Eric O. Freed, Ph.D.

Eric O. Freed, Ph.D.
Program Director
Senior Investigator
Head, Virus-Cell Interaction Section

Dr. Freed is recognized as a leader in the field of virus assembly who has made important strides in understanding the mechanisms of retroviral replication at the molecular level, with an emphasis on late stages of the HIV-1 replication cycle. As Director of the HIV DRP, he oversees a program of basic, translational, and clinical research aimed at developing a better understanding of HIV that can be used to generate more-effective treatment strategies. His research focuses on HIV-1 Gag trafficking, Env incorporation, virus assembly, budding, release, and maturation. Dr. Freed has a special interest in the complex relationship between viral proteins and cellular factors and pathways, believing that characterizing fundamental aspects of the retrovirus life cycle will suggest novel targets for the development of antiretroviral therapies.

Link to additional information about Dr. Freed

Areas of Expertise
1) HIV pathogenesis, 2) virus assembly, 3) retroviruses, 4) virology, 5) cell biology, 6) structural biology

Contact Info

Eric O. Freed, Ph.D.
Center for Cancer Research
National Cancer Institute
Building 535, Room 110
Frederick, MD 21702-1201
Ph: 301-846-6223 (office)
301-846-6483 (lab)
efreed@mail.nih.gov

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. 13: 484-496, 2015]. PR-mediated Gag processing leads to virus maturation, a morphological transition 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 Pr55Gag 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. Structural analysis of myristylated MA has indicated that the myristate moiety adopts both an exposed and a sequestered conformation and that the degree of myristate exposure regulates Gag–membrane association.
 
A large body of data indicates that HIV-1 assembly takes place predominantly at the PM [Freed, Nat. Rev. Microbiol. 13: 484-496, 2015]. In monocyte-derived macrophages (MDMs), assembly and budding can occur in an internal compartment that apparently represents deep, specialized invaginations that are connected to the PM via nanoscale channels [Bennett et al., PLoS Pathog. 5: e1000591, 2009]. We reported that single amino acid changes in the highly basic domain of MA cause virus assembly to be redirected to late endosomes or multivesicular bodies (MVBs). A focus of our work has involved characterizing the mechanism of virus spread from infected MDMs to T cells [Gousset et al., PLoS Pathog. 4: e1000015, 2008].
 
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 98: 13925-13930, 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. Depleting PI(4,5)P2 leads to the retargeting of HIV-1 assembly to MVBs and severely disrupts virus particle production [Ono et al., PNAS 101: 14889-14894, 2004]. Structural studies have shown that HIV-1 MA, as well as the MA domain of several other retroviruses, interacts directly with PI(4,5)P2 and that some of the basic residues we identified as being important for Gag targeting to the PM engage in electrostatic interactions with PI(4,5)P2. We hypothesize that host factors in addition to PI(4,5)P2 play a vital role in the trafficking of Gag to the PM. For example, we have demonstrated that the ADP ribosylation factors (Arfs) and Golgi-localized, gamma-ear-containing, Arf-binding (GGA) proteins function in directing Gag to the PM and modulating virus budding [Joshi et al., Mol. Cell 30: 227-238, 2008], as does the cellular SNARE pathway [Joshi et al., J. Biol. Chem. 286: 29861-29871, 2011]. We are currently working to define the role of host cell machinery in Gag trafficking to the PM and will seek to develop inhibitors of HIV-1 Gag trafficking, membrane binding, and assembly. In this project, we will also define viral and cellular determinants involved in directing HIV-1 assembly to the virological synapse, from which cell–cell transfer efficiently takes place.
 
Env incorporation. The envelope (Env) glycoproteins of HIV-1 are synthesized as a polyprotein precursor, gp160, that is proteolytically processed by a cellular protease to generate the surface (SU) glycoprotein gp120 and the transmembrane (TM) glycoprotein gp41. 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 97: 343-348, 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 recent findings highlight a requirement for MA trimers in Env incorporation [Tedbury et al., PNAS 113: E182-E190, 2016]. We are also defining host cell factors that play a role in HIV-1 Env glycoprotein incorporation [Checkley et al., J. Virol. 87: 3561-3570, 2013].
 
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 [Fujii et al., Nat. Rev. Microbiol. 5: 912-916, 2007]. Other retroviral Gag proteins also possess ‘late domains’ that, like the PTAP motif of p6, promote virus release. The HIV-1 accessory protein Vpu also promotes particle release after the completion of budding by counteracting the host cell restriction factor tetherin.
 
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. 13: 484-496, 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, 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. We are seeking to elucidate a number of key issues relating to lentivirus budding and are attempting to develop small-molecule inhibitors of the p6–Tsg101 interaction to disrupt HIV-1 budding. Of particular interest is the identification of cellular partners for the ESCRT-associated protein Alix [Keren-Kaplan et al., EMBO J. 32: 538-551, 2013].
 
