF-γ)-regulated factor that restricts HIV-1 infectivity, and has recently been found to possess broad-spectrum antiviral activities. Here we report that the expression of PSGL-1 in virus-producing cells impairs the incorporation of SARS-CoV and SARS-CoV-2 spike (S) glycoproteins into pseudovirions and blocks pseudovirus attachment and infection of target cells. These findings suggest that PSGL-1 may potentially inhibit coronavirus replication in PSGL-1+ cells
Sijia He; Abdul A. Waheed; Brian Hetrick; Deemah Dabbagh; Ivan V. Akhrymuk; Kylene Kehn-Hall; Eric O. Freed; Yuntao Wu. PSGL-1 Inhibits the Incorporation of SARS-CoV and SARS-CoV-2 Spike Glycoproteins into Pseudovirions and Impairs Pseudovirus Attachment and Infectivity. Viruses 2020, 13, 46 .
AMA StyleSijia He, Abdul A. Waheed, Brian Hetrick, Deemah Dabbagh, Ivan V. Akhrymuk, Kylene Kehn-Hall, Eric O. Freed, Yuntao Wu. PSGL-1 Inhibits the Incorporation of SARS-CoV and SARS-CoV-2 Spike Glycoproteins into Pseudovirions and Impairs Pseudovirus Attachment and Infectivity. Viruses. 2020; 13 (1):46.
Chicago/Turabian StyleSijia He; Abdul A. Waheed; Brian Hetrick; Deemah Dabbagh; Ivan V. Akhrymuk; Kylene Kehn-Hall; Eric O. Freed; Yuntao Wu. 2020. "PSGL-1 Inhibits the Incorporation of SARS-CoV and SARS-CoV-2 Spike Glycoproteins into Pseudovirions and Impairs Pseudovirus Attachment and Infectivity." Viruses 13, no. 1: 46.
Interactions between viral RNA and the integrase enzyme are required for HIV-1 particles to become infectious, a process that can be disrupted through multiple mechanisms.
Alex Kleinpeter; Eric O Freed. How to package the RNA of HIV-1. eLife 2020, 9, 1 .
AMA StyleAlex Kleinpeter, Eric O Freed. How to package the RNA of HIV-1. eLife. 2020; 9 ():1.
Chicago/Turabian StyleAlex Kleinpeter; Eric O Freed. 2020. "How to package the RNA of HIV-1." eLife 9, no. : 1.
Since the emergence of HIV and AIDS in the early 1980s, the development of safe and effective therapies has accompanied a massive increase in our understanding of the fundamental processes that drive HIV biology. As basic HIV research has informed the development of novel therapies, HIV inhibitors have been used as probes for investigating basic mechanisms of HIV-1 replication, transmission, and pathogenesis. This positive feedback cycle has led to the development of highly effective combination antiretroviral therapy (cART), which has helped stall the progression to AIDS, prolong lives, and reduce transmission of the virus. However, to combat the growing rates of virologic failure and toxicity associated with long-term therapy, it is important to diversify our repertoire of HIV-1 treatments by identifying compounds that block additional steps not targeted by current drugs. Most of the available therapeutics disrupt early events in the replication cycle, with the exception of the protease (PR) inhibitors, which act at the virus maturation step. HIV-1 maturation consists of a series of biochemical changes that facilitate the conversion of an immature, noninfectious particle to a mature infectious virion. These changes include proteolytic processing of the Gag polyprotein by the viral protease (PR), structural rearrangement of the capsid (CA) protein, and assembly of individual CA monomers into hexamers and pentamers that ultimately form the capsid. Here, we review the development and therapeutic potential of maturation inhibitors (MIs), an experimental class of anti-HIV-1 compounds with mechanisms of action distinct from those of the PR inhibitors. We emphasize the key insights into HIV-1 biology and structure that the study of MIs has provided. We will focus on three distinct groups of inhibitors that block HIV-1 maturation: (1) compounds that block the processing of the CA-spacer peptide 1 (SP1) cleavage intermediate, the original class of compounds to which the term MI was applied; (2) CA-binding inhibitors that disrupt capsid condensation; and (3) allosteric integrase inhibitors (ALLINIs) that block the packaging of the viral RNA genome into the condensing capsid during maturation. Although these three classes of compounds have distinct structures and mechanisms of action, they share the ability to block the formation of the condensed conical capsid, thereby blocking particle infectivity.
