Alan Rein, Ph.D.
- Center for Cancer Research
- National Cancer Institute
- Building 535, Room 211
- Frederick, MD 21702-1201
- 301-846-1361
- reina@mail.nih.gov
RESEARCH SUMMARY
Dr. Rein is a recognized expert in virus assembly, particularly with respect to human and murine retroviruses. Other research interests include structure-function relationships in viral RNA and cellular defense mechanisms against retroviruses. As Head of the Retrovirus Assembly Section, he studies molecular mechanisms of retroviral replication and pathogenesis, with the hope that this understanding will lead to new methods of combatting retrovirus-induced disease, including AIDS.
Areas of Expertise
Alan Rein, Ph.D.
Research
Mechanisms in Retroviral Replication and Pathogenesis
The goal of the research efforts in the Retroviral Assembly Section is to extend our understanding of basic mechanisms in retroviral replication and pathogenesis. Our hope is that this understanding will lead to new methods of combatting retrovirus-induced disease, including AIDS.
We have analyzed the properties of recombinant HIV-1 Gag protein in some detail. When nucleic acid is added to Gag, the protein assembles into virus-like particles. This is a very nonspecific interaction: almost any nucleic acid will support assembly. These particles are significantly smaller than authentic viral particles, but particles of the correct size are formed if inositol phosphates, as well as nucleic acid, are provided to the protein. In the absence of nucleic acid, the protein is in monomer-dimer equilibrium, using the previously described interface within the C-terminal domain of its capsid moiety. We generated a mutant of Gag that remains monomeric at relatively high concentrations. We have subjected this mutant protein to a series of hydrodynamic and biophysical analyses. These results, together with modeling studies, indicate that the protein is folded over in solution, with its N and C termini relatively close together in three-dimensional space. Since Gag molecules are extended rods in immature virus particles, they must undergo a drastic conformational change during particle assembly. Collaborative studies using neutron reflectivity and other techniques have now shown that Gag extends when preferred ligands are available for both its N-terminal ("matrix") domain and its C-terminal ("nucleocapsid") domain. Interestingly, unlike HIV-1 Gag protein, the Gag protein of the gammaretrovirus Moloney murine leukemia virus is rod-shaped in solution, just as it is in immature virus particles, and does not dimerize significantly in solution.
Gag is also a nucleic acid chaperone, and participates in the annealing of a cellular tRNA molecule to the viral RNA, where the tRNA will serve as primer for viral DNA synthesis.
There appear to be several different modes of interaction between retroviral proteins and nucleic acids, each with important functional consequences for viral replication. First, an exquisitely specific recognition by the Gag polyprotein (the structural protein of the virus particle) selects the viral RNA for packaging during virus assembly. This recognition involves zinc fingers in the protein. We are studying the mechanism by which the Gag protein recognizes and packages the genomic RNA of the virus during assembly in vivo. Our research strongly suggests that the recognition signal involves the three-dimensional structure formed by a dimer of genomic RNA molecules. We are studying the structure of the dimer linkage and its possible role in packaging of viral RNA. We have found that virus particles lacking genomic RNA contain cellular mRNA molecules in place of the viral RNA; in general, these mRNA molecules are packaged nonselectively. We developed methods enabling us to measure the affinity of recombinant Gag protein for different RNAs. Although we could detect preferential binding of the protein to RNA containing the packaging signal, this was only possible under highly unnatural conditions, such as supraphysiological ionic strengths. Nevertheless, this RNA supports particle assembly more efficiently than control RNAs. We are now testing the hypothesis that genomic RNA is selectively packaged because it initiates assembly with particularly high efficiency.
The mechanism by which nucleic acid promotes particle assembly by the Gag protein is not known. We recently reported that a very short region of Gag, termed "SP1," undergoes a conformational change when it is at high concentration. We proposed that this region "senses" the local Gag concentration, and that the increased Gag concentration resulting from cooperative binding to nucleic acid would thus lead to a change in Gag conformation, exposing new interfaces required for particle assembly. This hypothesis would explain the contribution of nucleic acid to particle assembly. Our subsequent experiments have provided extensive support for this overall hypothesis. We showed that juxtaposition of only two SP1 molecules is sufficient to induce the conformational change and additional association of these SP1 peptides. The data suggest that SP1-SP1 association can contribute significantly to virus particle assembly and stability. These experiments also uncovered the molecular mechanism underlying these phenomena.
