Alan Rein, Ph.D.
Alan  Rein, Ph.D.
Senior Investigator
Head, Retrovirus Assembly Section

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. Dr. Rein is also a collaborative member of the HIV Interaction and Viral Evolution Center, and a Fellow of the American Academy of Microbiology.

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

Contact Info

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

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.

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. While the majority of mRNA species are packaged into the virions nonselectively, there are a few species that are significantly enriched. We are currently analyzing the mechanism of this enrichment; it is possible that this will help us understand how Gag normally recognizes the viral RNA during wild-type particle assembly.

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, these properties of HIV-1 Gag are not shared with the Gag protein from another retrovirus, Moloney murine leukemia virus.

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.

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. Current studies are devoted to testing this hypothesis and exploring its implications.

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 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 angstroms. 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.

Scientific Focus Areas:
Molecular Biology and Biochemistry, Structural Biology, Virology
  1. Fang X, Wang J, O'Carroll IP, Mitchell M, Zuo X, Wang Y, Yu P, Liu Y, Rausch JW, Dyba MA, Kjems J, Schwieters CD, Seifert S, Winans RE, Watts NR, Stahl SJ, Wingfield PT, Byrd RA, Le Grice SF, Rein A, Wang Y.
    Cell. 155: 594-605, 2013. [ Journal Article ]
  2. Gherghe C, Lombo T, Leonard CW, Datta SAK, Bess JW Jr, Gorelick RJ, Rein A, Weeks, KM.
    Proc. Natl. Acad. Sci. U S A. 107: 19248-53, 2010. [ Journal Article ]
  3. Campbell S, Fisher RJ, Towler EM, Fox S, Issaq HJ, Wolfe T, Phillips LR, Rein A.
    Proc. Natl. Acad. Sci. U S A. 98: 5246-51, 2001. [ Journal Article ]
  4. Muriaux D, Mirro J, Harvin D, Rein A.
    Proc. Natl. Acad. Sci. U S A. 98: 5246-51, 2001. [ Journal Article ]
  5. Gorelick RJ, Henderson LE, Hanser JP, Rein A.
    Proc. Natl. Acad. Sci. U S A. 85: 8420-24, 1988. [ Journal Article ]

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 as 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 Edward Gabrielson, M.D., 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 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 Meeting on Retroviruses and the 2001 International Retroviral Nucleocapsid Symposium. He currently serves on the Editorial Boards of Virology, Journal of Virology, and the Journal of Biological Chemistry and is 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.

Name Position
Yadvinder Ahi Ph.D. Postdoctoral Fellow (Visiting)
Mauricio Comas-Garcia Ph.D. Postdoctoral Fellow (Visiting)
Siddhartha A.K. Datta, Ph.D. Staff Scientist
Demetria Harvin Research Biologist
Bridget Heeney Student Trainee
Jane Mirro M.S. Special Volunteer
Sofia Ryan Student Trainee
Yashna Thappeta Postbaccalaureate Fellow
Shu Zhang M.S. Predoctoral Fellow

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.

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. While the majority of mRNA species are packaged into the virions nonselectively, there are a few species that are significantly enriched. We are currently analyzing the mechanism of this enrichment; it is possible that this will help us understand how Gag normally recognizes the viral RNA during wild-type particle assembly.

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, these properties of HIV-1 Gag are not shared with the Gag protein from another retrovirus, Moloney murine leukemia virus.

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.

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. Current studies are devoted to testing this hypothesis and exploring its implications.

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 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 angstroms. 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.

Scientific Focus Areas:
Molecular Biology and Biochemistry, Structural Biology, Virology

Publications

  1. Fang X, Wang J, O'Carroll IP, Mitchell M, Zuo X, Wang Y, Yu P, Liu Y, Rausch JW, Dyba MA, Kjems J, Schwieters CD, Seifert S, Winans RE, Watts NR, Stahl SJ, Wingfield PT, Byrd RA, Le Grice SF, Rein A, Wang Y.
    Cell. 155: 594-605, 2013. [ Journal Article ]
  2. Gherghe C, Lombo T, Leonard CW, Datta SAK, Bess JW Jr, Gorelick RJ, Rein A, Weeks, KM.
    Proc. Natl. Acad. Sci. U S A. 107: 19248-53, 2010. [ Journal Article ]
  3. Campbell S, Fisher RJ, Towler EM, Fox S, Issaq HJ, Wolfe T, Phillips LR, Rein A.
    Proc. Natl. Acad. Sci. U S A. 98: 5246-51, 2001. [ Journal Article ]
  4. Muriaux D, Mirro J, Harvin D, Rein A.
    Proc. Natl. Acad. Sci. U S A. 98: 5246-51, 2001. [ Journal Article ]
  5. Gorelick RJ, Henderson LE, Hanser JP, Rein A.
    Proc. Natl. Acad. Sci. U S A. 85: 8420-24, 1988. [ Journal Article ]

Biography

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 as 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 Edward Gabrielson, M.D., 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 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 Meeting on Retroviruses and the 2001 International Retroviral Nucleocapsid Symposium. He currently serves on the Editorial Boards of Virology, Journal of Virology, and the Journal of Biological Chemistry and is 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.

Team

Name Position
Yadvinder Ahi Ph.D. Postdoctoral Fellow (Visiting)
Mauricio Comas-Garcia Ph.D. Postdoctoral Fellow (Visiting)
Siddhartha A.K. Datta, Ph.D. Staff Scientist
Demetria Harvin Research Biologist
Bridget Heeney Student Trainee
Jane Mirro M.S. Special Volunteer
Sofia Ryan Student Trainee
Yashna Thappeta Postbaccalaureate Fellow
Shu Zhang M.S. Predoctoral Fellow