Alex B. Kleinpeter; Eric O. Freed. HIV-1 Maturation: Lessons Learned from Inhibitors. Viruses 2020, 12, 940 .
AMA StyleAlex B. Kleinpeter, Eric O. Freed. HIV-1 Maturation: Lessons Learned from Inhibitors. Viruses. 2020; 12 (9):940.
Chicago/Turabian StyleAlex B. Kleinpeter; Eric O. Freed. 2020. "HIV-1 Maturation: Lessons Learned from Inhibitors." Viruses 12, no. 9: 940.
Despite the effectiveness of antiretroviral (ARV) therapy, virological failure can occur in some HIV-1 infected patients in the absence of mutations in the proteins targeted by these drugs. We previously reported that, in vitro, the lab-adapted NL4-3 strain of HIV-1 can acquire resistance to the integrase inhibitor dolutegravir (DTG) by acquiring mutations in the envelope glycoprotein (Env) that enhance the ability of HIV-1 to spread via cell-cell transmission. In this study, we investigated whether Env-mediated drug resistance extends to ARVs other than DTG and whether it occurs in other HIV-1 isolates. We demonstrate that Env mutations can broadly confer resistance to multiple classes of ARVs in the context of cell-cell but not cell-free infection and also increase resistance to ARVs when coupled with target-gene drug resistance mutations. To investigate the mechanism of Env-mediated drug resistance, we evaluated the impact of the Env mutations on Env stability and conformational dynamics. We observe that the NL4-3 Env mutants display a more stable and closed Env conformation compared to WT virus and reduced rates of gp120 shedding. We also selected for mutations in the gp41 ectodomain of clinically relevant, CCR5-tropic isolates in the presence of DTG. These Env mutants exhibit reduced susceptibility to DTG, with effects on replication kinetics and Env structure that are HIV-1 strain-dependent. Finally, to examine a possible in vivo relevance of Env-mediated drug resistance, we performed single-genome sequencing of plasma-derived virus from five patients failing an integrase inhibitor-containing regimen. This analysis revealed the presence of several mutations in the highly conserved gp120-gp41 interface despite low frequency of resistance mutations in integrase. These results suggest a “stepping-stone” model whereby mutations in Env that enhance the ability of HIV-1 to spread via a cell-cell route increase the opportunity for the virus to acquire high-level drug resistance mutations in ARV-target genes. Author summary Although combination antiretroviral (ARV) therapy has proven highly effective in controlling the progression of HIV disease, drug resistance can be a major obstacle to long-term treatment, particularly in resource-limited settings. In most cases, resistance arises from the accumulation of mutations in the ARV-target genes; however, in some cases, resistance develops without ARV target-gene mutations. We previously reported that mutations in the HIV-1 envelope glycoprotein (Env) confer resistance to an integrase inhibitor. Here we investigated the mechanism of Env-mediated drug resistance and the possible contribution of Env to virological failure in vivo. We demonstrate that Env mutations can confer broad resistance to multiple classes of ARVs and define the effect of the Env mutations on Env subunit interactions and sensitivity to neutralizing antibodies. We also selected for drug resistance mutations in Env in clinically relevant HIV-1 isolates. We observed that many Env mutations accumulated in individuals failing integrase inhibitor therapy despite a low frequency of resistance mutations in integrase. Our findings suggest that broad-based, Env-mediated drug resistance may impact current and possibly future therapeutic strategies. Our findings also provide clues towards understanding how ARV-treated patients can experience virological failure without acquiring drug resistance mutations in ARV-target genes.
Yuta Hikichi; Rachel Van Duyne; Phuong Pham; Jennifer L. Groebner; Ann Wiegand; John W. Mellors; Mary F. Kearney; Eric O. Freed. Mechanistic Analysis of the Broad Antiretroviral Resistance Conferred by HIV-1 Envelope Glycoprotein Mutations. 2020, 1 .
AMA StyleYuta Hikichi, Rachel Van Duyne, Phuong Pham, Jennifer L. Groebner, Ann Wiegand, John W. Mellors, Mary F. Kearney, Eric O. Freed. Mechanistic Analysis of the Broad Antiretroviral Resistance Conferred by HIV-1 Envelope Glycoprotein Mutations. . 2020; ():1.
