This page has only limited features, please log in for full access.

Dr. Judith Levin
Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, United States

Basic Info

Basic Info is private.

Research Keywords & Expertise

0 Reverse Transcription
0 HIV and related retroviruses
0 Genomic RNA-protein interactions
0 HIV assembly
0 Human APOBEC3 restriction proteins

Fingerprints

Reverse Transcription

Honors and Awards

The user has no records in this section


Career Timeline

The user has no records in this section.


Short Biography

The user biography is not available.
Following
Followers
Co Authors
The list of users this user is following is empty.
Following: 0 users

Feed

Journal article
Published: 29 December 2016 in Retrovirology
Reads 0
Downloads 0

The nucleocapsid (NC) domain of HIV-1 Gag is responsible for specific recognition and packaging of genomic RNA (gRNA) into new viral particles. This occurs through specific interactions between the Gag NC domain and the Psi packaging signal in gRNA. In addition to this critical function, NC proteins are also nucleic acid (NA) chaperone proteins that facilitate NA rearrangements during reverse transcription. Although the interaction with Psi and chaperone activity of HIV-1 NC have been well characterized in vitro, little is known about simian immunodeficiency virus (SIV) NC. Non-human primates are frequently used as a platform to study retroviral infection in vivo; thus, it is important to understand underlying mechanistic differences between HIV-1 and SIV NC. Here, we characterize SIV NC chaperone activity for the first time. Only modest differences are observed in the ability of SIV NC to facilitate reactions that mimic the minus-strand annealing and transfer steps of reverse transcription relative to HIV-1 NC, with the latter displaying slightly higher strand transfer and annealing rates. Quantitative single molecule DNA stretching studies and dynamic light scattering experiments reveal that these differences are due to significantly increased DNA compaction energy and higher aggregation capability of HIV-1 NC relative to the SIV protein. Using salt-titration binding assays, we find that both proteins are strikingly similar in their ability to specifically interact with HIV-1 Psi RNA. In contrast, they do not demonstrate specific binding to an RNA derived from the putative SIV packaging signal. Based on these studies, we conclude that (1) HIV-1 NC is a slightly more efficient NA chaperone protein than SIV NC, (2) mechanistic differences between the NA interactions of highly similar retroviral NC proteins are revealed by quantitative single molecule DNA stretching, and (3) SIV NC demonstrates cross-species recognition of the HIV-1 Psi RNA packaging signal.

ACS Style

Klara Post; Erik D. Olson; M. Nabuan Naufer; Robert J. Gorelick; Ioulia Rouzina; Mark C. Williams; Karin Musier-Forsyth; Judith G. Levin. Mechanistic differences between HIV-1 and SIV nucleocapsid proteins and cross-species HIV-1 genomic RNA recognition. Retrovirology 2016, 13, 89 .

AMA Style

Klara Post, Erik D. Olson, M. Nabuan Naufer, Robert J. Gorelick, Ioulia Rouzina, Mark C. Williams, Karin Musier-Forsyth, Judith G. Levin. Mechanistic differences between HIV-1 and SIV nucleocapsid proteins and cross-species HIV-1 genomic RNA recognition. Retrovirology. 2016; 13 (1):89.

Chicago/Turabian Style

Klara Post; Erik D. Olson; M. Nabuan Naufer; Robert J. Gorelick; Ioulia Rouzina; Mark C. Williams; Karin Musier-Forsyth; Judith G. Levin. 2016. "Mechanistic differences between HIV-1 and SIV nucleocapsid proteins and cross-species HIV-1 genomic RNA recognition." Retrovirology 13, no. 1: 89.

Research article
Published: 19 May 2016 in Biochemistry
Reads 0
Downloads 0

Human APOBEC3B (A3B) is a member of the APOBEC3 (A3) family of cytidine deaminases, which function as DNA mutators and restrict viral pathogens and endogenous retrotransposons. Recently, A3B was identified as a major source of genetic heterogeneity in several human cancers. Here, we determined the solution nuclear magnetic resonance structure of the catalytically active C-terminal domain (CTD) of A3B and performed detailed analyses of its deaminase activity. The core of the structure comprises a central five-stranded β-sheet with six surrounding helices, common to all A3 proteins. The structural fold is most similar to that of A3A and A3G-CTD, with the most prominent difference being found in loop 1. The catalytic activity of A3B-CTD is ∼15-fold lower than that of A3A, although both exhibit a similar pH dependence. Interestingly, A3B-CTD with an A3A loop 1 substitution had significantly increased deaminase activity, while a single-residue change (H29R) in A3A loop 1 reduced A3A activity to the level seen with A3B-CTD. This establishes that loop 1 plays an important role in A3-catalyzed deamination by precisely positioning the deamination-targeted C into the active site. Overall, our data provide important insights into the determinants of the activities of individual A3 proteins and facilitate understanding of their biological function.

ACS Style

In-Ja L. Byeon; Chang-Hyeock Byeon; Tiyun Wu; Mithun Mitra; Dustin Singer; Judith G. Levin; Angela M. Gronenborn. Nuclear Magnetic Resonance Structure of the APOBEC3B Catalytic Domain: Structural Basis for Substrate Binding and DNA Deaminase Activity. Biochemistry 2016, 55, 2944 -2959.

AMA Style

In-Ja L. Byeon, Chang-Hyeock Byeon, Tiyun Wu, Mithun Mitra, Dustin Singer, Judith G. Levin, Angela M. Gronenborn. Nuclear Magnetic Resonance Structure of the APOBEC3B Catalytic Domain: Structural Basis for Substrate Binding and DNA Deaminase Activity. Biochemistry. 2016; 55 (21):2944-2959.

