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Human immunodeficiency virus type 2 (HIV-2) accumulates fewer mutations during replication than HIV type 1 (HIV-1). Advanced studies of HIV-2 mutagenesis, however, have historically been confounded by high background error rates in traditional next-generation sequencing techniques. In this study, we describe the adaptation of the previously described maximum-depth sequencing (MDS) technique to studies of both HIV-1 and HIV-2 for the ultra-accurate characterization of viral mutagenesis. We also present the development of a user-friendly Galaxy workflow for the bioinformatic analyses of sequencing data generated using the MDS technique, designed to improve replicability and accessibility to molecular virologists. This adapted MDS technique and analysis pipeline were validated by comparisons with previously published analyses of the frequency and spectra of mutations in HIV-1 and HIV-2 and is readily expandable to studies of viral mutation across the genomes of both viruses. Using this novel sequencing pipeline, we observed that the background error rate was reduced 100-fold over standard Illumina error rates, and 10-fold over traditional unique molecular identifier (UMI)-based sequencing. This technical advancement will allow for the exploration of novel and previously unrecognized sources of viral mutagenesis in both HIV-1 and HIV-2, which will expand our understanding of retroviral diversity and evolution.
Morgan Meissner; Emily Julik; Jonathan Badalamenti; William Arndt; Lauren Mills; Louis Mansky. Development of a User-Friendly Pipeline for Mutational Analyses of HIV Using Ultra-Accurate Maximum-Depth Sequencing. Viruses 2021, 13, 1338 .
AMA StyleMorgan Meissner, Emily Julik, Jonathan Badalamenti, William Arndt, Lauren Mills, Louis Mansky. Development of a User-Friendly Pipeline for Mutational Analyses of HIV Using Ultra-Accurate Maximum-Depth Sequencing. Viruses. 2021; 13 (7):1338.
Chicago/Turabian StyleMorgan Meissner; Emily Julik; Jonathan Badalamenti; William Arndt; Lauren Mills; Louis Mansky. 2021. "Development of a User-Friendly Pipeline for Mutational Analyses of HIV Using Ultra-Accurate Maximum-Depth Sequencing." Viruses 13, no. 7: 1338.
5-aza-cytidine (5-aza-C) has been shown to be a potent human immunodeficiency virus type 1 (HIV-1) mutagen that induces G-to-C hypermutagenesis by incorporation of the reduced form (i.e., 5-aza-dC, 5-aza-dCTP). Evidence to date suggests that this lethal mutagenesis is the primary antiretroviral mechanism for 5-aza-C. To investigate the breadth of application of 5-aza-C as an antiretroviral mutagen, we have conducted a comparative, parallel analysis of the antiviral mechanism of 5-aza-C between HIV-1 and gammaretroviruses – i.e., murine leukemia virus (MuLV) and feline leukemia virus (FeLV). Intriguingly, in contrast to the hallmark G-to-C hypermutagenesis observed with HIV-1, MuLV and FeLV did not reveal the presence of a significant increase in mutational burden, particularly that of G-to-C transversion mutations. The effect of 5-aza-dCTP on DNA synthesis revealed that while HIV-1 RT was not inhibited by 5-aza-dCTP even at 100 µM, 5-aza-dCTP was incorporated and significantly inhibited MuLV RT, generating pause sites and reducing the fully extended product. 5-aza-dCTP was found to be incorporated into DNA by MuLV RT or HIV-1 RT, but only acted as a non-obligate chain terminator for MuLV RT. This biochemical data provides an independent line of experimental evidence in support of the conclusion that HIV-1 and MuLV have distinct primary mechanisms of antiretroviral action with 5-aza-C. Taken together, our data provides striking evidence that an antiretroviral mutagen can have strong potency via distinct mechanisms of action among closely related viruses, unlinking antiviral activity from antiviral mechanism of action.
Megan Roth; Yumeng Z. McDaniel; Michele B. Daly; Nathaniel Talledge; Willie M. Greggs; Steven E. Patterson; Baek Kim; Louis M. Mansky. Distinct Antiretroviral Mechanisms Elicited by a Viral Mutagen. Journal of Molecular Biology 2021, 433, 167111 .
AMA StyleMegan Roth, Yumeng Z. McDaniel, Michele B. Daly, Nathaniel Talledge, Willie M. Greggs, Steven E. Patterson, Baek Kim, Louis M. Mansky. Distinct Antiretroviral Mechanisms Elicited by a Viral Mutagen. Journal of Molecular Biology. 2021; 433 (18):167111.
Chicago/Turabian StyleMegan Roth; Yumeng Z. McDaniel; Michele B. Daly; Nathaniel Talledge; Willie M. Greggs; Steven E. Patterson; Baek Kim; Louis M. Mansky. 2021. "Distinct Antiretroviral Mechanisms Elicited by a Viral Mutagen." Journal of Molecular Biology 433, no. 18: 167111.
During the late phase of retroviral replication, the virally encoded Gag polyprotein is targeted to the plasma membrane (PM) for assembly. Gag–PM binding is mediated by the N-terminal matrix (MA) domain of Gag. For many retroviruses, Gag binding to the PM was found to be dependent on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. However, it was shown that for human T-cell leukemia virus type 1 (HTLV-1), Gag binding to membranes is less dependent on PI(4,5)P2, suggesting that other factors may modulate Gag assembly. To elucidate the mechanism by which HTLV-1 Gag binds to the PM, we employed NMR techniques to solve the structure of unmyristoylated MA (myr(–)MA) and to characterize its interactions with lipids and liposomes. The MA structure consists of four α-helices and unstructured N- and C-termini. We show that myr(–)MA binds to PI(4,5)P2via the polar head and that myr(–)MA binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups on the inositol ring, indicating that the MA–IP binding is governed by charge–charge interactions. The IP binding site was mapped to a well-defined basic patch formed by lysine and arginine residues. Using a sensitive NMR-based liposome binding assay, we show that myr(–)MA binding to membranes is significantly enhanced by phosphatidylserine (PS). Confocal microscopy data show that Gag is localized to the inner leaflet of the PM of infected cells, while the Gag G2A mutant, lacking myristoylation, is diffuse and cytoplasmic. These findings advance our understanding of a key mechanism in retroviral assembly.