Chicago/Turabian StyleYuta Hikichi; Rachel Van Duyne; Phuong Pham; Jennifer L. Groebner; Ann Wiegand; John W. Mellors; Mary F. Kearney; Eric O. Freed. 2020. "Mechanistic Analysis of the Broad Antiretroviral Resistance Conferred by HIV-1 Envelope Glycoprotein Mutations." , no. : 1.
HIV-1 encodes an envelope glycoprotein (Env) that contains a long cytoplasmic tail (CT) harboring trafficking motifs implicated in Env incorporation into virus particles and viral transmission. In most physiologically relevant cell types, the gp41 CT is required for HIV-1 replication, but in the MT-4 T-cell line the gp41 CT is not required for a spreading infection. To help elucidate the role of the gp41 CT in HIV-1 transmission, in this study we investigated the viral and cellular factors that contribute to the permissivity of MT-4 to gp41 CT truncation. We found that the kinetics of HIV-1 production are faster in MT-4 than in the other T-cell lines tested, but MT-4 express equivalent amounts of HIV-1 proteins on a per-cell basis relative to cells not permissive to CT truncation. MT-4 express higher levels of plasma-membrane-associated Env than non-permissive cells and Env internalization from the plasma membrane is slower compared to another T-cell line, SupT1. Paradoxically, despite the high levels of Env on the surface of MT-4, two-fold less Env is incorporated into virus particles in MT-4 compared to SupT1. Cell-to-cell transmission between co-cultured 293T and MT-4 is higher than in co-cultures of 293T with most other T-cell lines tested, indicating that MT-4 are highly susceptible to this mode of infection. These data help to clarify the long-standing question of how MT-4 cells overcome the requirement for the HIV-1 gp41 CT and support a role for gp41 CT-dependent trafficking in Env incorporation and cell-to-cell transmission in physiologically relevant cell lines.
Melissa Victoria Fernandez; Huxley K Hoffman; Nairi Pezeshkian; Philip R. Tedbury; Schuyler B Van Engelenburg; Eric O Freed. Elucidating the basis for permissivity of the MT-4 T-cell line to replication of an HIV-1 mutant lacking the gp41 cytoplasmic tail. 2020, 1 .
AMA StyleMelissa Victoria Fernandez, Huxley K Hoffman, Nairi Pezeshkian, Philip R. Tedbury, Schuyler B Van Engelenburg, Eric O Freed. Elucidating the basis for permissivity of the MT-4 T-cell line to replication of an HIV-1 mutant lacking the gp41 cytoplasmic tail. . 2020; ():1.
Chicago/Turabian StyleMelissa Victoria Fernandez; Huxley K Hoffman; Nairi Pezeshkian; Philip R. Tedbury; Schuyler B Van Engelenburg; Eric O Freed. 2020. "Elucidating the basis for permissivity of the MT-4 T-cell line to replication of an HIV-1 mutant lacking the gp41 cytoplasmic tail." , no. : 1.
Background The continued persistence of HIV-1 as a public health concern due to the lack of a cure calls for the development of new tools for studying replication of the virus. Here, we used NanoLuc, a small and extremely bright luciferase protein, to develop an HIV-1 bioluminescent reporter virus that simplifies functional measurement of virus particle production. Results The reporter virus encodes a Gag protein containing NanoLuc inserted between the matrix (MA) and capsid (CA) domains of Gag, thereby generating virus particles that package high levels of the NanoLuc reporter. We observe that inserting the NanoLuc protein within HIV-1 Gag has minimal impact on Gag expression and virus particle release. We show that the reporter virus recapitulates inhibition of HIV-1 particle release by Gag mutations, the restriction factor tetherin, and the small-molecule inhibitor amphotericin-B methyl ester. Conclusion These results demonstrate that this vector will provide a simple and rapid tool for functional studies of virus particle assembly and release and high-throughput screening for cellular factors and small-molecules that promote or inhibit HIV-1 particle production.
James Kirui; Eric Freed. Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release. 2020, 1 .
AMA StyleJames Kirui, Eric Freed. Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release. . 2020; ():1.
Chicago/Turabian StyleJames Kirui; Eric Freed. 2020. "Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release." , no. : 1.
