Eric O. Freed, Ph.D.

Assembly and Release of HIV-1 and Other Retroviruses

Retroviral Gag proteins are synthesized in the cytoplasm of the infected cell and assemble into virus particles that typically bud from the plasma membrane (PM). Expression of Gag proteins alone is generally sufficient for the assembly and release of noninfectious, virus-like particles (VLPs). The mature Gag proteins [matrix (MA), capsid (CA), and nucleocapsid (NC)] are generated concomitant with virus release upon cleavage of the Gag precursor by the viral protease (PR) [Freed, Nat. Rev. Microbiol. 2015]. PR-mediated Gag processing leads to virus maturation, a morphological transformation essential for virus infectivity. Retroviral gag genes often encode other domains and spacer peptides in addition to MA, CA, and NC. For example, the HIV-1 Gag precursor includes two spacer peptides (SP1 and SP2) and the p6 domain.

Retroviral Gag trafficking and assembly. After Gag synthesis, the MA domain directs Pr55^Gag to the PM. The affinity of the MA domain for membrane is provided in part by a myristic acid moiety covalently attached to the N-terminus of MA. Sequences in MA downstream of the myristate also contribute to membrane binding—in particular, a highly basic patch of amino acid residues.

A large body of data indicates that HIV-1 assembly takes place predominantly at the PM [Freed, Nat. Rev. Microbiol. 2015]. Although the cellular determinants that regulate the site of HIV-1 assembly remain to be fully defined, we demonstrated that lipid rafts serve as sites for assembly at the PM [Ono & Freed, PNAS 2001] and identified the phosphoinositide lipid phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂] as a host factor involved in directing Gag to the PM [Ono et al., PNAS 2004]. We are also working to elucidate the role of inositol-hexakisphosphate (IP₆) in HIV-1 assembly and maturation [Mallery et al., Cell Rep. 2019; Mallery et al., Sci. Adv. 2021; Renner et al., Nat Struct Mol Bio. 2023; Kleinpeter et al., J Mol Bio.2023] and have discovered that the cellular protein PSGL-1, when incorporated into virus particles, blocks the ability of those particles to bind to target cells [Fu et al., PNAS 2020]. We have recently discovered that the host cell enzyme neutral sphingomyelinase 2, which functions in the ceramide biosynthetic pathway, is required for the maturation of HIV-1 and other primate lentiviruses. Inhibitors of nSMase2 disrupt HIV-1 replication both in vivo and in humanized mouse models for HIV-1 infection [Waheed et al., PNAS 2023; Yoo et al., PNAS 2023].

Env incorporation and function. The envelope (Env) glycoproteins of HIV-1 are synthesized as a polyprotein precursor, gp160, that is proteolytically processed by the cellular protease furin to generate the surface (SU) glycoprotein gp120 and the transmembrane (TM) glycoprotein gp41 [Checkley et al., J. Mol. Biol. 2011]. Incorporation of Env glycoproteins into virions is an essential step in the replication process; however, the mechanism by which the Env glycoproteins are incorporated remains incompletely characterized. Several lines of evidence suggest that HIV-1 Env glycoproteins are actively recruited into virions via direct interactions between Env and MA; for example, mutations in both the MA domain of Gag and the cytoplasmic tail of gp41 can block HIV-1 Env incorporation. We showed that in most cell lines and in primary cell types, gp41 truncations severely disrupt Env incorporation but that in some cell lines these gp41 truncation mutants are efficiently incorporated [Murakami & Freed, PNAS 2000]. The cell-type-dependent requirement for the gp41 cytoplasmic tail in Env incorporation hints at the involvement of host cell factors. However, the identity of such factors remains to be defined. We are currently using a range of cell biology, virology, biochemical, and imaging approaches to characterize viral and cellular determinants of Env incorporation. Our findings highlight a requirement for trimerization of the MA domain of Gag in Env incorporation [Tedbury et al., PNAS 2016; Tedbury et al., J. Virol. 2020]. Our ongoing work has demonstrated that the cellular MARCH family of E3 ubiquitin ligases restricts the expression and incorporation of a range of viral glycoproteins including HIV-1 Env, VSV-G, Ebolavirus GP, and the spike protein of SARS-CoV-2 [Lun et al., mBio 2021].