Dominik Herrmann; Lynne W. Zhou; Heather M. Hanson; Nora A. Willkomm; Louis M. Mansky; Jamil S. Saad. Structural basis for human T-cell leukemia virus type 1 Gag targeting to the plasma membrane for assembly. 2021, 1 .
AMA StyleDominik Herrmann, Lynne W. Zhou, Heather M. Hanson, Nora A. Willkomm, Louis M. Mansky, Jamil S. Saad. Structural basis for human T-cell leukemia virus type 1 Gag targeting to the plasma membrane for assembly. . 2021; ():1.
Chicago/Turabian StyleDominik Herrmann; Lynne W. Zhou; Heather M. Hanson; Nora A. Willkomm; Louis M. Mansky; Jamil S. Saad. 2021. "Structural basis for human T-cell leukemia virus type 1 Gag targeting to the plasma membrane for assembly." , no. : 1.
The human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3, A3) family member proteins can deaminate cytosines in single-strand (ss) DNA, which restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons, and other viruses such as hepatitis B virus, but can cause a mutator phenotype in many cancers. While structural information exists for several A3 proteins, the precise details regarding deamination target selection are not fully understood. Here, we report the first parallel, comparative analysis of site selection of A3 deamination using six of the seven purified A3 member enzymes, oligonucleotides having 5′TC3′ or 5′CT3′ dinucleotide target sites, and different flanking bases within diverse DNA secondary structures. A3A, A3F and A3H were observed to have strong preferences toward the TC target flanked by A or T, while all examined A3 proteins did not show a preference for a TC target flanked by a G. We observed that the TC target was strongly preferred in ssDNA regions rather than dsDNA, loop or bulge regions, with flanking bases influencing the degree of preference. CT was also shown to be a potential deamination target. Taken together, our observations provide new insights into A3 enzyme target site selection and how A3 mutagenesis impacts mutation rates.
Yumeng Z McDaniel; Dake Wang; Robin P Love; Madison B Adolph; Nazanin Mohammadzadeh; Linda Chelico; Louis M Mansky. Deamination hotspots among APOBEC3 family members are defined by both target site sequence context and ssDNA secondary structure. Nucleic Acids Research 2020, 48, 1353 -1371.
AMA StyleYumeng Z McDaniel, Dake Wang, Robin P Love, Madison B Adolph, Nazanin Mohammadzadeh, Linda Chelico, Louis M Mansky. Deamination hotspots among APOBEC3 family members are defined by both target site sequence context and ssDNA secondary structure. Nucleic Acids Research. 2020; 48 (3):1353-1371.
Chicago/Turabian StyleYumeng Z McDaniel; Dake Wang; Robin P Love; Madison B Adolph; Nazanin Mohammadzadeh; Linda Chelico; Louis M Mansky. 2020. "Deamination hotspots among APOBEC3 family members are defined by both target site sequence context and ssDNA secondary structure." Nucleic Acids Research 48, no. 3: 1353-1371.
Reverse transcriptase (RT) is an essential enzyme for the replication of retroviruses and hepadnaviruses. Current therapies do not eliminate the intracellular viral replication intermediate termed covalently closed circular (ccc) DNA, which has enhanced interest in hepatitis B virus (HBV) reverse transcription and cccDNA formation. The HBV cccDNA is generated as a plasmid-like episome in the host cell nucleus from the protein-linked relaxed circular (rc) DNA genome in incoming virions during HBV replication. The creation of the cccDNA via conversion from rcDNA remains not fully understood. Here, we sought to investigate whether viral mutagens can effect HBV replication. In particular, we investigated whether nucleoside analogs that act as viral mutagens with retroviruses could impact hepadnaviral DNA synthesis. We observed that a viral mutagen (e.g., 5-aza-2′-deoxycytidine, 5-aza-dC or 5-azacytidine, 5-aza-C) severely diminished the ability of a HBV vector to express a reporter gene following virus transfer and infection of target cells. As predicted, the treatment of 5-aza-dC or 5-aza-C elevated the HBV rcDNA mutation frequency, primarily by increasing the frequency of G-to-C transversion mutations. A reduction in rcDNA synthesis was also observed. Intriguingly, the cccDNA nick/gap region transcription was diminished by 5-aza-dC, but did not enhance viral mutagenesis. Taken together, our results demonstrate that viral mutagens can impact HBV reverse transcription, and propose a model in which viral mutagens can induce mutagenesis during rcDNA formation and diminish viral DNA synthesis during both rcDNA formation and the conversion of rcDNA to cccDNA.
Yumeng Z. McDaniel; Steven E. Patterson; Louis M. Mansky. Distinct dual antiviral mechanism that enhances hepatitis B virus mutagenesis and reduces viral DNA synthesis. Antiviral Research 2019, 170, 104540 -104540.
AMA StyleYumeng Z. McDaniel, Steven E. Patterson, Louis M. Mansky. Distinct dual antiviral mechanism that enhances hepatitis B virus mutagenesis and reduces viral DNA synthesis. Antiviral Research. 2019; 170 ():104540-104540.
Chicago/Turabian StyleYumeng Z. McDaniel; Steven E. Patterson; Louis M. Mansky. 2019. "Distinct dual antiviral mechanism that enhances hepatitis B virus mutagenesis and reduces viral DNA synthesis." Antiviral Research 170, no. : 104540-104540.
In the original publication, the names of the second and third authors were incorrectly published.
Wei Zhang; Luiza M. Mendonça; Louis M. Mansky. Correction to: The Retrovirus Capsid Core. Prokaryotic Cytoskeletons 2018, 88, E1 -E1.