All retroviruses encode a Gag polyprotein containing an N-terminal matrix domain (MA) that anchors Gag to the plasma membrane and recruits envelope glycoproteins to virus assembly sites. Membrane binding by the Gag protein of HIV-1 and most other lentiviruses is dependent on N-terminal myristoylation of MA by host N-myristoyltransferase enzymes (NMTs), which recognize a six-residue “myristoylation signal” with consensus sequence: M1GXXX[ST]. For unknown reasons, the feline immunodeficiency virus (FIV), which infects both domestic and wild cats, encodes a non-consensus myristoylation sequence not utilized by its host or by other mammals (most commonly: M1GNGQG). To explore the evolutionary basis for this sequence, we compared the structure, dynamics, and myristoylation properties of native FIV MA with a mutant protein containing a consensus feline myristoylation motif (MANOS) and examined the impact of MA mutations on virus assembly and ability to support spreading infection. Unexpectedly, myristoylation efficiency of MANOS in E. coli by co-expressed mammalian NMT was reduced by ~ 70% compared to the wild-type protein. NMR studies revealed that residues of the N-terminal myristoylation signal are fully exposed and mobile in the native protein but partially sequestered in the MANOS chimera, suggesting that the unusual FIV sequence is conserved to promote exposure and efficient myristoylation of the MA N-terminus. In contrast, virus assembly studies indicate that the MANOS mutation does not affect virus assembly, but does prevent virus spread, in feline kidney cells. Our findings indicate that residues of the FIV myristoylation sequence play roles in replication beyond NMT recognition and Gag–membrane binding.
Janae B. Brown; Holly R. Summers; Lola A. Brown; Jan Marchant; Paige N. Canova; Colin T. O'Hern; Sophia T. Abbott; Constance Nyaunu; Simon Maxwell; Talayah Johnson; Morgan Moser; Sherimay D. Ablan; Hannah Carter; Eric O. Freed; Michael F. Summers. Structural and Mechanistic Studies of the Rare Myristoylation Signal of the Feline Immunodeficiency Virus. Journal of Molecular Biology 2020, 432, 4076 -4091.
AMA StyleJanae B. Brown, Holly R. Summers, Lola A. Brown, Jan Marchant, Paige N. Canova, Colin T. O'Hern, Sophia T. Abbott, Constance Nyaunu, Simon Maxwell, Talayah Johnson, Morgan Moser, Sherimay D. Ablan, Hannah Carter, Eric O. Freed, Michael F. Summers. Structural and Mechanistic Studies of the Rare Myristoylation Signal of the Feline Immunodeficiency Virus. Journal of Molecular Biology. 2020; 432 (14):4076-4091.
Chicago/Turabian StyleJanae B. Brown; Holly R. Summers; Lola A. Brown; Jan Marchant; Paige N. Canova; Colin T. O'Hern; Sophia T. Abbott; Constance Nyaunu; Simon Maxwell; Talayah Johnson; Morgan Moser; Sherimay D. Ablan; Hannah Carter; Eric O. Freed; Michael F. Summers. 2020. "Structural and Mechanistic Studies of the Rare Myristoylation Signal of the Feline Immunodeficiency Virus." Journal of Molecular Biology 432, no. 14: 4076-4091.
Background The continued persistence of HIV-1 as a public health concern due to the lack of a cure calls for the development of new tools for studying replication of the virus. Here, we used NanoLuc, a small and extremely bright luciferase protein, to develop an HIV-1 bioluminescent reporter virus that simplifies functional measurement of virus particle production. Results The reporter virus encodes a Gag protein containing NanoLuc inserted between the matrix (MA) and capsid (CA) domains of Gag, thereby generating virus particles that package high levels of the NanoLuc reporter. We observe that inserting the NanoLuc protein within HIV-1 Gag has minimal impact on Gag expression and virus particle release. We show that the reporter virus recapitulates inhibition of HIV-1 particle release by Gag mutations, the restriction factor tetherin, and the small-molecule inhibitor amphotericin-B methyl ester. Conclusion These results demonstrate that this vector will provide a simple and rapid tool for functional studies of virus particle assembly and release and high-throughput screening for cellular factors and small molecules that promote or inhibit HIV-1 particle production.
James Kirui; Eric O. Freed. Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release. Retrovirology 2020, 17, 1 -11.