More than two dozen drugs, most of which target the viral enzymes reverse transcriptase (RT), protease (PR), or integrase (IN), are currently available to treat HIV-1-infected individuals. These drugs, when used in combination (combination antiretroviral therapy or cART), effectively suppress virus replication in most treated individuals. However, resistance to cART does arise in some individuals, particularly in the context of poor adherence, suboptimal drug regimens, or lack of viral load monitoring. Resistance is usually associated with mutations in the viral genes targeted by antiretrovirals (ARVs). However, in some cases, resistance arises without mutations in the drug-target gene. For example, many patients fail PR inhibitor (PI)-based therapy without acquiring mutations in PR, and in a number of individuals treated with the highly potent IN strand-transfer inhibitors (INSTIs) dolutegravir (DTG) and raltegravir (RTG), patients fail therapy without the virus acquiring mutations in IN. We performed selections for DTG resistance and identified Env mutations that conferred resistance to DTG. Resistance was linked to the ability of the Env mutations to significantly enhance virus spread through cell-to-cell contact. These results demonstrate that, at least in vitro, mutations in Env can confer resistance to an ARV [Van Duyne et al., PNAS 2019]. Recent work has demonstrated that the Env mutations can confer resistance not only to the INSTI DTG but also to RT and PR inhibitors [Hikichi et al., mBio 2021]. Ultimately, we plan to fully define the mechanism by which the identified mutations in Env confer escape from ARVs and determine whether Env-mediated escape also occurs in vivo.

Retrovirus budding. The p6 domain of HIV-1 Gag is required for efficient virus budding. We mapped the virus release function of p6 to a highly conserved Pro-Thr-Ala-Pro (PTAP) motif near the N-terminus of p6 [Huang et al., J. Virol. 1995; Demirov et al., J. Virol. 2002; Fujii et al., Nat. Rev. Microbiol. 2007]. Other retroviral Gag proteins also possess "late domains" that, like the PTAP motif of p6, promote virus release.

Numerous lines of evidence support the hypothesis that retroviral late domains function by interacting with host factors—specifically, components of the cellular endosomal sorting complex required for transport (ESCRT) machinery [Freed, Nat. Rev. Microbiol. 2015]. The ESCRT machinery is composed of four multiprotein complexes (ESCRT-0, I, II, and III) and a variety of accessory proteins including Alix and the ATPase Vps4. This cellular apparatus functions in the sorting and release of cargo proteins into vesicles that bud into late endosomes and also plays a key role in the abscission step of cytokinesis. Retroviruses have evolved to usurp this cellular budding machinery to promote their release from the PM. Work from several labs, including ours [Demirov et al., PNAS. 2002], demonstrated that HIV-1 recruits the ESCRT machinery primarily through a direct interaction between the PTAP motif in p6 and the ESCRT-I component tumor susceptibility gene 101 (Tsg101). Secondary interactions occur between p6 and Alix.

Virion maturation. PR-mediated cleavage of the Gag and Gag–Pol precursors leads to a dramatic change in virion morphology, a process known as maturation. The highly ordered nature of Gag processing and the strict dependence on complete processing for proper virion maturation make the Gag processing cascade a potential target for drug development. Indeed, the betulinic acid derivative dimethylsuccinyl betulinic acid [PA-457 or bevirimat (BVM)] potently inhibits HIV-1 infectivity by targeting a late Gag processing event: the cleavage of the CA–SP1 processing intermediate to mature CA [Li et al., PNAS 2003]. By specifically disrupting this step in Gag processing, BVM treatment leads to the formation of poorly infectious viral particles with aberrantly condensed cores. We have selected and characterized a number of single amino acid mutations in the CA–SP1 boundary region that confer resistance to BVM, establishing this region of Gag as the target of the inhibitor [Adamson et al., J. Virol. 2006]. Clinical trials conducted with BVM provided mixed results; in some patients significant reductions in viral loads were achieved, whereas in other patients no benefit of BVM therapy was observed. We and others were able to attribute this lack of response to polymorphisms in SP1. We also performed studies on a second HIV-1 maturation inhibitor, PF-46396, developed by Pfizer. While structurally distinct from BVM, PF-46396 also inhibits CA–SP1 processing. Particularly interesting is our observation that a number of PF-46396-resistant mutants are severely defective for assembly and infectivity in the absence of the compound but produce infectious virions in its presence [Waki et al., PLoS Pathog. 2012]. With collaborators at DFH Pharma, we have identified a series of highly potent BVM derivatives that display broad activity against strains of HIV-1 that are resistant to BVM [Urano et al., Antimicrob. Agents Chemother. 2016; Urano et al., J. Virol. 2019]. We have also been working with structural biologists to solve the structure of maturation inhibitors bound to their target in Gag [Sarkar et al., NatComm 2023]. This work advances our efforts to develop virus maturation inhibitors as an antiviral strategy.