AMA StyleWei Zhang, Luiza M. Mendonça, Louis M. Mansky. Correction to: The Retrovirus Capsid Core. Prokaryotic Cytoskeletons. 2018; 88 ():E1-E1.
Chicago/Turabian StyleWei Zhang; Luiza M. Mendonça; Louis M. Mansky. 2018. "Correction to: The Retrovirus Capsid Core." Prokaryotic Cytoskeletons 88, no. : E1-E1.
This report describes the results of experiments examining the pathway by which the human retroviral Gag protein is recruited to sites along the inner leaflet of the plasma membrane where Gag punctum biogenesis occurs. In particular, clever and sensitive experimental methods were devised to image in living cells fluorescently labeled Gag protein derivatives from human T-cell leukemia virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1) at the plasma membrane. The photoconvertible fluorescent protein mEos2 was strategically utilized, as the fluorescence emission of Gag at the plasma membrane could be differentiated from that of cytosolic Gag. This experimental strategy allowed for the determination of the Gag recruitment pathway into Gag puncta. For HTLV-1 Gag, puncta recruited Gag primarily from the plasma membrane, while HIV-1 Gag was recruited from the cytoplasm. These observations represent the first report of HTLV-1 particle biogenesis and its contrast to that of HIV-1. The observed differences in the Gag recruitment pathways used by HTLV-1 and HIV-1 Gag provide key information that is useful for informing the discovery of novel targets for antiretroviral therapies directed at eliminating virus infectivity and spread.
John P. Eichorst; Yan Chen; Joachim D. Mueller; Louis M. Mansky. Distinct Pathway of Human T-Cell Leukemia Virus Type 1 Gag Punctum Biogenesis Provides New Insights into Enveloped Virus Assembly. mBio 2018, 9, e00758-18 .
AMA StyleJohn P. Eichorst, Yan Chen, Joachim D. Mueller, Louis M. Mansky. Distinct Pathway of Human T-Cell Leukemia Virus Type 1 Gag Punctum Biogenesis Provides New Insights into Enveloped Virus Assembly. mBio. 2018; 9 (5):e00758-18.
Chicago/Turabian StyleJohn P. Eichorst; Yan Chen; Joachim D. Mueller; Louis M. Mansky. 2018. "Distinct Pathway of Human T-Cell Leukemia Virus Type 1 Gag Punctum Biogenesis Provides New Insights into Enveloped Virus Assembly." mBio 9, no. 5: e00758-18.
Human T-cell leukemia virus type 1 (HTLV-1) is the first retrovirus that has conclusively been shown to cause human diseases. In HIV-1, specific interactions between the nucleocapsid (NC) domain of the Gag protein and genomic RNA (gRNA) mediate gRNA dimerization and selective packaging; however, the mechanism for gRNA packaging in HTLV-1, a deltaretrovirus, is unclear. In other deltaretroviruses, the matrix (MA) and NC domains of Gag are both involved in gRNA packaging, but MA binds nucleic acids with higher affinity and has more robust chaperone activity, suggesting that this domain may play a primary role. Here, we show that the MA domain of HTLV-1, but not the NC domain, binds short hairpin RNAs derived from the putative gRNA packaging signal. RNA probing of the HTLV-1 5′leader and crosslinking studies revealed that the primer-binding site and a region within the putative packaging signal form stable hairpins that interact with MA. In addition to a previously identified palindromic dimerization initiation site (DIS), we identified a new DIS in HTLV-1 gRNA and found that both palindromic sequences bind specifically the NC domain. Surprisingly, a mutant partially defective in dimer formation in vitro exhibited a significant increase in RNA packaging into HTLV-1–like particles, suggesting that efficient RNA dimerization may not be strictly required for RNA packaging in HTLV-1. Moreover, the lifecycle of HTLV-1 and other deltaretroviruses may be characterized by NC and MA functions that are distinct from those of the corresponding HIV-1 proteins, but together provide the functions required for viral replication.
Weixin Wu; Joshua Hatterschide; Yu-Ci Syu; William A. Cantara; Ruth Blower; Heather M. Hanson; Louis M. Mansky; Karin Musier-Forsyth. Human T-cell leukemia virus type 1 Gag domains have distinct RNA-binding specificities with implications for RNA packaging and dimerization. Journal of Biological Chemistry 2018, 293, 16261 -16276.
AMA StyleWeixin Wu, Joshua Hatterschide, Yu-Ci Syu, William A. Cantara, Ruth Blower, Heather M. Hanson, Louis M. Mansky, Karin Musier-Forsyth. Human T-cell leukemia virus type 1 Gag domains have distinct RNA-binding specificities with implications for RNA packaging and dimerization. Journal of Biological Chemistry. 2018; 293 (42):16261-16276.
Chicago/Turabian StyleWeixin Wu; Joshua Hatterschide; Yu-Ci Syu; William A. Cantara; Ruth Blower; Heather M. Hanson; Louis M. Mansky; Karin Musier-Forsyth. 2018. "Human T-cell leukemia virus type 1 Gag domains have distinct RNA-binding specificities with implications for RNA packaging and dimerization." Journal of Biological Chemistry 293, no. 42: 16261-16276.