Chicago/Turabian Style

In-Ja L. Byeon; Chang-Hyeock Byeon; Tiyun Wu; Mithun Mitra; Dustin Singer; Judith G. Levin; Angela M. Gronenborn. 2016. "Nuclear Magnetic Resonance Structure of the APOBEC3B Catalytic Domain: Structural Basis for Substrate Binding and DNA Deaminase Activity." Biochemistry 55, no. 21: 2944-2959.

Journal article
Published: 22 January 2015 in Retrovirology
Reads 0
Downloads 0

Human APOBEC3H (A3H) belongs to the A3 family of host restriction factors, which are cytidine deaminases that catalyze conversion of deoxycytidine to deoxyuridine in single-stranded DNA. A3 proteins contain either one (A3A, A3C, A3H) or two (A3B, A3D, A3F, A3G) Zn-binding domains. A3H has seven haplotypes (I-VII) that exhibit diverse biological phenotypes and geographical distribution in the human population. Its single Zn-coordinating deaminase domain belongs to a phylogenetic cluster (Z3) that is different from the Z1- and Z2-type domains in other human A3 proteins. A3H HapII, unlike A3A or A3C, has potent activity against HIV-1. Here, we sought to identify the determinants of A3H HapII deaminase and antiviral activities, using site-directed sequence- and structure-guided mutagenesis together with cell-based, biochemical, and HIV-1 infectivity assays. We have constructed a homology model of A3H HapII, which is similar to the known structures of other A3 proteins. The model revealed a large cluster of basic residues (not present in A3A or A3C) that are likely to be involved in nucleic acid binding. Indeed, RNase A pretreatment of 293T cell lysates expressing A3H was shown to be required for detection of deaminase activity, indicating that interaction with cellular RNAs inhibits A3H catalytic function. Similar observations have been made with A3G. Analysis of A3H deaminase substrate specificity demonstrated that a 5′ T adjacent to the catalytic C is preferred. Changing the putative nucleic acid binding residues identified by the model resulted in reduction or abrogation of enzymatic activity, while substituting Z3-specific residues in A3H to the corresponding residues in other A3 proteins did not affect enzyme function. As shown for A3G and A3F, some A3H mutants were defective in catalysis, but retained antiviral activity against HIV-1vif (−) virions. Furthermore, endogenous reverse transcription assays demonstrated that the E56A catalytic mutant inhibits HIV-1 DNA synthesis, although not as efficiently as wild type. The molecular and biological activities of A3H are more similar to those of the double-domain A3 proteins than to those of A3A or A3C. Importantly, A3H appears to use both deaminase-dependent and -independent mechanisms to target reverse transcription and restrict HIV-1 replication.

ACS Style

Mithun Mitra; Dustin Singer; Yu Mano; Jozef Hritz; Gabriel Nam; Robert J Gorelick; In-Ja L Byeon; Angela M Gronenborn; Yasumasa Iwatani; Judith G Levin. Sequence and structural determinants of human APOBEC3H deaminase and anti-HIV-1 activities. Retrovirology 2015, 12, 1 -15.

AMA Style

Mithun Mitra, Dustin Singer, Yu Mano, Jozef Hritz, Gabriel Nam, Robert J Gorelick, In-Ja L Byeon, Angela M Gronenborn, Yasumasa Iwatani, Judith G Levin. Sequence and structural determinants of human APOBEC3H deaminase and anti-HIV-1 activities. Retrovirology. 2015; 12 (1):1-15.

Chicago/Turabian Style

Mithun Mitra; Dustin Singer; Yu Mano; Jozef Hritz; Gabriel Nam; Robert J Gorelick; In-Ja L Byeon; Angela M Gronenborn; Yasumasa Iwatani; Judith G Levin. 2015. "Sequence and structural determinants of human APOBEC3H deaminase and anti-HIV-1 activities." Retrovirology 12, no. 1: 1-15.

Journal article
Published: 20 June 2014 in Virus Research
Reads 0
Downloads 0

The mature HIV-1 nucleocapsid protein (NCp7) is generated by sequential proteolytic cleavage of precursor proteins containing additional C-terminal peptides: NCp15 (NCp7-spacer peptide 2 (SP2)-p6); and NCp9 (NCp7-SP2). Here, we compare the nucleic acid chaperone activities of the three proteins, using reconstituted systems that model the annealing and elongation steps in tRNALys3-primed (−) strong-stop DNA synthesis and subsequent minus-strand transfer. The maximum levels of annealing are similar for all of the proteins, but there are important differences in their ability to facilitate reverse transcriptase (RT)-catalyzed DNA extension. Thus, at low concentrations, NCp9 has the greatest activity, but with increasing concentrations, DNA synthesis is significantly reduced. This finding reflects NCp9's strong nucleic acid binding affinity (associated with the highly basic SP2 domain) as well as its slow dissociation kinetics, which together limit the ability of RT to traverse the nucleic acid template. NCp15 has the poorest activity of the three proteins due to its acidic p6 domain. Indeed, mutants with alanine substitutions for the acidic residues in p6 have improved chaperone function. Collectively, these data can be correlated with the known biological properties of NCp9 and NCp15 mutant virions and help to explain why mature NC has evolved as the critical cofactor for efficient virus replication and long-term viral fitness.

ACS Style

Tiyun Wu; Robert J. Gorelick; Judith G. Levin. Selection of fully processed HIV-1 nucleocapsid protein is required for optimal nucleic acid chaperone activity in reverse transcription. Virus Research 2014, 193, 52 -64.

AMA Style

Tiyun Wu, Robert J. Gorelick, Judith G. Levin. Selection of fully processed HIV-1 nucleocapsid protein is required for optimal nucleic acid chaperone activity in reverse transcription. Virus Research. 2014; 193 ():52-64.

Chicago/Turabian Style

Tiyun Wu; Robert J. Gorelick; Judith G. Levin. 2014. "Selection of fully processed HIV-1 nucleocapsid protein is required for optimal nucleic acid chaperone activity in reverse transcription." Virus Research 193, no. : 52-64.