We are also studying the mechanism by which the restriction factor mouse APOBEC3 blocks infection by murine retroviruses. This restriction, unlike retroviral restriction by human APOBEC3G, does not involve hypermutation of G to A in the viral genome.
We are also performing a collaborative study on the HIV-1 Rev Response Element (RRE), a structure in HIV-1 RNA that is recognized by the virus-coded protein Rev in the export of unspliced viral RNAs from the nucleus. Using small-angle X-ray scattering, our colleague Dr. Yun-Xing Wang showed that the RRE folds into an "A" shape, and that the two primary Rev binding sites are opposite each other on the legs of the A. They are separated by a distance of 55 Å. This distance corresponds to the length of a dimer of Rev, suggesting that this unusual topology can explain the specificity of Rev for the RRE. We tested this hypothesis by assaying the functional capabilities of a number of RRE mutants; all of the results supported the idea that the distance between the two sites is the key to RRE function. We are continuing to dissect the RRE and to explore the implications of these results for antiviral therapy.
In collaboration with Dr. Edward Gabrielson (Johns Hopkins Medical Institution), we have developed sensitive assays for the presence of mouse mammary tumor virus-related sequences in human breast cancers. However, to date we have found no evidence for these sequences in the human samples.
Many gammaretroviruses such as MLV encode an alternative form of the Gag protein called "glycogag." Although multiple functions of this protein have been proposed, its significance in MLV replication is still not clear. We have developed reagents for the independent expression and detection of Gag and glycogag and are analyzing the functional effects of glycogag expression. Our results to date show that glycoGag has a very significant effect on the infectivity of an MLV virus particle under certain very specific conditions. We have found that with some viral envelope proteins, glycogag expression enhances the ability of MLV particles to penetrate into new target cells. However, glycogag impairs the ability of MLV with Ebola glycoprotein to penetrate new cells. Our data imply that all of these glycogag effects represent antagonism towards the cellular protein Serinc5. Another retroviral “accessory protein”, the S2 protein of equine infectious anemia virus, acts very similarly to MLV glycogag, although the two proteins are completely unrelated.
Publications
Efficient support of virus-like particle assembly by the HIV-1 packaging signal
Dissection of specific binding of HIV-1 Gag to the “packaging signal” in viral RNA
An unusual topological structure of the HIV-1 Rev response element
Modulation of HIV-like particle assembly in vitro by inositol phosphates
Point mutants of Moloney murine leukemia virus that fail to package viral RNA: Evidence for specific RNA recognition by a "zinc finger-like" protein sequence
Biography
Alan Rein, Ph.D.
Dr. Alan Rein obtained his Ph.D. with Dr. Harry Rubin at the University of California at Berkeley and did postdoctoral research as an American Cancer Society Fellow under the direction of Dr. Sheldon Penman at the Massachusetts Institute of Technology. Dr. Rein has been associated with the National Cancer Institute (NCI) since 1976 and served as Head of the Retroviral Genetics Section in the ABL-Basic Research Program from 1984 to 1999. In 1999 he joined the HIV Drug Resistance Program (renamed the HIV Dynamics and Replication Program in 2015) as Head of the Retrovirus Assembly Section and was appointed to the NCI Senior Biomedical Research Service. Dr. Rein received the NIH Director's Award in 2012, and in 2014 he was elected a Fellow of the American Academy of Microbiology. His research has dealt with a number of aspects of the biology and molecular biology of murine and human retroviruses, including virus assembly and maturation, viral envelope function, translational suppression, and pathogenesis. In 2011 he received a two-year Breast Cancer Research Program grant from the Avon Foundation for Women to support a collaborative project on "Testing for MMTV and Related Retroviruses in Breast Cancer" with Dr. Edward Gabrielson at Johns Hopkins Medical Institution. In 2012 Dr. Rein