Emergence of human immunodeficiency virus type 1 (HIV-1) drug resistance arises from mutation fixation in the viral genome during antiretroviral therapy. Primary mutations directly confer antiviral drug resistance, while secondary mutations arise that do not confer drug resistance. The A62V amino acid substitution in HIV-1 reverse transcriptase (RT) was observed to be associated with multi-drug resistance, but is not known to be a resistance-conferring mutation. In particular, A62V was observed in various multi-dideoxynucleoside resistant (MDR) mutation complexes, including the Q151M complex (i.e., A62V, V75I, F77L, F116Y, and Q151M), and the T69SSS insertion complex, which has a serine–serine insertion between amino acid positions 69 and 70 (i.e., M41L, A62V, T69SSS, K70R, and T215Y). However, what selective advantage is conferred to the virus remains unresolved. In this study, we hypothesized that A62V could influence replication fidelity and viral fitness with viruses harboring the Q151M and T69SSS MDR mutation complexes. A single-cycle replication assay and a dual-competition fitness assay were used to assess viral mutant frequency and viral fitness, respectively. A62V was found to increase the observed lower mutant frequency identified with each of the viruses harboring the MDR mutation complexes in the single-cycle assay. Furthermore, A62V was observed to improve viral fitness of replication-competent MDR viruses. Taken together, these observations indicate an adaptive role of A62V in virus replication fidelity and viral fitness, which would likely enhance virus persistence during drug-selective pressure.
José O. Maldonado; Louis M. Mansky. The HIV-1 Reverse Transcriptase A62V Mutation Influences Replication Fidelity and Viral Fitness in the Context of Multi-Drug-Resistant Mutations. Viruses 2018, 10, 376 .
AMA StyleJosé O. Maldonado, Louis M. Mansky. The HIV-1 Reverse Transcriptase A62V Mutation Influences Replication Fidelity and Viral Fitness in the Context of Multi-Drug-Resistant Mutations. Viruses. 2018; 10 (7):376.
Chicago/Turabian StyleJosé O. Maldonado; Louis M. Mansky. 2018. "The HIV-1 Reverse Transcriptase A62V Mutation Influences Replication Fidelity and Viral Fitness in the Context of Multi-Drug-Resistant Mutations." Viruses 10, no. 7: 376.
Retrovirus particle assembly and release from infected cells is driven by the Gag structural protein. Gag-Gag interactions, which form an oligomeric lattice structure at a particle budding site, are essential to the biogenesis of an infectious virus particle. The CA domain of Gag is generally thought to possess the key determinants for Gag-Gag interactions, and the present study has discovered several critical amino acid residues in the CA domain of HTLV-1 Gag, an important cancer-causing human retrovirus, which are distinct from that of HIV-1 as well as other retroviruses studied to date. Altogether, our results provide important new insights into a poorly understood aspect of HTLV-1 replication that significantly enhances our understanding of the molecular nature of Gag-Gag interaction determinants crucial for virus particle assembly.
Jessica L. Martin; Luiza M. Mendonça; Rachel Marusinec; Jennifer Zuczek; Isaac Angert; Ruth J. Blower; Joachim D. Mueller; Juan R. Perilla; Wei Zhang; Louis M. Mansky. Critical Role of the Human T-Cell Leukemia Virus Type 1 Capsid N-Terminal Domain for Gag-Gag Interactions and Virus Particle Assembly. Journal of Virology 2018, 92, e00333-18 .
AMA StyleJessica L. Martin, Luiza M. Mendonça, Rachel Marusinec, Jennifer Zuczek, Isaac Angert, Ruth J. Blower, Joachim D. Mueller, Juan R. Perilla, Wei Zhang, Louis M. Mansky. Critical Role of the Human T-Cell Leukemia Virus Type 1 Capsid N-Terminal Domain for Gag-Gag Interactions and Virus Particle Assembly. Journal of Virology. 2018; 92 (14):e00333-18.
Chicago/Turabian StyleJessica L. Martin; Luiza M. Mendonça; Rachel Marusinec; Jennifer Zuczek; Isaac Angert; Ruth J. Blower; Joachim D. Mueller; Juan R. Perilla; Wei Zhang; Louis M. Mansky. 2018. "Critical Role of the Human T-Cell Leukemia Virus Type 1 Capsid N-Terminal Domain for Gag-Gag Interactions and Virus Particle Assembly." Journal of Virology 92, no. 14: e00333-18.
The retrovirus capsid core is a metastable structure that disassembles during the early phase of viral infection after membrane fusion. The core is intact and permeable to essential nucleotides during reverse transcription, but it undergoes disassembly for nuclear entry and genome integration. Increasing or decreasing the stability of the capsid core has a substantial negative impact on virus infectivity, which makes the core an attractive anti-viral target. The retrovirus capsid core also encounters a variety of virus- and organism-specific host cellular factors that promote or restrict viral replication. This review describes the structural elements fundamental to the formation and stability of the capsid core. The physical and chemical properties of the capsid core that are critical to its functional role in reverse transcription and interaction with host cellular factors are highlighted to emphasize areas of current research.
Wei Zhang; Luiza M. Mendonça; Louis M. Mansky. The Retrovirus Capsid Core. Prokaryotic Cytoskeletons 2018, 88, 169 -187.
AMA StyleWei Zhang, Luiza M. Mendonça, Louis M. Mansky. The Retrovirus Capsid Core. Prokaryotic Cytoskeletons. 2018; 88 ():169-187.
Chicago/Turabian StyleWei Zhang; Luiza M. Mendonça; Louis M. Mansky. 2018. "The Retrovirus Capsid Core." Prokaryotic Cytoskeletons 88, no. : 169-187.
Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 cell-to-cell transmission is dependent on the release of infectious virus particles into the virological synapse. The HTLV-1 particle structure is still poorly understood, and previous studies analyzed viruses produced by transformed lymphocytic cell lines chronically infected with HTLV-1, particularly the MT-2 cell line, which harbors truncated proviruses and expresses aberrant forms of the Gag protein. In this study, we demonstrate that the chronically infected SP cell line harbors a relatively low number of proviruses, making it a more promising experimental system for the study of the HTLV-1 particle structure. We first identified the genomic sites of integration and characterized the genetic structure of the gag region in each provirus. We also determined that despite encoding a truncated Gag protein, only the full-length Gag protein was incorporated into virus particles. Cryo-transmission electron microscopy analyses of the purified virus particles revealed three classes of particles based upon capsid core morphology: complete cores, incomplete cores, and particles without distinct electron densities that would correlate with the capsid region of a core structure. Observed cores were generally polygonal, and virus particles were on average 115 nm in diameter. These data corroborate particle morphologies previously observed for MT-2 cells and provide evidence that the known poor infectivity of HTLV-1 particles may correlate with HTLV-1 particle populations containing few virus particles possessing a complete capsid core structure. IMPORTANCE Studies of retroviral particle core morphology have demonstrated a correlation between capsid core stability and the relative infectivity of the virus. In this study, we used cryo-transmission electron microscopy to demonstrate that HTLV-1 particles produced from a distinct chronically infected cell line are polymorphic in nature, with many particles lacking organized electron densities that would correlate with a complete core structure. These findings have important implications for infectious HTLV-1 spread, particularly in the context of cell-to-cell transmission, a critical step in HTLV-1 transmission and pathogenesis.