Journal article
Published: 24 October 2013 in Nucleic Acids Research
Reads 0
Downloads 0

Human APOBEC3A (A3A) is a single-domain cytidine deaminase that converts deoxycytidine residues to deoxyuridine in single-stranded DNA (ssDNA). It inhibits a wide range of viruses and endogenous retroelements such as LINE-1, but it can also edit genomic DNA, which may play a role in carcinogenesis. Here, we extend our recent findings on the NMR structure of A3A and report structural, biochemical and cell-based mutagenesis studies to further characterize A3A’s deaminase and nucleic acid binding activities. We find that A3A binds ssRNA, but the RNA and DNA binding interfaces differ and no deamination of ssRNA is detected. Surprisingly, with only one exception (G105A), alanine substitution mutants with changes in residues affected by specific ssDNA binding retain deaminase activity. Furthermore, A3A binds and deaminates ssDNA in a length-dependent manner. Using catalytically active and inactive A3A mutants, we show that the determinants of A3A deaminase activity and anti-LINE-1 activity are not the same. Finally, we demonstrate A3A’s potential to mutate genomic DNA during transient strand separation and show that this process could be counteracted by ssDNA binding proteins. Taken together, our studies provide new insights into the molecular properties of A3A and its role in multiple cellular and antiviral functions.

ACS Style

Mithun Mitra; Kamil Hercík; In-Ja L. Byeon; Jinwoo Ahn; Shawn Hill; Kathyrn Hinchee-Rodriguez; Dustin Singer; Chang-Hyeock Byeon; Lisa M. Charlton; Gabriel Nam; Gisela Heidecker; Angela M. Gronenborn; Judith G. Levin. Structural determinants of human APOBEC3A enzymatic and nucleic acid binding properties. Nucleic Acids Research 2013, 42, 1095 -1110.

AMA Style

Mithun Mitra, Kamil Hercík, In-Ja L. Byeon, Jinwoo Ahn, Shawn Hill, Kathyrn Hinchee-Rodriguez, Dustin Singer, Chang-Hyeock Byeon, Lisa M. Charlton, Gabriel Nam, Gisela Heidecker, Angela M. Gronenborn, Judith G. Levin. Structural determinants of human APOBEC3A enzymatic and nucleic acid binding properties. Nucleic Acids Research. 2013; 42 (2):1095-1110.

Chicago/Turabian Style

Mithun Mitra; Kamil Hercík; In-Ja L. Byeon; Jinwoo Ahn; Shawn Hill; Kathyrn Hinchee-Rodriguez; Dustin Singer; Chang-Hyeock Byeon; Lisa M. Charlton; Gabriel Nam; Gisela Heidecker; Angela M. Gronenborn; Judith G. Levin. 2013. "Structural determinants of human APOBEC3A enzymatic and nucleic acid binding properties." Nucleic Acids Research 42, no. 2: 1095-1110.

Journal article
Published: 28 February 2013 in Virus Research
Reads 0
Downloads 0

During (−) strong-stop DNA [(−) SSDNA] synthesis, RNase H cleavage of genomic viral RNA generates small 5′-terminal RNA fragments (14–18 nt) that remain annealed to the DNA. Unless these fragments are removed, the minus-strand transfer reaction, required for (−) SSDNA elongation, cannot occur. Here, we describe the mechanism of 5′-terminal RNA removal and the roles of HIV-1 nucleocapsid protein (NC) and RNase H cleavage in this process. Using an NC-dependent system that models minus-strand transfer, we show that the presence of short terminal fragments pre-annealed to (−) SSDNA has no impact on strand transfer, implying efficient fragment removal. Moreover, in reactions with an RNase H− reverse transcriptase mutant, NC alone is able to facilitate fragment removal, albeit less efficiently than in the presence of both RNase H activity and NC. Results obtained from novel electrophoretic gel mobility shift and Förster Resonance Energy Transfer assays, which each directly measure RNA fragment release from a duplex in the absence of DNA synthesis, demonstrate for the first time that the architectural integrity of NC's zinc finger (ZF) domains is absolutely required for this reaction. This suggests that NC's helix destabilizing activity (associated with the ZFs) facilitates strand exchange through the displacement of these short terminal RNAs by the longer 3′ acceptor RNA, which forms a more stable duplex with (−) SSDNA. Taken together with previously published results, we conclude that NC-mediated fragment removal is linked mechanistically with selection of the correct primer for plus-strand DNA synthesis and tRNA removal step prior to plus-strand transfer. Thus, HIV-1 has evolved a single mechanism for these RNA removal reactions that are critical for successful reverse transcription.

ACS Style

Christopher B. Hergott; Mithun Mitra; Jianhui Guo; Tiyun Wu; Jennifer T. Miller; Yasumasa Iwatani; Robert J. Gorelick; Judith G. Levin. Zinc finger function of HIV-1 nucleocapsid protein is required for removal of 5′-terminal genomic RNA fragments: A paradigm for RNA removal reactions in HIV-1 reverse transcription. Virus Research 2013, 171, 346 -355.

AMA Style

Christopher B. Hergott, Mithun Mitra, Jianhui Guo, Tiyun Wu, Jennifer T. Miller, Yasumasa Iwatani, Robert J. Gorelick, Judith G. Levin. Zinc finger function of HIV-1 nucleocapsid protein is required for removal of 5′-terminal genomic RNA fragments: A paradigm for RNA removal reactions in HIV-1 reverse transcription. Virus Research. 2013; 171 (2):346-355.

Chicago/Turabian Style

Christopher B. Hergott; Mithun Mitra; Jianhui Guo; Tiyun Wu; Jennifer T. Miller; Yasumasa Iwatani; Robert J. Gorelick; Judith G. Levin. 2013. "Zinc finger function of HIV-1 nucleocapsid protein is required for removal of 5′-terminal genomic RNA fragments: A paradigm for RNA removal reactions in HIV-1 reverse transcription." Virus Research 171, no. 2: 346-355.