Dr. Eric Freed received his Ph.D. in 1990 in the laboratories of Drs. Rex Risser and Howard Temin at the University of Wisconsin-Madison and did postdoctoral work with Dr. Temin at UW-Madison in 1991. His work in Madison focused on the function of the murine leukemia virus and HIV envelope glycoproteins in membrane fusion and virus entry. He joined the Laboratory of Molecular Microbiology at the National Institute of Allergy and Infectious Diseases (LMM/NIAID) in 1992, where he worked with Dr. Malcolm Martin on HIV assembly and entry/post-entry events in the HIV replication cycle. In 1997 Dr. Freed was appointed as a Tenure-Track Investigator in LMM/NIAID, and he was promoted to a tenured Senior Investigator position in 2002. In 2003 he joined the HIV Drug Resistance Program (HIV DRP, renamed the HIV Dynamics and Replication Program in 2015) as Head of the Virus-Cell Interaction Section and was appointed to the NCI Senior Biomedical Research Service in 2011. Dr. Freed was appointed Deputy Director of the HIV DRP in 2014 and Director of the Program in 2015. He was an Organizer of the 2004 Cold Spring Harbor Meeting on Retroviruses, 2006 ASCB Conference "Cell Biology of HIV-1 and Other Retroviruses," 2012 Keystone Symposium "Frontiers in HIV Pathogenesis, Therapy and Eradication," 2014 Keystone Symposium "The Ins and Outs of Viral Infection: Entry, Assembly, Exit and Spread," Viruses 2016 Conference "At the Forefront of Virus–Host Interactions," Viruses 2018 Conference "Breakthroughs in Virus Replication," 2018 gp41 Cytoplasmic Tail Structure and Function Workshop, and Viruses 2020 “Novel Concepts in Virology.” He also served on the Scientific Committee of the International Retroviral Nucleocapsid Protein and Assembly Symposium in 2013, 2016, and 2019 and the Organizing Committee of the Annual Meeting of the American Society for Virology in 2018. He has served as Co-Organizer of the Annual HIV DRP Conference and Annual David Derse Memorial Lecture and Award since 2012 and Organizer of the Annual George Khoury Memorial Lecture since 2014. Dr. Freed is the founding Editor-in-Chief of Viruses and was appointed Editor of Journal of Molecular Biology in 2012 and Editor of Recent Advances in HIV-1 Assembly and Release in 2013. He also currently serves on the Editorial Boards of a number of journals, including Journal of Virology, Retrovirology, and Frontiers in Virology, and is an Associate Editor for Science Advances and Fields Virology. He was a sitting member of the NIH AIDS Discovery and Development of Therapeutics (ADDT) study section (2012-2017) and served as ADDT Chair from 2015 to 2017. He is currently Chair of the CCR Center for Molecular Microscopy Advisory Panel and CCR Cryo-EM Expert Users Committee, Co-Chair of the NIH Virology Interest Group, and Scientific Advisory Board Member of the NIH Center for HIV-1 RNA Studies. He is also Co-Director of the University of Maryland Virology Program and an Adjunct Professor in the Department of Cell Biology and Molecular Genetics at the University of Maryland, College Park. Dr. Freed is a member of the Pittsburgh Center for HIV Protein Interactions (PCHPI) and the Behavior of HIV in Viral Environments (B-HIVE) Center and is a member of the Scientific Advisory Board of the Center for Structural Biology of HIV RNA (CRNA). Dr. Freed received the NCI Mentor of Merit Award in 2010 and the NCI Women Scientists Advisors Mentoring and Leadership Award in 2021 for excellence in mentoring and guiding the careers of scientists in all stages of their careers. He received the Outstanding Science Alumni Award from Penn State University in 2014 and the NCI Research Highlights Award in 2016. In recognition of his outstanding contributions to the field of retrovirology, Dr. Freed was awarded the KT Jeang Retrovirology Prize in 2018 and the Ohio State University Center for Retrovirus Research Distinguished Research Career Award in 2020. He was elected to Fellowship in the American Academy of Microbiology in 2019. He will deliver a plenary lecture at the 2024 Conference on Retroviruses and Opportunistic Infections (CROI) and a keynote address at the 2024 Cold Spring Harbor Retroviruses Meeting.