Morgan E. Meissner; Luiza M. Mendonça; Wei Zhang; Louis M. Mansky. Polymorphic Nature of Human T-Cell Leukemia Virus Type 1 Particle Cores as Revealed through Characterization of a Chronically Infected Cell Line. Journal of Virology 2017, 91, 1 .
AMA StyleMorgan E. Meissner, Luiza M. Mendonça, Wei Zhang, Louis M. Mansky. Polymorphic Nature of Human T-Cell Leukemia Virus Type 1 Particle Cores as Revealed through Characterization of a Chronically Infected Cell Line. Journal of Virology. 2017; 91 (16):1.
Chicago/Turabian StyleMorgan E. Meissner; Luiza M. Mendonça; Wei Zhang; Louis M. Mansky. 2017. "Polymorphic Nature of Human T-Cell Leukemia Virus Type 1 Particle Cores as Revealed through Characterization of a Chronically Infected Cell Line." Journal of Virology 91, no. 16: 1.
Human T-cell leukemia virus type 1 (HTLV-1) is an important cancer-causing human retrovirus that has infected approximately 15 million individuals worldwide. Many aspects of HTLV-1 replication, including virus particle structure and assembly, are poorly understood. Group-specific antigen (Gag) proteins labeled at the carboxy terminus with a fluorophore protein have been used extensively as a surrogate for fluorescence studies of retroviral assembly. How these tags affect Gag stoichiometry and particle morphology has not been reported in detail. In this study, we used an HTLV-1 Gag expression construct with the yellow fluorescence protein (YFP) fused to the carboxy-terminus as a surrogate for the HTLV-1 Gag-Pol to assess the effects of co-packaging of Gag and a Gag-YFP on virus-like particle (VLP) morphology and analyzed particles by cryogenic transmission electron microscopy (cryo-TEM). Scanning transmission electron microscopy (STEM) and fluorescence fluctuation spectroscopy (FFS) were also used to determine the Gag stoichiometry. We found that ratios of 3:1 (Gag:Gag-YFP) or greater resulted in a particle morphology indistinguishable from that of VLPs produced with the untagged HTLV-1 Gag, i.e., a mean diameter of ~113 nm and a mass of 220 MDa as determined by cryo-TEM and STEM, respectively. Furthermore, FFS analysis indicated that HTLV-1 Gag-YFP was incorporated into VLPs in a predictable manner at the 3:1 Gag:Gag-YFP ratio. Both STEM and FFS analyses found that the Gag copy number in VLPs produced with a 3:1 ratio of Gag:Gag-YFP was is in the range of 1500–2000 molecules per VLP. The observations made in this study indicate that biologically relevant Gag–Gag interactions occur between Gag and Gag-YFP at ratios of 3:1 or higher and create a Gag lattice structure in VLPs that is morphologically indistinguishable from that of VLPs produced with just untagged Gag. This information is useful for the quantitative analysis of Gag–Gag interactions that occur during virus particle assembly and in released immature particles.
José O. Maldonado; Isaac Angert; Sheng Cao; Serkan Berk; Wei Zhang; Joachim D. Mueller; Louis M. Mansky. Perturbation of Human T-Cell Leukemia Virus Type 1 Particle Morphology by Differential Gag Co-Packaging. Viruses 2017, 9, 191 .
AMA StyleJosé O. Maldonado, Isaac Angert, Sheng Cao, Serkan Berk, Wei Zhang, Joachim D. Mueller, Louis M. Mansky. Perturbation of Human T-Cell Leukemia Virus Type 1 Particle Morphology by Differential Gag Co-Packaging. Viruses. 2017; 9 (7):191.
Chicago/Turabian StyleJosé O. Maldonado; Isaac Angert; Sheng Cao; Serkan Berk; Wei Zhang; Joachim D. Mueller; Louis M. Mansky. 2017. "Perturbation of Human T-Cell Leukemia Virus Type 1 Particle Morphology by Differential Gag Co-Packaging." Viruses 9, no. 7: 191.
The capsid domain (CA) of the retroviral Gag protein is a primary determinant of Gag oligomerization, which is a critical step for immature Gag lattice formation and virus particle budding. Although the human immunodeficiency virus type 1 (HIV-1) CA carboxy-terminal domain (CTD) is essential for CA-CA interactions, the CA CTD has been suggested to be largely dispensable for human T-cell leukemia virus type 1 (HTLV-1) particle biogenesis. To more clearly define the roles of the HTLV-1 CA amino-terminal domain (NTD) and CA CTD in particle biogenesis, we generated and analyzed a panel of Gag proteins with chimeric HIV-1/HTLV-1 CA domains. Subcellular distribution and protein expression levels indicated that Gag proteins with a chimeric HIV-1 CA NTD/HTLV-1 CA CTD did not result in Gag oligomerization regardless of the parent Gag background. Furthermore, chimeric Gag proteins with the HTLV-1 CA NTD produced particles phenotypically similar to HTLV-1 immature particles, highlighting the importance of the HTLV-1 CA NTD in HTLV-1 immature particle morphology. Taken together, these observations support the conclusion that the HTLV-1 CA NTD can functionally replace the HIV-1 CA CTD, but the HIV-1 CA NTD cannot replace the HTLV-1 CA CTD, indicating that the HTLV-1 CA subdomains provide distinct contributions to Gag-Gag oligomerization, particle morphology, and biogenesis. Furthermore, we have shown for the first time that HIV-1 and HTLV-1 Gag domains outside the CA (e.g., matrix and nucleocapsid) impact Gag oligomerization as well as immature particle size and morphology. IMPORTANCE A key aspect in virus replication is virus particle assembly, which is a poorly understood process for most viruses. For retroviruses, the Gag structural protein is the primary driver of virus particle biogenesis, and the CA CTD is the primary determinant of Gag-Gag interactions for HIV-1. In this study, the HTLV-1 capsid amino-terminal domain was found to provide distinct contributions to Gag-Gag oligomerization, particle morphology, and biogenesis. This study provides information that will aid efforts for discovery of therapeutic targets for intervention.