Biography
Published: 01 February 2013 in Virus Research
Reads 0
Downloads 0
ACS Style

Judith G. Levin. Obituary. Virus Research 2013, 171, 356 -150.

AMA Style

Judith G. Levin. Obituary. Virus Research. 2013; 171 (2):356-150.

Chicago/Turabian Style

Judith G. Levin. 2013. "Obituary." Virus Research 171, no. 2: 356-150.

Journal article
Published: 01 January 2012 in Biophysical Journal
Reads 0
Downloads 0
ACS Style

Kathy R. Chaurasiya; Hylkje Geertsema; Minic F. Qualley; Tiyun Wu; Yasumasa Iwatani; Denise Chan; Amber Hertz; Judith G. Levin; Karin Musier-Forsyth; Ioulia Rouzina; Mark C. Williams. Oligomerization of HIV-1 Restriction Factor APOBEC3G Transforms it from a Fast Enzyme to a Slow Nucleic Acid Binding Protein. Biophysical Journal 2012, 102, 601a .

AMA Style

Kathy R. Chaurasiya, Hylkje Geertsema, Minic F. Qualley, Tiyun Wu, Yasumasa Iwatani, Denise Chan, Amber Hertz, Judith G. Levin, Karin Musier-Forsyth, Ioulia Rouzina, Mark C. Williams. Oligomerization of HIV-1 Restriction Factor APOBEC3G Transforms it from a Fast Enzyme to a Slow Nucleic Acid Binding Protein. Biophysical Journal. 2012; 102 (3):601a.

Chicago/Turabian Style

Kathy R. Chaurasiya; Hylkje Geertsema; Minic F. Qualley; Tiyun Wu; Yasumasa Iwatani; Denise Chan; Amber Hertz; Judith G. Levin; Karin Musier-Forsyth; Ioulia Rouzina; Mark C. Williams. 2012. "Oligomerization of HIV-1 Restriction Factor APOBEC3G Transforms it from a Fast Enzyme to a Slow Nucleic Acid Binding Protein." Biophysical Journal 102, no. 3: 601a.

Journal article
Published: 20 December 2011 in Virology
Reads 0
Downloads 0

The HIV-1 capsid protein consists of two independently folded domains connected by a flexible peptide linker (residues 146–150), the function of which remains to be defined. To investigate the role of this region in virus replication, we made alanine or leucine substitutions in each linker residue and two flanking residues. Three classes of mutants were identified: (i) S146A and T148A behave like wild type (WT); (ii) Y145A, I150A, and L151A are noninfectious, assemble unstable cores with aberrant morphology, and synthesize almost no viral DNA; and (iii) P147L and S149A display a poorly infectious, attenuated phenotype. Infectivity of P147L and S149A is rescued specifically by pseudotyping with vesicular stomatitis virus envelope glycoprotein. Moreover, despite having unstable cores, these mutants assemble WT-like structures and synthesize viral DNA, although less efficiently than WT. Collectively, these findings demonstrate that the linker region is essential for proper assembly and stability of cores and efficient replication.

ACS Style

Jiyang Jiang; Sherimay D. Ablan; Suchitra Derebail; Kamil Hercík; Ferri Soheilian; James A. Thomas; Shixing Tang; Indira Hewlett; Kunio Nagashima; Robert J. Gorelick; Eric O. Freed; Judith G. Levin. The interdomain linker region of HIV-1 capsid protein is a critical determinant of proper core assembly and stability. Virology 2011, 421, 253 -265.

AMA Style

Jiyang Jiang, Sherimay D. Ablan, Suchitra Derebail, Kamil Hercík, Ferri Soheilian, James A. Thomas, Shixing Tang, Indira Hewlett, Kunio Nagashima, Robert J. Gorelick, Eric O. Freed, Judith G. Levin. The interdomain linker region of HIV-1 capsid protein is a critical determinant of proper core assembly and stability. Virology. 2011; 421 (2):253-265.

Chicago/Turabian Style

Jiyang Jiang; Sherimay D. Ablan; Suchitra Derebail; Kamil Hercík; Ferri Soheilian; James A. Thomas; Shixing Tang; Indira Hewlett; Kunio Nagashima; Robert J. Gorelick; Eric O. Freed; Judith G. Levin. 2011. "The interdomain linker region of HIV-1 capsid protein is a critical determinant of proper core assembly and stability." Virology 421, no. 2: 253-265.

Conference abstract
Published: 02 February 2011 in Biophysical Journal
Reads 0
Downloads 0
ACS Style

Ioulia Rouzina; Minic F. Qualley; Tiyun Wu; Yasumasa Iwatani; Denise S.B. Chan; Amber Hertz; Kathy Chaurasiya; Judith G. Levin; Mark C. Williams; Karin Musier-Forsyth. Complex Kinetics of Apobec3g Interaction with Single-Stranded Nucleic Acids. Biophysical Journal 2011, 100, 192a .

AMA Style

Ioulia Rouzina, Minic F. Qualley, Tiyun Wu, Yasumasa Iwatani, Denise S.B. Chan, Amber Hertz, Kathy Chaurasiya, Judith G. Levin, Mark C. Williams, Karin Musier-Forsyth. Complex Kinetics of Apobec3g Interaction with Single-Stranded Nucleic Acids. Biophysical Journal. 2011; 100 (3):192a.

Chicago/Turabian Style

Ioulia Rouzina; Minic F. Qualley; Tiyun Wu; Yasumasa Iwatani; Denise S.B. Chan; Amber Hertz; Kathy Chaurasiya; Judith G. Levin; Mark C. Williams; Karin Musier-Forsyth. 2011. "Complex Kinetics of Apobec3g Interaction with Single-Stranded Nucleic Acids." Biophysical Journal 100, no. 3: 192a.