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 100: 13555-13560, 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. 80: 10957-10971, 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 have recently initiated 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. Ongoing studies will define the target and mechanism of action of PF-46396. 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. 8(11): e1002997, 2012]. We are also engaged in studies aimed at elucidating the structure of the maturation inhibitor-binding site in assembled Gag. We believe that increased structural information on the HIV-1 maturation inhibitor-binding site will lead to the discovery of new, structurally distinct inhibitors. Finally, 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. 60: 190-197, 2016]. This work advances our efforts to develop virus maturation inhibitors as an antiviral strategy.
 
Scientific Focus Areas:
Cell Biology, Molecular Biology and Biochemistry, Structural Biology, Virology
  1. Tedbury PR, Novikova M, Ablan SD, Freed EO.
    Proc Natl Acad Sci U S A . 113(2): E182-90, 2016. [ Journal Article ]
  2. Waki K, Durell SR, Soheilian F, Nagashima K, Butler SL, Freed EO.
    PLoS Pathog. 8(11): Epub 2012 Nov 8, 2012. [ Journal Article ]
  3. Joshi A, Garg H, Nagashima K, Bonifacino JS, Freed EO.
    Mol Cell. 30(2): 227-38, 2008. [ Journal Article ]
  4. Ono A, Ablan SD, Lockett SJ, Nagashima K, Freed EO.
    Proc Natl Acad Sci U S A. 101(41): 14889-94, 2004. [ Journal Article ]
  5. Demirov DG, Ono A, Orenstein JM, Freed EO.
    Proc Natl Acad Sci U S A. 99(2): 955-60, 2002. [ Journal Article ]

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 a variety of topics relating to virus assembly and entry/post-entry events in the HIV replication cycle.  In 1997 he was appointed as a Tenure-Track Investigator in LMM/NIAID, and he was promoted to a tenured Senior Investigator position in 2002.  In 2003 Dr. Freed joined the HIV Drug Resistance Program (HIV DRP, renamed as the HIV Dynamics and Replication Program in 2015).  He was an Organizer of the 2004 Cold Spring Harbor Meeting on Retroviruses, 2006 ASCB Cell Biology of Retroviruses Conference, 2012 Keystone Conference "Frontiers in HIV Pathogenesis, Therapy and Eradication," 2014 Keystone Conference "Viral Entry, Assembly, Exit and Spread," and Viruses 2016 Conference "At the Forefront of Virus–Host Interactions."  In 2009 he was appointed as the first Editor-in-Chief of Viruses; he also currently serves on the Editorial Boards of a number of journals, including Journal of Virology, Retrovirology, Frontiers in Virology, and Journal of Molecular Biology.  He was selected as an NCI Mentor of Merit in 2010 for excellence in mentoring and guiding the careers of trainees in cancer research, and in 2011 he was appointed to the NCI Senior Biomedical Research Service.  In 2014 Dr. Freed was appointed as the Deputy Director of the HIV DRP and received the 2014 Outstanding Science Alumni Award from Penn State University.  He became the Director of the HIV DRP in 2015.  In 2016 Dr. Freed received the NCI Research Highlights Award and served on the Scientific Committee of the International Retroviral Nucleocapsid and Assembly Symposium.  He is currently serving as Chair of the Collaboratory Advisory Panel of the CCR Center for Molecular Microscopy and Co-Chair of the NIH Virology Interest Group and the Annual Norman P. Salzman Memorial Symposium in Virology.  He is also a member of the NIH AIDS Discovery and Development of Therapeutics (ADDT) study section and currently serves as ADDT Chair.  Dr. Freed is the Organizer of the Viruses 2018 Conference "Breakthroughs in Virus Biology" and is serving on the Organizing Committee of the 2018 Annual Meeting of the American Society for Virology.  He is a 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.

Name Position
Sherimay D. Ablan Research Biologist
Hannah Carter Postbaccalaureate Fellow (CRTA)
Melissa Fernandez Ph.D. Postdoctoral Fellow (CRTA)
Ali Kahn Special Volunteer
James Kirui Ph.D. Postdoctoral Fellow (Visiting)
Cheng Man (Bonnie) Lun Ph.D. Postdoctoral Fellow (CRTA)
Mariia Novikova Ph.D. Postdoctoral Fellow (Visiting)
Phuong Pham Postbaccalaureate Fellow (CRTA)
Rachel Van Duyne Ph.D. Postdoctoral Fellow (CRTA)
Abdul A. Waheed, Ph.D. Staff Scientist
Chin-Tien Wang M.D., Ph.D. Special Volunteer

 

Travel Awards, HIV DRP Think Tank Meeting

Rachel Van Duyne received a $1000 travel award from the HIV DRP for one of the two best presentations by NCI fellows at the 2017 HIV DRP Think Tank Meeting.  Members of the Freed Lab who received travel awards in previous years include Mariia Novikova (2016), Emiko Urano (2015), Kayoko Waki (2011), and Muthukumar Balasubramaniam (2010).