Jessica L. Martin; Luiza M. Mendonça; Isaac Angert; Joachim D. Mueller; Wei Zhang; Louis M. Mansky. Disparate Contributions of Human Retrovirus Capsid Subdomains to Gag-Gag Oligomerization, Virus Morphology, and Particle Biogenesis. Journal of Virology 2017, 91, e00298-17 .
AMA StyleJessica L. Martin, Luiza M. Mendonça, Isaac Angert, Joachim D. Mueller, Wei Zhang, Louis M. Mansky. Disparate Contributions of Human Retrovirus Capsid Subdomains to Gag-Gag Oligomerization, Virus Morphology, and Particle Biogenesis. Journal of Virology. 2017; 91 (14):e00298-17.
Chicago/Turabian StyleJessica L. Martin; Luiza M. Mendonça; Isaac Angert; Joachim D. Mueller; Wei Zhang; Louis M. Mansky. 2017. "Disparate Contributions of Human Retrovirus Capsid Subdomains to Gag-Gag Oligomerization, Virus Morphology, and Particle Biogenesis." Journal of Virology 91, no. 14: e00298-17.
A long-standing question of human immunodeficiency virus (HIV) genetic variation and evolution has been whether differences exist in mutation rate and/or mutation spectra among HIV types (i.e., HIV-1 versus HIV-2) and among HIV groups (i.e., HIV-1 groups M–P and HIV-2 groups A–H) and HIV-1 Group M subtypes (i.e., subtypes A–D, F–H, and J–K). To address this, we developed a new single-strand consensus sequencing assay for the determination of HIV mutation frequencies and spectra using the Illumina sequencing platform. This assay enables parallel and standardized comparison of HIV mutagenesis among various viral vectors with lower background error than traditional methods of Illumina library preparation. We found significant differences in viral mutagenesis between HIV types but intriguingly no significant differences among HIV-1 Group M subtypes. More specifically, HIV-1 exhibited higher transition frequencies than HIV-2, due mostly to single G-to-A mutations and (to a lesser extent) G-to-A hypermutation. These data suggest that HIV-2 RT exhibits higher fidelity during viral replication, and taken together, these findings demonstrate that HIV type but not subtype significantly affects viral mutation frequencies and spectra. These differences may inform antiviral and vaccine strategies.
Jonathan M.O. Rawson; Daryl M. Gohl; Sean R. Landman; Megan E. Roth; Morgan E. Meissner; Tara S. Peterson; James S. Hodges; Kenneth B. Beckman; Louis M. Mansky. Single-Strand Consensus Sequencing Reveals that HIV Type but not Subtype Significantly Impacts Viral Mutation Frequencies and Spectra. Journal of Molecular Biology 2017, 429, 2290 -2307.
AMA StyleJonathan M.O. Rawson, Daryl M. Gohl, Sean R. Landman, Megan E. Roth, Morgan E. Meissner, Tara S. Peterson, James S. Hodges, Kenneth B. Beckman, Louis M. Mansky. Single-Strand Consensus Sequencing Reveals that HIV Type but not Subtype Significantly Impacts Viral Mutation Frequencies and Spectra. Journal of Molecular Biology. 2017; 429 (15):2290-2307.
Chicago/Turabian StyleJonathan M.O. Rawson; Daryl M. Gohl; Sean R. Landman; Megan E. Roth; Morgan E. Meissner; Tara S. Peterson; James S. Hodges; Kenneth B. Beckman; Louis M. Mansky. 2017. "Single-Strand Consensus Sequencing Reveals that HIV Type but not Subtype Significantly Impacts Viral Mutation Frequencies and Spectra." Journal of Molecular Biology 429, no. 15: 2290-2307.
The Gag protein is the main retroviral structural protein, and its expression alone is usually sufficient for production of virus-like particles (VLPs). In this study, we sought to investigate—in parallel comparative analyses—Gag cellular distribution, VLP size, and basic morphological features using Gag expression constructs (Gag or Gag-YFP, where YFP is yellow fluorescent protein) created from all representative retroviral genera: Alpharetrovirus , Betaretrovirus , Deltaretrovirus , Epsilonretrovirus , Gammaretrovirus , Lentivirus , and Spumavirus . We analyzed Gag cellular distribution by confocal microscopy, VLP budding by thin-section transmission electron microscopy (TEM), and general morphological features of the VLPs by cryogenic transmission electron microscopy (cryo-TEM). Punctate Gag was observed near the plasma membrane for all Gag constructs tested except for the representative Beta - and Epsilonretrovirus Gag proteins. This is the first report of Epsilonretrovirus Gag localizing to the nucleus of HeLa cells. While VLPs were not produced by the representative Beta - and Epsilonretrovirus Gag proteins, the other Gag proteins produced VLPs as confirmed by TEM, and morphological differences were observed by cryo-TEM. In particular, we observed Deltaretrovirus -like particles with flat regions of electron density that did not follow viral membrane curvature, Lentivirus -like particles with a narrow range and consistent electron density, suggesting a tightly packed Gag lattice, and Spumavirus -like particles with large envelope protein spikes and no visible electron density associated with a Gag lattice. Taken together, these parallel comparative analyses demonstrate for the first time the distinct morphological features that exist among retrovirus-like particles. Investigation of these differences will provide greater insights into the retroviral assembly pathway. IMPORTANCE Comparative analysis among retroviruses has been critically important in enhancing our understanding of retroviral replication and pathogenesis, including that of important human pathogens such as human T-cell leukemia virus type 1 (HTLV-1) and HIV-1. In this study, parallel comparative analyses have been used to study Gag expression and virus-like particle morphology among representative retroviruses in the known retroviral genera. Distinct differences were observed, which enhances current knowledge of the retroviral assembly pathway.