Review
Published: 01 November 2010 in RNA Biology
Reads 0
Downloads 0

The HIV -1 nucleocapsid protein (NC) is a nucleic acid chaperone, which remodels nucleic acid structures so that the most thermodynamically stable conformations are formed. This activity is essential for virus replication and has a critical role in mediating highly specific and efficient reverse transcription. NC's function in this process depends upon three properties: (1) ability to aggregate nucleic acids; (2) moderate duplex destabilization activity; and (3) rapid on-off binding kinetics. Here, we present a detailed molecular analysis of the individual events that occur during viral DNA synthesis and show how NC's properties are important for almost every step in the pathway. Finally, we also review biological aspects of reverse transcription during infection and the interplay between NC, reverse transcriptase and human APOBEC3G, an HIV-1 restriction factor that inhibits reverse transcription and virus replication in the absence of the HIV-1 Vif protein.

ACS Style

Judith G Levin; Mithun Mitra; Anjali Mascarenhas; Karin Musier-Forsyth. Role of HIV-1 nucleocapsid protein in HIV-1 reverse transcription. RNA Biology 2010, 7, 754 -774.

AMA Style

Judith G Levin, Mithun Mitra, Anjali Mascarenhas, Karin Musier-Forsyth. Role of HIV-1 nucleocapsid protein in HIV-1 reverse transcription. RNA Biology. 2010; 7 (6):754-774.

Chicago/Turabian Style

Judith G Levin; Mithun Mitra; Anjali Mascarenhas; Karin Musier-Forsyth. 2010. "Role of HIV-1 nucleocapsid protein in HIV-1 reverse transcription." RNA Biology 7, no. 6: 754-774.

Journal article
Published: 30 September 2010 in Virology
Reads 0
Downloads 0

The HIV-1 Gag polyprotein precursor has multiple domains including nucleocapsid (NC). Although mature NC and NC embedded in Gag are nucleic acid chaperones (proteins that remodel nucleic acid structure), few studies include detailed analysis of the chaperone activity of partially processed Gag proteins and comparison with NC and Gag. Here we address this issue by using a reconstituted minus-strand transfer system. NC and NC-containing Gag proteins exhibited annealing and duplex destabilizing activities required for strand transfer. Surprisingly, unlike NC, with increasing concentrations, Gag proteins drastically inhibited the DNA elongation step. This result is consistent with “nucleic acid-driven multimerization” of Gag and the reported slow dissociation of Gag from bound nucleic acid, which prevent reverse transcriptase from traversing the template (“roadblock” mechanism). Our findings illustrate one reason why NC (and not Gag) has evolved as a critical cofactor in reverse transcription, a paradigm that might also extend to other retrovirus systems.

ACS Style

Tiyun Wu; Siddhartha A.K. Datta; Mithun Mitra; Robert J. Gorelick; Alan Rein; Judith G. Levin. Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: Biological implications. Virology 2010, 405, 556 -567.

AMA Style

Tiyun Wu, Siddhartha A.K. Datta, Mithun Mitra, Robert J. Gorelick, Alan Rein, Judith G. Levin. Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: Biological implications. Virology. 2010; 405 (2):556-567.

Chicago/Turabian Style

Tiyun Wu; Siddhartha A.K. Datta; Mithun Mitra; Robert J. Gorelick; Alan Rein; Judith G. Levin. 2010. "Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: Biological implications." Virology 405, no. 2: 556-567.

Journal article
Published: 21 January 2009 in Nucleic Acids Research
Reads 0
Downloads 0

During minus-strand DNA synthesis, RNase H degrades viral RNA sequences, generating potential plus-strand DNA primers. However, selection of the 3′ polypurine tract (PPT) as the exclusive primer is required for formation of viral DNA with the correct 5′-end and for subsequent integration. Here we show a new function for the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NC) in reverse transcription: blocking mispriming by non-PPT RNAs. Three representative 20-nt RNAs from the PPT region were tested for primer extension. Each primer had activity in the absence of NC, but less than the PPT. NC reduced priming by these RNAs to essentially base-line level, whereas PPT priming was unaffected. RNase H cleavage and zinc coordination by NC were required for maximal inhibition of mispriming. Biophysical properties, including thermal stability, helical structure and reverse transcriptase (RT) binding affinity, showed significant differences between PPT and non-PPT duplexes and the trends were generally correlated with the biochemical data. Binding studies in reactions with both NC and RT ruled out a competition binding model to explain NC's observed effects on mispriming efficiency. Taken together, these results demonstrate that NC chaperone activity has a major role in ensuring the fidelity of plus-strand priming.

ACS Style

Klara Post; Besik Kankia; Swathi Gopalakrishnan; Victoria Yang; Elizabeth Cramer; Pilar Saladores; Robert J. Gorelick; Jianhui Guo; Karin Musier-Forsyth; Judith G. Levin. Fidelity of plus-strand priming requires the nucleic acid chaperone activity of HIV-1 nucleocapsid protein. Nucleic Acids Research 2009, 37, 1755 -1766.

AMA Style

Klara Post, Besik Kankia, Swathi Gopalakrishnan, Victoria Yang, Elizabeth Cramer, Pilar Saladores, Robert J. Gorelick, Jianhui Guo, Karin Musier-Forsyth, Judith G. Levin. Fidelity of plus-strand priming requires the nucleic acid chaperone activity of HIV-1 nucleocapsid protein. Nucleic Acids Research. 2009; 37 (6):1755-1766.

Chicago/Turabian Style

Klara Post; Besik Kankia; Swathi Gopalakrishnan; Victoria Yang; Elizabeth Cramer; Pilar Saladores; Robert J. Gorelick; Jianhui Guo; Karin Musier-Forsyth; Judith G. Levin. 2009. "Fidelity of plus-strand priming requires the nucleic acid chaperone activity of HIV-1 nucleocapsid protein." Nucleic Acids Research 37, no. 6: 1755-1766.