AMA StyleJames Kirui, Eric O. Freed. Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release. Retrovirology. 2020; 17 (1):1-11.
Chicago/Turabian StyleJames Kirui; Eric O. Freed. 2020. "Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release." Retrovirology 17, no. 1: 1-11.
P-selectin glycoprotein ligand-1 (PSGL-1) is a cell surface glycoprotein that binds to P-, E-, and L-selectins to mediate the tethering and rolling of immune cells on the surface of the endothelium for cell migration into inflamed tissues. PSGL-1 has been identified as an interferon-γ (INF-γ)-regulated factor that restricts HIV-1 infectivity, and has recently been found to possess broad-spectrum antiviral activities. Here we report that the expression of PSGL-1 in virus-producing cells impairs the incorporation of SARS-CoV and SARS-CoV-2 spike (S) glycoproteins into pseudovirions and blocks virus attachment and infection of target cells. These findings suggest that PSGL-1 may potentially inhibit coronavirus replication in PSGL-1+ cells.
Sijia He; Abdul A. Waheed; Brian Hetrick; Deemah Dabbagh; Ivan V. Akhrymuk; Kylene Kehn-Hall; Eric O. Freed; Yuntao Wu. PSGL-1 inhibits the virion incorporation of SARS-CoV and SARS-CoV-2 spike glycoproteins and impairs virus attachment and infectivity. 2020, 1 .
AMA StyleSijia He, Abdul A. Waheed, Brian Hetrick, Deemah Dabbagh, Ivan V. Akhrymuk, Kylene Kehn-Hall, Eric O. Freed, Yuntao Wu. PSGL-1 inhibits the virion incorporation of SARS-CoV and SARS-CoV-2 spike glycoproteins and impairs virus attachment and infectivity. . 2020; ():1.
Chicago/Turabian StyleSijia He; Abdul A. Waheed; Brian Hetrick; Deemah Dabbagh; Ivan V. Akhrymuk; Kylene Kehn-Hall; Eric O. Freed; Yuntao Wu. 2020. "PSGL-1 inhibits the virion incorporation of SARS-CoV and SARS-CoV-2 spike glycoproteins and impairs virus attachment and infectivity." , no. : 1.
Background The continued persistence of HIV-1 as a public health concern due to the lack of a cure calls for the development of new tools for studying replication of the virus. Here, we used NanoLuc, a small and extremely bright luciferase protein, to develop an HIV-1 bioluminescent reporter virus that simplifies functional measurement of virus particle production. Results The reporter virus encodes a Gag protein containing NanoLuc inserted between the matrix (MA) and capsid (CA) domains of Gag, thereby generating virus particles that package high levels of the NanoLuc reporter. We observe that inserting the NanoLuc protein within HIV-1 Gag has minimal impact on Gag expression and virus particle release. We show that the reporter virus recapitulates inhibition of HIV-1 particle release by Gag mutations, the restriction factor tetherin, and the small-molecule inhibitor amphotericin-B methyl ester. Conclusion These results demonstrate that this vector will provide a simple and rapid tool for functional studies of virus particle assembly and release and high-throughput screening for cellular factors and small-molecules that promote or inhibit HIV-1 particle production.
James Kirui; Eric Freed. Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release. 2020, 1 .
AMA StyleJames Kirui, Eric Freed. Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release. . 2020; ():1.
Chicago/Turabian StyleJames Kirui; Eric Freed. 2020. "Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release." , no. : 1.
P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric, mucin-like, 120-kDa glycoprotein that binds to P-, E-, and L-selectins. PSGL-1 is expressed primarily on the surface of lymphoid and myeloid cells and is up-regulated during inflammation to mediate leukocyte tethering and rolling on the surface of endothelium for migration into inflamed tissues. Although it has been reported that PSGL-1 expression inhibits HIV-1 replication, the mechanism of PSGL-1–mediated anti-HIV activity remains to be elucidated. Here we report that PSGL-1 in virions blocks the infectivity of HIV-1 particles by preventing the binding of particles to target cells. This inhibitory activity is independent of the viral glycoprotein present on the virus particle; the binding of particles bearing the HIV-1 envelope glycoprotein or vesicular stomatitis virus G glycoprotein or even lacking a viral glycoprotein is impaired by PSGL-1. Mapping studies show that the extracellular N-terminal domain of PSGL-1 is necessary for its anti–HIV-1 activity, and that the PSGL-1 cytoplasmic tail contributes to inhibition. In addition, we demonstrate that the PSGL-1–related monomeric E-selectin–binding glycoprotein CD43 also effectively blocks HIV-1 infectivity. HIV-1 infection, or expression of either Vpu or Nef, down-regulates PSGL-1 from the cell surface; expression of Vpu appears to be primarily responsible for enabling the virus to partially escape PSGL-1–mediated restriction. Finally, we show that PSGL-1 inhibits the infectivity of other viruses, such as murine leukemia virus and influenza A virus. These findings demonstrate that PSGL-1 is a broad-spectrum antiviral host factor with a unique mechanism of action.