Travel Awards, Fall HIV/AIDS & Cancer Virology Think Tank Meeting

Mariia Novikova and Rachel Van Duyne won $1000 travel awards for their outstanding talks at the 2016 Fall HIV/AIDS & Cancer Virology Think Tank Meeting.  This annual Think Tank meeting on the NIH-Bethesda campus provides a venue for students, postdoctoral fellows, and staff scientists to present emerging work and hypotheses in the field of cancer virology.  The travel awards were provided by the Center of Excellence in HIV/AIDS & Cancer Virology, Center for Cancer Research, NCI.  Members of the Freed Lab who received travel awards in previous years include Philip Tedbury (2014), Angelica Martins (2013), and Emiko Urano (2013).  

Young Investigator Awards, Conference on Retroviruses and Opportunistic Infections

Justin Kaplan, Mariia Novikova, and Rachel Van Duyne received Young Investigator Awards to present their research findings at the 2017 Conference on Retroviruses and Opportunistic Infections (CROI).  Recipients of this highly selective CROI travel scholarship in previous years include Mariia Novikova and Emiko Urano in 2016, Lillian Kuo in 2013, and Catherine Adamson in 2007. 

Intramural AIDS Research Fellowships

The following Postdoctoral Fellows in the Freed Lab received Intramural AIDS Research Fellowship (IARF) awards from the Office of AIDS Research, Office of Intramural Research, and Office of Intramural Research & Training in the National Institutes of Health to support their proposed research projects:

Emiko Urano:  "Development of Potent and Broadly Active HIV-1 Maturation Inhibitors" (2016)
Mariia Novikova:  "Mechanisms of HIV-1 Gag Lattice Formation and Env Incorporation" (2016)
Rachel Van Duyne:  "Characterizing the Host Cell Factors Involved in HIV-1 Gag Trafficking to Sites of Virus Assembly" (2015)
Robert Buckheit:  "The Effect of Host Antiviral Restriction Factors on Viral Budding and Maturation" (2014)
Lillian Kuo:  "Characterizing the Role of HIV-1 p6-Alix Binding in HIV-1 Cell-to-Cell Infectivity" (2011, 2012)
Philip Tedbury:  "HIV-1 Gag in Assembly and Release: Interactions with gp41 and Cellular Host Factors" (2011, 2012)

IARF awards include full stipend support to successful candidates who demonstrate outstanding scientific potential through both an imaginative and thoughtful research plan and a well thought out career development plan.

Poster Awards, Spring Research Festival

Maya Swiderski was awarded "Outstanding Poster - Infectious Pathogens and Epidemiology" for her presentation at the 2016 Spring Research Festival, which is sponsored by the National Cancer Institute at Frederick and the other eight agencies of the National Interagency Confederation for Biological Research. Maya's presentation highlighted the research she conducted as a student trainee in the Freed Lab over the past year. Other members of the Freed Lab who won poster awards at the Spring Research Festival in previous years include Megan Mounts (2014), Scott MacDonald (2013), Darren D'Souza (2012), and Nishani Kuruppu (2011). All five of these award recipients were undergraduate students or Werner H. Kirsten student interns under the mentorship of Abdul Waheed.

NIH Postbac Poster Day Awards

Nishani Kuruppu and Justin Kaplan were honored for presenting two of the best posters on NIH Postbac Poster Day in 2016. Established in 2001, NIH Postbac Poster Day added a judging component in 2011 to highlight the contributions of postbaccalaureate fellows to the NIH Intramural Research Program. Awardees are recognized for having authored posters that score in the top 20% of all posters presented.

Research Highlights Award at 2016 NCI Scientific Retreat

In 2016, Eric Freed received a Research Highlights Award for his presentation on "Development of Potent and Broadly Active HIV-1 Maturation Inhibitors" at the NCI Annual Intramural Scientific Retreat.

Sallie Rosen Kaplan Postdoctoral Fellowship for Women Scientists

Melissa Fernandez was selected for the Sallie Rosen Kaplan (SRK) Postdoctoral Fellowship for Women Scientists at the National Cancer Institute in 2015.  The SRK Fellowship is a highly competitive annual program that provides additional mentoring opportunities, networking, seminars, and workshops to help prepare NCI’s female postdoctoral fellows for the competitive nature of the job market and help them to transition to independent research careers.  The highlight of this selective program is a 30-week course entitled "Career Building for Women in Science," which includes two day-long workshops.  The SRK Fellowship also includes mentoring opportunities with successful women scientists from government, industry, and academia.