Journal article
Published: 16 October 2007 in Nucleic Acids Research
Reads 0
Downloads 0

APOBEC3G (A3G), a host protein that inhibits HIV-1 reverse transcription and replication in the absence of Vif, displays cytidine deaminase and single-stranded (ss) nucleic acid binding activities. HIV-1 nucleocapsid protein (NC) also binds nucleic acids and has a unique property, nucleic acid chaperone activity, which is crucial for efficient reverse transcription. Here we report the interplay between A3G, NC and reverse transcriptase (RT) and the effect of highly purified A3G on individual reactions that occur during reverse transcription. We find that A3G did not affect the kinetics of NC-mediated annealing reactions, nor did it inhibit RNase H cleavage. In sharp contrast, A3G significantly inhibited all RT-catalyzed DNA elongation reactions with or without NC. In the case of ( − ) strong-stop DNA synthesis, the inhibition was independent of A3G's catalytic activity. Fluorescence anisotropy and single molecule DNA stretching analyses indicated that NC has a higher nucleic acid binding affinity than A3G, but more importantly, displays faster association/disassociation kinetics. RT binds to ssDNA with a much lower affinity than either NC or A3G. These data support a novel mechanism for deaminase-independent inhibition of reverse transcription that is determined by critical differences in the nucleic acid binding properties of A3G, NC and RT.

ACS Style

Yasumasa Iwatani; Denise S.B. Chan; F. Wang; Kristen Stewart-Maynard; Wataru Sugiura; Angela M Gronenborn; Ioulia Rouzina; Mark C. Williams; Karin Musier-Forsyth; Judith G. Levin. Deaminase-independent inhibition of HIV-1 reverse transcription by APOBEC3G. Nucleic Acids Research 2007, 35, 7096 -7108.

AMA Style

Yasumasa Iwatani, Denise S.B. Chan, F. Wang, Kristen Stewart-Maynard, Wataru Sugiura, Angela M Gronenborn, Ioulia Rouzina, Mark C. Williams, Karin Musier-Forsyth, Judith G. Levin. Deaminase-independent inhibition of HIV-1 reverse transcription by APOBEC3G. Nucleic Acids Research. 2007; 35 (21):7096-7108.

Chicago/Turabian Style

Yasumasa Iwatani; Denise S.B. Chan; F. Wang; Kristen Stewart-Maynard; Wataru Sugiura; Angela M Gronenborn; Ioulia Rouzina; Mark C. Williams; Karin Musier-Forsyth; Judith G. Levin. 2007. "Deaminase-independent inhibition of HIV-1 reverse transcription by APOBEC3G." Nucleic Acids Research 35, no. 21: 7096-7108.

Journal article
Published: 06 June 2007 in Nucleic Acids Research
Reads 0
Downloads 0

HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone, which is required for highly specific and efficient reverse transcription. Here, we demonstrate that local structure of acceptor RNA at a potential nucleation site, rather than overall thermodynamic stability, is a critical determinant for the minus-strand transfer step (annealing of acceptor RNA to (−) strong-stop DNA followed by reverse transcriptase (RT)-catalyzed DNA extension). In our system, destabilization of a stem-loop structure at the 5′ end of the transactivation response element (TAR) in a 70-nt RNA acceptor (RNA 70) appears to be the major nucleation pathway. Using a mutational approach, we show that when the acceptor has a weak local structure, NC has little or no effect. In this case, the efficiencies of both annealing and strand transfer reactions are similar. However, when NC is required to destabilize local structure in acceptor RNA, the efficiency of annealing is significantly higher than that of strand transfer. Consistent with this result, we find that Mg2+ (required for RT activity) inhibits NC-catalyzed annealing. This suggests that Mg2+ competes with NC for binding to the nucleic acid substrates. Collectively, our findings provide new insights into the mechanism of NC-dependent and -independent minus-strand transfer.

ACS Style

Tiyun Wu; Susan L. Heilman-Miller; Judith G. Levin. Effects of nucleic acid local structure and magnesium ions on minus-strand transfer mediated by the nucleic acid chaperone activity of HIV-1 nucleocapsid protein. Nucleic Acids Research 2007, 35, 3974 -3987.

AMA Style

Tiyun Wu, Susan L. Heilman-Miller, Judith G. Levin. Effects of nucleic acid local structure and magnesium ions on minus-strand transfer mediated by the nucleic acid chaperone activity of HIV-1 nucleocapsid protein. Nucleic Acids Research. 2007; 35 (12):3974-3987.

Chicago/Turabian Style

Tiyun Wu; Susan L. Heilman-Miller; Judith G. Levin. 2007. "Effects of nucleic acid local structure and magnesium ions on minus-strand transfer mediated by the nucleic acid chaperone activity of HIV-1 nucleocapsid protein." Nucleic Acids Research 35, no. 12: 3974-3987.

Journal article
Published: 01 March 2007 in Virology
Reads 0
Downloads 0

The HIV-1 capsid (CA) protein plays an important role in virus assembly and infectivity. Previously, we showed that Ala substitutions in the N-terminal residues Trp23 and Phe40 cause a severely defective phenotype. In searching for mutations at these positions that result in a non-lethal phenotype, we identified one candidate, W23F. Mutant virions contained aberrant cores, but unlike W23A, also displayed some infectivity in a single-round replication assay and delayed replication kinetics in MT-4 cells. Following long-term passage in MT-4 cells, two second-site mutations were isolated. In particular, the W23F/V26I mutation partially restored the wild-type phenotype, including production of particles with conical cores and wild-type replication kinetics in MT-4 cells. A structural model is proposed to explain the suppressor phenotype. These findings describe a novel occurrence, namely suppression of a mutation in a hydrophobic residue that is critical for maintaining the structural integrity of CA and proper core assembly.