Yajing Fu; Sijia He; Abdul A. Waheed; Deemah Dabbagh; Zheng Zhou; Benjamin Trinité; Zhao Wang; Jieshi Yu; Dan Wang; Feng Li; David N. Levy; Hong Shang; Eric O. Freed; Yuntao Wu. PSGL-1 restricts HIV-1 infectivity by blocking virus particle attachment to target cells. Proceedings of the National Academy of Sciences 2020, 117, 9537 -9545.
AMA StyleYajing Fu, Sijia He, Abdul A. Waheed, Deemah Dabbagh, Zheng Zhou, Benjamin Trinité, Zhao Wang, Jieshi Yu, Dan Wang, Feng Li, David N. Levy, Hong Shang, Eric O. Freed, Yuntao Wu. PSGL-1 restricts HIV-1 infectivity by blocking virus particle attachment to target cells. Proceedings of the National Academy of Sciences. 2020; 117 (17):9537-9545.
Chicago/Turabian StyleYajing Fu; Sijia He; Abdul A. Waheed; Deemah Dabbagh; Zheng Zhou; Benjamin Trinité; Zhao Wang; Jieshi Yu; Dan Wang; Feng Li; David N. Levy; Hong Shang; Eric O. Freed; Yuntao Wu. 2020. "PSGL-1 restricts HIV-1 infectivity by blocking virus particle attachment to target cells." Proceedings of the National Academy of Sciences 117, no. 17: 9537-9545.
Background The continued persistence of HIV-1 as a public health concern due to the lack of a cure calls for the development of new tools for studying replication of the virus. Here, we used NanoLuc, a small and extremely bright luciferase protein, to develop an HIV-1 bioluminescent reporter virus that simplifies functional measurement of virus particle production. Results The reporter virus encodes a Gag protein containing NanoLuc inserted between the matrix (MA) and capsid (CA) domains of Gag, thereby generating virus particles that package high levels of the NanoLuc reporter. We observe that inserting the NanoLuc protein within HIV-1 Gag has minimal impact on Gag expression and virus particle release. We show that the reporter virus recapitulates inhibition of HIV-1 particle release by Gag mutations, the restriction factor tetherin, and the small-molecule inhibitor amphotericin-B methyl ester. Conclusion These results demonstrate that this vector will provide a simple and rapid tool for functional studies of virus particle assembly and release and high-throughput screening for cellular factors and small-molecules that promote or inhibit HIV-1 particle production.
James Kirui; Eric Freed. Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release. 2020, 1 .
AMA StyleJames Kirui, Eric Freed. Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release. . 2020; ():1.
Chicago/Turabian StyleJames Kirui; Eric Freed. 2020. "Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release." , no. : 1.
The HIV-1 accessory protein Vpu enhances virus release by down-regulating cell surface expression of the host restriction factor tetherin. To further understand the role of host proteins in Vpu function, we carried out yeast two-hybrid screening and identified the V0 subunit C of vacuolar ATPase (ATP6V0C) as a Vpu-binding protein. To examine the role of ATP6V0C in Vpu-mediated tetherin degradation and HIV-1 release, we knocked down ATP6V0C expression in HeLa cells and observed that ATP6V0C depletion impairs Vpu-mediated tetherin degradation, resulting in a defect in HIV-1 release. We also observed that overexpression of ATP6V0C stabilizes tetherin expression. This stabilization is specific to ATP6V0C, as overexpression of another subunit of the vacuolar ATPase, ATP6V0C”, had no effect on tetherin expression. ATP6V0C overexpression did not stabilize CD4, another target of Vpu-mediated degradation. Immunofluorescence localization studies showed that the ATP6V0C-stabilized tetherin is sequestered in a CD63- and LAMP1-positive intracellular compartment. These data demonstrate that the Vpu-interacting protein ATP6V0C plays a role in regulating tetherin expression and HIV-1 assembly and release.