Jessica L. Martin; Sheng Cao; Jose O. Maldonado; Wei Zhang; Louis M. Mansky. Distinct Particle Morphologies Revealed through Comparative Parallel Analyses of Retrovirus-Like Particles. Journal of Virology 2016, 90, 8074 -8084.
AMA StyleJessica L. Martin, Sheng Cao, Jose O. Maldonado, Wei Zhang, Louis M. Mansky. Distinct Particle Morphologies Revealed through Comparative Parallel Analyses of Retrovirus-Like Particles. Journal of Virology. 2016; 90 (18):8074-8084.
Chicago/Turabian StyleJessica L. Martin; Sheng Cao; Jose O. Maldonado; Wei Zhang; Louis M. Mansky. 2016. "Distinct Particle Morphologies Revealed through Comparative Parallel Analyses of Retrovirus-Like Particles." Journal of Virology 90, no. 18: 8074-8084.
Although many compounds have been approved for the treatment of human immunodeficiency type-1 (HIV-1) infection, additional anti-HIV-1 drugs (particularly those belonging to new drug classes) are still needed due to issues such as long-term drug-associated toxicities, transmission of drug-resistant variants, and development of multi-class resistance. Lethal mutagenesis represents an antiviral strategy that has not yet been clinically translated for HIV-1 and is based on the use of small molecules to induce excessive levels of deleterious mutations within the viral genome. Here, we show that 5-azacytidine (5-aza-C), a ribonucleoside analog that induces the lethal mutagenesis of HIV-1, and multiple inhibitors of the enzyme ribonucleotide reductase (RNR) interact in a synergistic fashion to more effectively reduce the infectivity of HIV-1. In these drug combinations, RNR inhibitors failed to significantly inhibit the conversion of 5-aza-C to 5-aza-2’-deoxycytidine, suggesting that 5-aza-C acts primarily as a deoxyribonucleoside even in the presence of RNR inhibitors. The mechanism of antiviral synergy was further investigated for the combination of 5-aza-C and one specific RNR inhibitor, resveratrol, as this combination improved the selectivity index of 5-aza-C to the greatest extent. Antiviral synergy was found to be primarily due to the reduced accumulation of reverse transcription products rather than the enhancement of viral mutagenesis. To our knowledge, these observations represent the first demonstration of antiretroviral synergy between a ribonucleoside analog and RNR inhibitors, and encourage the development of additional ribonucleoside analogs and RNR inhibitors with improved antiretroviral activity.
Jonathan M.O. Rawson; Megan E. Roth; Jiashu Xie; Michele B. Daly; Christine L. Clouser; Sean R. Landman; Cavan S. Reilly; Laurent Bonnac; Baek Kim; Steven E. Patterson; Louis M. Mansky. Synergistic reduction of HIV-1 infectivity by 5-azacytidine and inhibitors of ribonucleotide reductase. Bioorganic & Medicinal Chemistry 2016, 24, 2410 -2422.
AMA StyleJonathan M.O. Rawson, Megan E. Roth, Jiashu Xie, Michele B. Daly, Christine L. Clouser, Sean R. Landman, Cavan S. Reilly, Laurent Bonnac, Baek Kim, Steven E. Patterson, Louis M. Mansky. Synergistic reduction of HIV-1 infectivity by 5-azacytidine and inhibitors of ribonucleotide reductase. Bioorganic & Medicinal Chemistry. 2016; 24 (11):2410-2422.
Chicago/Turabian StyleJonathan M.O. Rawson; Megan E. Roth; Jiashu Xie; Michele B. Daly; Christine L. Clouser; Sean R. Landman; Cavan S. Reilly; Laurent Bonnac; Baek Kim; Steven E. Patterson; Louis M. Mansky. 2016. "Synergistic reduction of HIV-1 infectivity by 5-azacytidine and inhibitors of ribonucleotide reductase." Bioorganic & Medicinal Chemistry 24, no. 11: 2410-2422.
The Gag polyprotein is the main retroviral structural protein and is essential for the assembly and release of virus particles. In this study, we have analyzed the morphology and Gag stoichiometry of human T-cell leukemia virus type 1 (HTLV-1)-like particles and authentic, mature HTLV-1 particles by using cryogenic transmission electron microscopy (cryo-TEM) and scanning transmission electron microscopy (STEM). HTLV-1-like particles mimicked the morphology of immature authentic HTLV-1 virions. Importantly, we have observed for the first time that the morphology of these virus-like particles (VLPs) has the unique local feature of a flat Gag lattice that does not follow the curvature of the viral membrane, resulting in an enlarged distance between the Gag lattice and the viral membrane. Other morphological features that have been previously observed with other retroviruses include: (1) a Gag lattice with multiple discontinuities; (2) membrane regions associated with the Gag lattice that exhibited a string of bead-like densities at the inner leaflet; and (3) an arrangement of the Gag lattice resembling a railroad track. Measurement of the average size and mass of VLPs and authentic HTLV-1 particles suggested a consistent range of size and Gag copy numbers in these two groups of particles. The unique local flat Gag lattice morphological feature observed suggests that HTLV-1 Gag could be arranged in a lattice structure that is distinct from that of other retroviruses characterized to date.