In 2014, Rachel Van Duyne and Mariia Novikova were selected for SRK Fellowships (2nd and 4th from left in photo below).

                                          Photo of Rachel Van Duyne and Mariia Novikova with other SRK Fellows in 2015             

                                                                            2015 SRK Fellows 

 

NIH Fellows Awards for Research Excellence

Rachel Van Duyne won a 2015 NIH Fellows Award for Research Excellence (FARE) for travel to attend and present her work at a scientific meeting in the U.S.  This award, which acknowledges outstanding scientific research performed by intramural postdoctoral fellows, is sponsored by the NIH Fellows Committee, Scientific Directors, and Office of Intramural Training and Education and is funded by the Scientific Directors.  FARE awards are based on scientific merit, originality, experimental design, and overall quality/presentation of the abstracts.  Members of the Freed Lab who were FARE awardees in previous years include Lillian Kuo (2012), Kayoko Waki (2012), Angelica Martins (2012), Muthukumar Balasubramaniam (2010), Benjamin Luttge (2010), Catherine Adamson (2009), and Karine Gousset (2007).

2014 Outstanding Science Alumni Award, Penn State University

Eric Freed was selected by the Eberly College of Science to receive the 2014 Outstanding Science Alumni Award at Penn State University.  This award recognizes alumni who have a record of significant professional achievements in their field and are outstanding role models for the current students in the college.

Award for Outstanding Scientific Presentation, 9th International Retroviral Nucleocapsid Protein and Assembly Symposium

    Philip Tedbury received an award for one of the best posters at the 2013 International Retroviral Nucleocapsid Protein and Assembly Symposium in Montreal, Canada.  His presentation on "Identification of a matrix mutation that globally rescues Env incorporation defects: implications for matrix structure and Env recruitment" highlighted the latest findings from his research project supported by an Intramural AIDS Research Fellowship.

    Postdoctoral Fellowship, Japan Society for the Promotion of Science

    Emiko Urano was awarded a Postdoctoral Fellowship from 2013 to 2015 by the Japan Society for the Promotion of Science. The fellowship program sponsored by this society supports meritorious biomedical research projects undertaken in NIH laboratories by Japanese postdoctoral researchers. Fellowships are awarded after a competitive review of research proposals.

    Special Recognition as a Top Peer Reviewer for the Journal of Virology

      In 2011, Eric Freed merited special recognition as one of the 25 top peer reviewers (among 1570 reviewers) for the Journal of Virology.  This recognition was based on his outstanding attention to a high number of manuscripts during the year, providing insightful reviews within an exceptionally short time frame.

      2010 NCI Mentor of Merit Award

      Eric Freed was selected as an NCI Mentor of Merit in 2010 for excellence in mentoring and guiding the careers of trainees in cancer research.  Dr. Freed was nominated by present and past trainees who view his mentorship as outstanding.  NCI Director Harold Varmus presented the Mentor of Merit citation to him at the NCI Director's Awards ceremony in November 2010.
       

      Photo of NCI Director Harold Varmus with Eric Freed and other 2010 Mentor of Merit awardees

      2010 Multi-Investigator Award Sponsored by Center for Cancer Research (CCR) and Office of AIDS Research (OAR)

      In 2010, Eric Freed and Vineet KewalRamani of the HIV DRP, NCI-Frederick, and Sriram Subramaniam of the Laboratory of Cell Biology, NCI-Bethesda, successfully competed for a $150,000 CCR/OAR-sponsored multi-investigator award with their research proposal "Catching HIV in the act: Structural studies of HIV transport from antigen-presenting cells into the T-cell nucleus."  In total, four CCR/OAR-sponsored awards were made to 11 investigators within and between the Frederick and Bethesda campuses of the National Cancer Institute.

      Young Investigator Award, 2008 International Feline Retrovirus Research Symposium

      Benjamin Luttge was awarded the Young Investigator Award for his oral presentation at the 2008 International Feline Retrovirus Research Symposium in Vienna, Austria.

      Travel Award, 2008 NCI-Frederick Spring Research Festival Symposium

        Benjamin Luttge won a travel award for the best oral presentation at the 2008 NCI-Frederick Spring Research Festival Symposium on "Virology: from Genetic Vehicles to Human Pathogens."

        2006 Travel Fellowship, HIV & Cancer Virology Faculty, Center for Cancer Research

        In 2006, Catherine Adamson won one of the three available travel fellowships awarded by the HIV & Cancer Virology Faculty, Center for Cancer Research, National Cancer Institute.