Abdul A. Waheed; Maya Swiderski; Ali Khan; Ariana Gitzen; Ahlam Majadly; Eric O. Freed. The Vpu-interacting protein ATP6V0C regulates expression of tetherin and HIV-1 release. 2020, 1 .
AMA StyleAbdul A. Waheed, Maya Swiderski, Ali Khan, Ariana Gitzen, Ahlam Majadly, Eric O. Freed. The Vpu-interacting protein ATP6V0C regulates expression of tetherin and HIV-1 release. . 2020; ():1.
Chicago/Turabian StyleAbdul A. Waheed; Maya Swiderski; Ali Khan; Ariana Gitzen; Ahlam Majadly; Eric O. Freed. 2020. "The Vpu-interacting protein ATP6V0C regulates expression of tetherin and HIV-1 release." , no. : 1.
T cell SERINC proteins were recently identified as human immunodeficiency virus (HIV) restriction factors that diminish viral infectivity by incorporation into virions. Here we provide evidence that SERINC3 and SERINC5 perform additional antiviral activity by enhancing the type I interferon (IFN-I) and NF-κB signaling pathways. SERINC5 interacts with the mitochondrial antiviral-signaling (MAVS) and TRAF6 proteins, resulting in MAVS aggregation and TRAF6 polyubiquitination. Knockdown of SERINC5 in the target cell increases single-round HIV-1 infectivity, as well as infection by recombinant vesicular stomatitis virus (rVSV) bearing VSV-G or Ebola virus (EBOV) glycoprotein (GP). Infection by an endemic Asian strain of Zika virus (ZIKV) FSS13025 is also enhanced by SERINC5 knockdown, suggesting that SERINC5 has direct antiviral activity. Further experiments indicated that the antiviral activity of SERINC5 is IFN-I dependent. Altogether, our work uncovered a new function of SERINC proteins that promotes IFN-I and NF-B inflammatory signaling, thus contributing to SERINC-mediated antiviral activity.
Cong Zeng; Abdul A. Waheed; Tianliang Li; Jingyou Yu; Yi-Min Zheng; Jacob Yount; Haitao Wen; Eric O. Freed; Shan-Lu Liu. SERINC Proteins Potentiate Antiviral Type I IFN Induction and Proinflammatory Signaling Pathways. Proceedings 2020, 50, 1 .
AMA StyleCong Zeng, Abdul A. Waheed, Tianliang Li, Jingyou Yu, Yi-Min Zheng, Jacob Yount, Haitao Wen, Eric O. Freed, Shan-Lu Liu. SERINC Proteins Potentiate Antiviral Type I IFN Induction and Proinflammatory Signaling Pathways. Proceedings. 2020; 50 (1):1.
Chicago/Turabian StyleCong Zeng; Abdul A. Waheed; Tianliang Li; Jingyou Yu; Yi-Min Zheng; Jacob Yount; Haitao Wen; Eric O. Freed; Shan-Lu Liu. 2020. "SERINC Proteins Potentiate Antiviral Type I IFN Induction and Proinflammatory Signaling Pathways." Proceedings 50, no. 1: 1.