was awarded a three-year Program Grant from the Human Frontier Science Program (HFSP) to support his international collaboration with Drs. Bogdan Dragnea, Dmitri Svergun, and Paul Van der Schoot on "Physical Principles in the Self-Assembly of Immature HIV-1 Particles." Dr. Rein was an Organizer of the 1995 Cold Spring Harbor Laboratory Retroviruses Meeting and the 2001 International Retroviral Nucleocapsid Symposium and a Keynote Speaker at the 1998 International Congress of Medical Virology, 2000 West Coast Retrovirus Meeting, 2014 Symposium on Mathematical Virology, and 2016 Cold Spring Harbor Laboratory Retroviruses Meeting Since 2019, he has served on the Organizing Committee of the Annual Norman P. Salzman Memorial Award and Symposium in Virology. He has been an Editorial Board Member of the Journal of Virology since 1990 and served on the Editorial Boards of Virology (1990-2016) and Journal of Biological Chemistry (2008-2012, 2014-2018). In 2021, he was appointed as Guest Editor of the Viruses Special Issue "In Memory of Stephen Oroszlan" and Co-Editor of the Viruses Special Issue "Molecular Genetics of Retrovirus Replication." He served on the Center for Cancer Research (CCR) Tenure Review Panel from 2006 to 2017 and currently serves as a member of the CCR RNA Biology Initiative. He is also a collaborative member of the HIV Interaction and Viral Evolution (HIVE) Center, a consortium of leaders in the field of HIV research who are applying their structural and functional knowledge of drug resistance evolution to the design of more effective anti-HIV treatments.
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Team
News
NIH Intramural AIDS Research Fellowship
Tomas Kroupa (postdoctoral fellow, 2017-2021) received an Intramural AIDS Research Fellowship (IARF) award in 2020 to support his research project on "Kinetics of Specific and Nonspecific Interactions of HIV-1 Gag with RNA." Awarded by the Office of AIDS Research, Office of Intramural Research, and Office of Intramural Research & Training in the National Institutes of Health, IARFs 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.
Journal of Biological Chemistry Editors’ Pick: Nucleic Acid-Induced Dimerization of HIV-1 Gag Protein
A publication resulting from a collaboration between the Rein lab and that of Peter Schuck, National Institute of Biomedical Imaging and Bioengineering (Zhao et al., J. Biol. Chem. 294: 16480, 2019) was selected as an Editors’ Pick in the November 8, 2019 issue of Journal of Biological Chemistry.
Journal of Virology Spotlight Feature: Functional Contributions of Individual Domains in the HIV-1 Rev Response Element
A publication by the Rein lab on “Contributions of individual domains to function of the HIV-1 Rev response element” (O'Carroll et al., J. Virol. 91:e00746-17, 2017) was selected as a Spotlight feature in the November 2017 issue of Journal of Virology.
Alan Rein Presented Keynote Lecture at 2016 Cold Spring Harbor Laboratory Retroviruses Meeting
The keynote lecture by Alan Rein ("Retroviruses: Some Perspectives") at the 2016 Cold Spring Harbor Laboratory (CSHL) Retroviruses Meeting is featured on The Leading Strand, a website spotlighting the keynote presentations at CSHL meetings. Dr. Rein’s lecture is also publicly available through YouTube and Apple's iTunes University.
Travel Awards, Think Tank Meetings
Mauricio Comas-Garcia (postdoctoral fellow, 2013-2018) received $1,000 travel awards for presenting one of the two most meritorious talks by NCI fellows at the 2017 and 2016 HIV DRP Think Tank Meetings.
Ina O'Carroll (research fellow, 2014-2015) received a travel award for one of the best presentations at the Center for Cancer Research Fall HIV/AIDS & Cancer Virology Think Tank Meeting in 2015.
Alan Rein Elected to American Academy of Microbiology
Alan Rein was elected to Fellowship in the American Academy of Microbiology (AAM) in 2014. AAM Fellows are recognized as distinguished scientists who are "elected through a highly selective, annual, peer review process, based on their records of scientific achievement and original contributions that have advanced microbiology....Each elected Fellow has built an exemplary career in basic and applied research, teaching, clinical and public health, industry or government service."