ACS Style

Shixing Tang; Sherimay Ablan; Megan Dueck; Wilfredo Ayala-López; Brenda Soto; Margaret Caplan; Kunio Nagashima; Indira K. Hewlett; Eric O. Freed; Judith G. Levin. A second-site suppressor significantly improves the defective phenotype imposed by mutation of an aromatic residue in the N-terminal domain of the HIV-1 capsid protein. Virology 2007, 359, 105 -115.

AMA Style

Shixing Tang, Sherimay Ablan, Megan Dueck, Wilfredo Ayala-López, Brenda Soto, Margaret Caplan, Kunio Nagashima, Indira K. Hewlett, Eric O. Freed, Judith G. Levin. A second-site suppressor significantly improves the defective phenotype imposed by mutation of an aromatic residue in the N-terminal domain of the HIV-1 capsid protein. Virology. 2007; 359 (1):105-115.

Chicago/Turabian Style

Shixing Tang; Sherimay Ablan; Megan Dueck; Wilfredo Ayala-López; Brenda Soto; Margaret Caplan; Kunio Nagashima; Indira K. Hewlett; Eric O. Freed; Judith G. Levin. 2007. "A second-site suppressor significantly improves the defective phenotype imposed by mutation of an aromatic residue in the N-terminal domain of the HIV-1 capsid protein." Virology 359, no. 1: 105-115.

Journal article
Published: 15 June 2006 in Journal of Virology
Reads 0
Downloads 0

APOBEC3G (APO3G), a cytidine deaminase with two zinc finger domains, inhibits human immunodeficiency virus type 1 replication in the absence of Vif. Here, we provide a comprehensive molecular analysis of the deaminase and nucleic acid binding activities of human APO3G using a pure system containing only one protein component, i.e., highly purified, catalytically active enzyme expressed in a baculovirus system. We demonstrate that APO3G deaminates cytosines in single-stranded DNA (ssDNA) only, whereas it binds efficiently to ssDNA and ssRNA, about half as well to a DNA/RNA hybrid, and poorly to double-stranded DNA and RNA. In addition, the base specificities for deamination and binding of ssDNA are not correlated. The minimum length required for detection of APO3G binding to an ssDNA oligonucleotide in an electrophoretic mobility shift assay is 16 nucleotides. Interestingly, if nucleocapsid protein and APO3G are present in the same reaction, we find that they do not interfere with each other's binding to RNA and a complex containing the RNA and both proteins is formed. Finally, we also identify the functional activities of each zinc finger domain. Thus, although both zinc finger domains have the ability to bind nucleic acids, the first zinc finger contributes more to binding and APO3G encapsidation into virions than finger two. In contrast, deamination is associated exclusively with the second zinc finger. Moreover, zinc finger two is more important than finger one for the antiviral effect, demonstrating a correlation between deaminase and antiviral activities.

ACS Style

Yasumasa Iwatani; Hiroaki Takeuchi; Klaus Strebel; Judith G. Levin. Biochemical Activities of Highly Purified, Catalytically Active Human APOBEC3G: Correlation with Antiviral Effect. Journal of Virology 2006, 80, 5992 -6002.

AMA Style

Yasumasa Iwatani, Hiroaki Takeuchi, Klaus Strebel, Judith G. Levin. Biochemical Activities of Highly Purified, Catalytically Active Human APOBEC3G: Correlation with Antiviral Effect. Journal of Virology. 2006; 80 (12):5992-6002.

Chicago/Turabian Style

Yasumasa Iwatani; Hiroaki Takeuchi; Klaus Strebel; Judith G. Levin. 2006. "Biochemical Activities of Highly Purified, Catalytically Active Human APOBEC3G: Correlation with Antiviral Effect." Journal of Virology 80, no. 12: 5992-6002.

Journal article
Published: 01 December 2003 in Journal of Virology
Reads 0
Downloads 0

We previously described the phenotype associated with three alanine substitution mutations in conserved residues (Trp23, Phe40, and Asp51) in the N-terminal domain of human immunodeficiency virus type 1 capsid protein (CA). All of the mutants produce noninfectious virions that lack conical cores and, despite having a functional reverse transcriptase (RT), are unable to initiate reverse transcription in vivo. Here, we have focused on elucidating the mechanism by which these CA mutations disrupt virus infectivity. We also report that cyclophilin A packaging is severely reduced in W23A and F40A virions, even though these residues are distant from the cyclophilin A binding loop. To correlate loss of infectivity with a possible defect in an early event preceding reverse transcription, we modeled disassembly by generating viral cores from particles treated with mild nonionic detergent; cores were isolated by sedimentation in sucrose density gradients. In general, fractions containing mutant cores exhibited a normal protein profile. However, there were two striking differences from the wild-type pattern: mutant core fractions displayed a marked deficiency in RT protein and enzymatic activity (<5% of total RT in gradient fractions) and a substantial increase in the retention of CA. The high level of core-associated CA suggests that mutant cores may be unable to undergo proper disassembly. Thus, taken together with the almost complete absence of RT in mutant cores, these findings can account for the failure of the three CA mutants to synthesize viral DNA following virus entry into cells.

ACS Style

Shixing Tang; Tsutomu Murakami; Naiqian Cheng; Alasdair C. Steven; Eric O. Freed; Judith G. Levin. Human Immunodeficiency Virus Type 1 N-Terminal Capsid Mutants Containing Cores with Abnormally High Levels of Capsid Protein and Virtually No Reverse Transcriptase. Journal of Virology 2003, 77, 12592 -12602.

AMA Style

Shixing Tang, Tsutomu Murakami, Naiqian Cheng, Alasdair C. Steven, Eric O. Freed, Judith G. Levin. Human Immunodeficiency Virus Type 1 N-Terminal Capsid Mutants Containing Cores with Abnormally High Levels of Capsid Protein and Virtually No Reverse Transcriptase. Journal of Virology. 2003; 77 (23):12592-12602.