The MARCH family of RING-finger E3 ubiquitin ligases comprise 11 members that have been reported to play a variety of roles in the downregulation of cell-surface proteins involved in adaptive immunity. The RING-CH domain of MARCH proteins is thought to ubiquitinate the cytoplasmic tails (CTs) of target proteins, leading to protein degradation through either lysosomal or proteasomal pathways. Three MARCH proteins (MARCH1, 2, and 8) have recently been reported to target the HIV-1 envelope glycoprotein (Env) and vesicular stomatitis virus G glycoprotein (VSV-G), thereby impairing the infectivity of HIV-1 virions bearing HIV-1 Env or VSV-G. However, the mechanism of antiviral activity remains poorly defined. Our data show that MARCH proteins antagonize the full-length forms of HIV-1 Env, VSV-G, and Ebola glycoprotein (GP), and impair the infectivity of HIV-1 virions bearing these viral glycoproteins. This Env-targeting activity of the MARCH proteins requires the E3 ubiquitin ligase activity of the RING-CH domain. We observe that the MARCH protein targeting of VSV-G is, to a large extent, CT-dependent. In striking contrast, the MARCH-protein targeting of HIV-1 Env and Ebola GP does not require the CT. Confocal microscopy data demonstrate that MARCH proteins are able to trap the viral glycoproteins in an intracellular compartment. We observe that the endogenous expression of MARCH8 in T-cell lines and PBMCs is inducible by type I interferons (a and b) and is also upregulated by HIV-1 infection. Current studies are aimed at identifying the cellular target for MARCH-mediated ubiquitination in the context of their antiviral activity. These results will clarify the mechanism by which MARCH proteins antagonize viral glycoproteins and provide insights into the antiviral role of cellular inhibitory factors in Env biogenesis, trafficking, and virion incorporation.
Cheng Man Lun; Abdul A. Waheed; Eric O. Freed. Does the Cytoplasmic Tail Matter? Mechanism of Viral Envelope Glycoprotein Targeting by Membrane-Associated-RING-CH (MARCH) Proteins. Proceedings 2020, 50, 21 .
AMA StyleCheng Man Lun, Abdul A. Waheed, Eric O. Freed. Does the Cytoplasmic Tail Matter? Mechanism of Viral Envelope Glycoprotein Targeting by Membrane-Associated-RING-CH (MARCH) Proteins. Proceedings. 2020; 50 (1):21.
Chicago/Turabian StyleCheng Man Lun; Abdul A. Waheed; Eric O. Freed. 2020. "Does the Cytoplasmic Tail Matter? Mechanism of Viral Envelope Glycoprotein Targeting by Membrane-Associated-RING-CH (MARCH) Proteins." Proceedings 50, no. 1: 21.
HIV-1 encodes an envelope glycoprotein complex (Env) containing a long cytoplasmic tail (CT) harboring trafficking motifs implicated in Env incorporation into virions. Although the requirement for the Env CT in viral transmission is known, the precise mechanism by which Env is incorporated into nascent virions and localizes to the virological synapse remains poorly defined. To further elucidate the mechanism of Env trafficking, we examined three HIV-1 strains: the lab-adapted clade B strain, NL4-3, and a transmitted/founder (T/F) clade C virus, K3016, and a T/F clade B virus, CH077. The HIV-1 Env CT contains two invariant trafficking motifs: tyrosine endocytosis motif, Y712SPL, and C-terminal dileucine motif, LL855. Virion Env incorporation analysis revealed that Y712SPL is necessary for efficient Env incorporation, while LL855 is dispensable. Spreading infection kinetics were analyzed in various T-cell lines and primary human PBMCs; the results indicated that both endocytic motifs contribute to efficient viral spread in culture. Analysis of Env localization to the T-cell uropod, the portion of the plasma membrane that forms a virological synapse with uninfected cells, was found to be dependent on the Env CT and the Y712SPL motif. Cell-to-cell and cell-free transmission assays using T cells infected with HIV-1 bearing Y712A or LL855AA Env CT mutations are ongoing to establish a role for these motifs in both modes of viral transmission. These studies will significantly enhance our understanding of Env trafficking and viral transmission, providing insights into viral Env–host interactions in physiologically relevant cells.
Melissa Victoria Fernandez; Lwar N Naing; David A Scheiblin; Sherimay D Ablan; Jennifer A Simmons; Eric O Freed. HIV-1 Envelope Glycoprotein Trafficking and Viral Transmission. Proceedings 2020, 50, 17 .
AMA StyleMelissa Victoria Fernandez, Lwar N Naing, David A Scheiblin, Sherimay D Ablan, Jennifer A Simmons, Eric O Freed. HIV-1 Envelope Glycoprotein Trafficking and Viral Transmission. Proceedings. 2020; 50 (1):17.
Chicago/Turabian StyleMelissa Victoria Fernandez; Lwar N Naing; David A Scheiblin; Sherimay D Ablan; Jennifer A Simmons; Eric O Freed. 2020. "HIV-1 Envelope Glycoprotein Trafficking and Viral Transmission." Proceedings 50, no. 1: 17.