José O. Maldonado; Sheng Cao; Wei Zhang; Louis M. Mansky. Distinct Morphology of Human T-Cell Leukemia Virus Type 1-Like Particles. Viruses 2016, 8, 132 .
AMA StyleJosé O. Maldonado, Sheng Cao, Wei Zhang, Louis M. Mansky. Distinct Morphology of Human T-Cell Leukemia Virus Type 1-Like Particles. Viruses. 2016; 8 (5):132.
Chicago/Turabian StyleJosé O. Maldonado; Sheng Cao; Wei Zhang; Louis M. Mansky. 2016. "Distinct Morphology of Human T-Cell Leukemia Virus Type 1-Like Particles." Viruses 8, no. 5: 132.
5-Azacytidine (5-aza-C) is a ribonucleoside analog that induces the lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1) by causing predominantly G-to-C transversions during reverse transcription. 5-Aza-C could potentially act primarily as a ribonucleotide (5-aza-CTP) or as a deoxyribonucleotide (5-aza-2′-deoxycytidine triphosphate [5-aza-dCTP]) during reverse transcription. In order to determine the primary form of 5-aza-C that is active against HIV-1, Illumina sequencing was performed using proviral DNA from cells treated with 5-aza-C or 5-aza-dC. 5-Aza-C and 5-aza-dC were found to induce highly similar patterns of mutation in HIV-1 in terms of the types of mutations observed, the magnitudes of effects, and the distributions of mutations at individual sequence positions. Further, 5-aza-dCTP was detected by liquid chromatography–tandem mass spectrometry in cells treated with 5-aza-C, demonstrating that 5-aza-C was a substrate for ribonucleotide reductase. Notably, levels of 5-aza-dCTP were similar in cells treated with equivalent effective concentrations of 5-aza-C or 5-aza-dC. Lastly, HIV-1 reverse transcriptase was found to incorporate 5-aza-CTP in vitro at least 10,000-fold less efficiently than 5-aza-dCTP. Taken together, these data support the model that 5-aza-C enhances the mutagenesis of HIV-1 primarily after reduction to 5-aza-dC, which can then be incorporated during reverse transcription and lead to G-to-C hypermutation. These findings may have important implications for the design of new ribonucleoside analogs directed against retroviruses.
Jonathan M. O. Rawson; Michele B. Daly; Jiashu Xie; Christine L. Clouser; Sean R. Landman; Cavan S. Reilly; Laurent Bonnac; Baek Kim; Steven E. Patterson; Louis M. Mansky. 5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2′-Deoxycytidine. Antimicrobial Agents and Chemotherapy 2016, 60, 2318 -2325.
AMA StyleJonathan M. O. Rawson, Michele B. Daly, Jiashu Xie, Christine L. Clouser, Sean R. Landman, Cavan S. Reilly, Laurent Bonnac, Baek Kim, Steven E. Patterson, Louis M. Mansky. 5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2′-Deoxycytidine. Antimicrobial Agents and Chemotherapy. 2016; 60 (4):2318-2325.
Chicago/Turabian StyleJonathan M. O. Rawson; Michele B. Daly; Jiashu Xie; Christine L. Clouser; Sean R. Landman; Cavan S. Reilly; Laurent Bonnac; Baek Kim; Steven E. Patterson; Louis M. Mansky. 2016. "5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2′-Deoxycytidine." Antimicrobial Agents and Chemotherapy 60, no. 4: 2318-2325.
HIV-1 replication kinetics inherently depends on the availability of cellular dNTPs for viral DNA synthesis. In activated CD4(+) T cells and other rapidly dividing cells, the concentrations of dNTPs are high and HIV-1 reverse transcription occurs in an efficient manner. In contrast, nondividing cells such as macrophages have lower dNTP pools, which restricts efficient reverse transcription. Clofarabine is an FDA approved ribonucleotide reductase inhibitor, which has shown potent antiretroviral activity in transformed cell lines. Here, we explore the potency, toxicity and mechanism of action of clofarabine in the human primary HIV-1 target cells: activated CD4(+) T cells and macrophages. Clofarabine is a potent HIV-1 inhibitor in both activated CD4(+) T cells and macrophages. Due to its minimal toxicity in macrophages, clofarabine displays a selectivity index over 300 in this nondividing cell type. The anti-HIV-1 activity of clofarabine correlated with a significant decrease in both cellular dNTP levels and viral DNA synthesis. Additionally, we observed that clofarabine triphosphate was directly incorporated into DNA by HIV-1 reverse transcriptase and blocked processive DNA synthesis, particularly at the low dNTP levels found in macrophages. Taken together, these data provide strong mechanistic evidence that clofarabine is a dual action inhibitor of HIV-1 replication that both limits dNTP substrates for viral DNA synthesis and directly inhibits the DNA polymerase activity of HIV-1 reverse transcriptase.
Michele B. Daly; Megan E. Roth; Laurent Bonnac; José O. Maldonado; Jiashu Xie; Christine L. Clouser; Steven E. Patterson; Baek Kim; Louis M. Mansky. Dual anti-HIV mechanism of clofarabine. Retrovirology 2016, 13, 20 .
AMA StyleMichele B. Daly, Megan E. Roth, Laurent Bonnac, José O. Maldonado, Jiashu Xie, Christine L. Clouser, Steven E. Patterson, Baek Kim, Louis M. Mansky. Dual anti-HIV mechanism of clofarabine. Retrovirology. 2016; 13 (1):20.
Chicago/Turabian StyleMichele B. Daly; Megan E. Roth; Laurent Bonnac; José O. Maldonado; Jiashu Xie; Christine L. Clouser; Steven E. Patterson; Baek Kim; Louis M. Mansky. 2016. "Dual anti-HIV mechanism of clofarabine." Retrovirology 13, no. 1: 20.