Chicago/Turabian Style

Shixing Tang; Tsutomu Murakami; Naiqian Cheng; Alasdair C. Steven; Eric O. Freed; Judith G. Levin. 2003. "Human Immunodeficiency Virus Type 1 N-Terminal Capsid Mutants Containing Cores with Abnormally High Levels of Capsid Protein and Virtually No Reverse Transcriptase." Journal of Virology 77, no. 23: 12592-12602.

Journal article
Published: 01 July 2003 in Journal of Virology
Reads 0
Downloads 0

Human immunodeficiency virus type 2 (HIV-2) infection is a serious problem in West Africa and Asia. However, there have been relatively few studies of HIV-2 reverse transcriptase (RT), a potential target for antiviral therapy. Detailed knowledge of HIV-2 RT activities is critical for development of specific high-throughput screening assays of potential inhibitors. Here, we have conducted a systematic evaluation of HIV-2 RT function, using assays that model specific steps in reverse transcription. Parallel studies were performed with HIV-1 RT. In general, under standard assay conditions, the polymerase and RNase H activities of the two enzymes were comparable. However, when the RT concentration was significantly reduced, HIV-2 RT was less active than the HIV-1 enzyme. HIV-2 RT was also impaired in its ability to catalyze secondary RNase H cleavage in assays that mimic tRNA primer removal during plus-strand transfer and degradation of genomic RNA fragments during minus-strand DNA synthesis. In addition, initiation of plus-strand DNA synthesis was much less efficient with HIV-2 RT than with HIV-1 RT. This may reflect architectural differences in the primer grip regions in the p66 (HIV-1) and p68 (HIV-2) palm subdomains of the two enzymes. The implications of our findings for antiviral therapy are discussed.

ACS Style

Klara Post; Jianhui Guo; Kathryn J. Howard; Michael D. Powell; Jennifer T. Miller; Amnon Hizi; Stuart F. J. Le Grice; Judith G. Levin. Human Immunodeficiency Virus Type 2 Reverse Transcriptase Activity in Model Systems That Mimic Steps in Reverse Transcription. Journal of Virology 2003, 77, 7623 -7634.

AMA Style

Klara Post, Jianhui Guo, Kathryn J. Howard, Michael D. Powell, Jennifer T. Miller, Amnon Hizi, Stuart F. J. Le Grice, Judith G. Levin. Human Immunodeficiency Virus Type 2 Reverse Transcriptase Activity in Model Systems That Mimic Steps in Reverse Transcription. Journal of Virology. 2003; 77 (13):7623-7634.

Chicago/Turabian Style

Klara Post; Jianhui Guo; Kathryn J. Howard; Michael D. Powell; Jennifer T. Miller; Amnon Hizi; Stuart F. J. Le Grice; Judith G. Levin. 2003. "Human Immunodeficiency Virus Type 2 Reverse Transcriptase Activity in Model Systems That Mimic Steps in Reverse Transcription." Journal of Virology 77, no. 13: 7623-7634.

Journal article
Published: 01 May 2002 in Journal of Virology
Reads 0
Downloads 0

The nucleocapsid protein (NC) of human immunodeficiency virus type 1 has two zinc fingers, each containing the invariant CCHC zinc-binding motif; however, the surrounding amino acid context is not identical in the two fingers. Recently, we demonstrated that zinc coordination is required when NC unfolds complex secondary structures in RNA and DNA minus- and plus-strand transfer intermediates; this property of NC reflects its nucleic acid chaperone activity. Here we have analyzed the chaperone activities of mutants having substitutions of alternative zinc-coordinating residues, i.e., CCHH or CCCC, for the wild-type CCHC motif. We also investigated the activities of mutants that retain the CCHC motifs but have mutations that exchange or duplicate the zinc fingers (mutants 1-1, 2-1, and 2-2); these changes affect amino acid context. Our results indicate that in general, for optimal activity in an assay that measures stimulation of minus-strand transfer and inhibition of nonspecific self-priming, the CCHC motif in the zinc fingers cannot be replaced by CCHH or CCCC and the amino acid context of the fingers must be conserved. Context changes also reduce the ability of NC to facilitate primer removal in plus-strand transfer. In addition, we found that the first finger is a more crucial determinant of nucleic acid chaperone activity than the second finger. Interestingly, comparison of the in vitro results with earlier in vivo replication data raises the possibility that NC may adopt multiple conformations that are responsible for different NC functions during virus replication.

ACS Style

Jianhui Guo; Tiyun Wu; Bradley F. Kane; Donald G. Johnson; Louis E. Henderson; Robert J. Gorelick; Judith G. Levin. Subtle Alterations of the Native Zinc Finger Structures Have Dramatic Effects on the Nucleic Acid Chaperone Activity of Human Immunodeficiency Virus Type 1 Nucleocapsid Protein. Journal of Virology 2002, 76, 4370 -4378.

AMA Style

Jianhui Guo, Tiyun Wu, Bradley F. Kane, Donald G. Johnson, Louis E. Henderson, Robert J. Gorelick, Judith G. Levin. Subtle Alterations of the Native Zinc Finger Structures Have Dramatic Effects on the Nucleic Acid Chaperone Activity of Human Immunodeficiency Virus Type 1 Nucleocapsid Protein. Journal of Virology. 2002; 76 (9):4370-4378.

Chicago/Turabian Style

Jianhui Guo; Tiyun Wu; Bradley F. Kane; Donald G. Johnson; Louis E. Henderson; Robert J. Gorelick; Judith G. Levin. 2002. "Subtle Alterations of the Native Zinc Finger Structures Have Dramatic Effects on the Nucleic Acid Chaperone Activity of Human Immunodeficiency Virus Type 1 Nucleocapsid Protein." Journal of Virology 76, no. 9: 4370-4378.