NIH Fellows Award for Research Excellence
Ina O’Carroll (postdoctoral fellow, 2009-2014) received an NIH Fellows Award for Research Excellence in 2013 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 and presentation of the abstracts.
Alan Rein Received NIH Director's Award
Presentations at the 2009 Cold Spring Harbor Retroviruses Meeting in May 2009 suggested that xenotropic murine leukemia virus-related virus (XMRV), a novel gammaretrovirus with a potential link to prostate cancer and chronic fatigue syndrome, might be present in ~3% of the U.S. population, raising both public health issues and concern for contamination of the nation's blood supply. In response, the Intramural Research Program (IRP) of the National Cancer Institute immediately formed a multidisciplinary XMRV Working Group and charged the group with developing, implementing, and making available diagnostic reagents for rapid, accurate, and reliable detection of XMRV nucleic acids, antigens, and infectious virus. The group developed an action plan, and within three months, the SAIC Protein Expression Laboratory reported construction of 40 recombinant clones expressing all XMRV antigens and their subsequent purification for use as immunological reagents in December 2009. Importantly, these reagents were also made available (through the NIH AIDS Reagent Program) to the extramural community to accelerate XMRV research and allow sharing of a common set of reagents. A parallel effort in the HIV Dynamics and Replication Program resulted in establishing an assay to quantify XMRV DNA (from tissue) and RNA (from plasma) in November and December 2009, respectively. Since ultrasensitive XMRV nucleic acid detection methods were not available, this required in-house development and standardization, using the existing manpower and financial resources of the HIV DRP. In response to the need for "authentic" viral antigens for the development and standardization of immunological reagents by the Viral Technology Laboratory, the large-scale virus culture facilities of the SAIC AIDS and Cancer Virus Program were recruited for XMRV production. Finally, researchers of the HIV DRP developed the DERSE indicator cell line for detection of infectious XMRV. In contrast to traditional virological methods, this novel assay reduced the time needed to detect low levels of replicating XMRV in cell culture from months to a matter of weeks.
Subsequent studies have demonstrated that XMRV does not pose a threat to public health. Despite this, events between October 2009 and October 2010 highlighted the ability of dedicated scientists of the IRP to respond very quickly to a potential public health crisis by assembling a multidisciplinary team with a single goal of rapidly preparing, standardizing, and making available reagents for diagnostic virology. In every instance, reagents were prepared with existing manpower and resources, and without a serious interruption in the normal work flow or productivity of each group involved. Their non-XMRV work continued unimpeded. The success of this effort relied on close cooperation between all groups to establish and meet important deadlines. In addition to their individual contributions, the XMRV Working Group made reagents and technologies available to the general scientific community, and performed additional diagnostic analysis of samples supplied by federal, intramural, and extramural laboratories. In February 2012, the external XMRV Working Group (the Blood XMRV Scientific Research Working Group) received a Special Recognition Award from the Department of Health and Human Services, recognizing their exemplary team performance for "evaluating XMRV, a potential threat to the blood supply." In July 2012, members of the IRP XMRV Working Group were similarly recognized for their outstanding work by receiving the NIH Director's Award.
The IRP XMRV Working Group included:
Stuart Le Grice, HIV DRP
Alan Rein, HIV DRP
Vineet KewalRamani, HIV DRP
Mary Kearney, HIV DRP
James Hartley, Protein Expression Laboratory, SAIC-Frederick
Rachel Bagni, Viral Technology Laboratory, SAIC-Frederick
Jeffrey Lifson, AIDS and Cancer Virus Program, SAIC-Frederick
Journal of Virology Spotlight Feature: Inositol Phosphates Modulate the Nucleic Acid Chaperone Activity of Gag
A publication by the Rein lab showing that “Matrix domain modulates HIV-1 Gag's nucleic acid chaperone activity via inositol phosphate binding” (Jones et al., J. Virol. 85:1594-603, 2011) was selected as a Spotlight feature in the February 2011 issue of Journal of Virology.
Alan Rein Featured on “This Week in Virology” Podcast
Alan Rein was featured as the guest investigator on the December 26, 2010 podcast of "This Week in Virology" to discuss retroviruses and the newly reported virus XMRV.