HIV-1 Gag co-opts a cellular complex containing DDX6, a helicase that facilitates capsid assembly.

The Journal of Cell Biology, Volume 198:439-56, 2012.
Reed JC, Molter B, Geary CD, McNevin J, McElrath J, Giri S, Klein KC, Lingappa JR.

ABSTRACT: To produce progeny virus, human immunodeficiency virus type I (HIV-1) Gag assembles into capsids that package the viral genome and bud from the infected cell. During assembly of immature capsids, Gag traffics through a pathway of assembly intermediates (AIs) that contain the cellular adenosine triphosphatase ABCE1 (ATP-binding cassette protein E1). In this paper, we showed by coimmunoprecipitation and immunoelectron microscopy (IEM) that these Gag-containing AIs also contain endogenous processing body (PB)-related proteins, including AGO2 and the ribonucleic acid (RNA) helicase DDX6. Moreover, we found a similar complex containing ABCE1 and PB proteins in uninfected cells. Additionally, knockdown and rescue studies demonstrated that the RNA helicase DDX6 acts enzymatically to facilitate capsid assembly independent of RNA packaging. Using IEM, we localized the defect in DDX6-depleted cells to Gag multimerization at the plasma membrane. We also confirmed that DDX6 depletion reduces production of infectious HIV-1 from primary human T cells. Thus, we propose that assembling HIV-1 co-opts a preexisting host complex containing cellular facilitators such as DDX6, which the virus uses to catalyze capsid assembly.


HIV Gag-leucine zipper chimeras form ABCE1-containing intermediates and RNase-resistant immature capsids similar to those formed by wild-type HIV-1 Gag.

The Journal of Virology, Volume 85:7419-35, 2011.
Klein KC, Reed JC, Tanaka M, Nguyen VT, Giri S, Lingappa JR.

ABSTRACT: During HIV-1 assembly, Gag polypeptides multimerize to form an immature capsid and also package HIV-1 genomic RNA. Assembling Gag forms immature capsids by progressing through a stepwise pathway of assembly intermediates containing the cellular ATPase ABCE1, which facilitates capsid formation. The NC domain of Gag is required for ABCE1 binding, acting either directly or indirectly. NC is also critical for Gag multimerization and RNA binding. Previous studies of GagZip chimeric proteins in which NC was replaced with a heterologous leucine zipper that promotes protein dimerization but not RNA binding established that the RNA binding properties of NC are dispensable for capsid formation per se. Here we utilized GagZip proteins to address the question of whether the RNA binding properties of NC are required for ABCE1 binding and for the formation of ABCE1-containing capsid assembly intermediates. We found that assembly-competent HIV-1 GagZip proteins formed ABCE1-containing intermediates, while assembly-incompetent HIV-1 GagZip proteins harboring mutations in residues critical for leucine zipper dimerization did not. Thus, these data suggest that ABCE1 does not bind to NC directly or through an RNA bridge, and they support a model in which dimerization of Gag, mediated by NC or a zipper, results in exposure of an ABCE1-binding domain located elsewhere in Gag, outside NC. Additionally, we demonstrated that immature capsids formed by GagZip proteins are insensitive to RNase A, as expected. However, unexpectedly, immature HIV-1 capsids were almost as insensitive to RNase A as GagZip capsids, suggesting that RNA is not a structural element holding together immature wild-type HIV-1 capsids.


Comparing Capsid Assembly of Primate Lentiviruses and Hepatitis B Virus Using Cell-Free Systems

Virology, Volume 333: 114-123, 2005
Jaisri R. Lingappa, Michael A. Newman, Kevin C. Klein, Julia E. Dooher

ABSTRACT: Many viruses that assemble their capsids in the eukaryotic cytoplasm require a threshold concentration of capsid protein to achieve capsid assembly. Strategies for achieving this include maintaining high levels of capsid protein synthesis and targeting to specific sites to raise the effective concentration of capsid polypeptides. To understand how different viruses achieve the threshold capsid protein concentration required for assembly, we used cell-free systems to compare capsid assembly of hepatitis B virus (HBV) and three primate lentiviruses. Capsid formation of these diverse viruses in a common eukaryotic extract was dependent on capsid protein concentration. HBV capsid assembly was also dependent on the presence of intact membrane surfaces. Surprisingly, not all of the primate lentiviral capsid proteins examined required myristoylation and intact membranes for assembly, even though all contain a myristoylation signal. These findings reveal significant diversity in how different capsid proteins assemble in the same cellular extract.


Identification of Residues in the Hepatitis C Virus Core Protein That Are Critical for Capsid Assembly in a Cell-Free System

Journal of Virology, Volume 79: 6814-6826, 2005
Kevin C. Klein, Sheri R. Dellos, and Jaisri R. Lingappa

ABSTRACT: Significant advances have been made in understanding hepatitis C virus (HCV) replication through development of replicon systems. However, neither replicon systems nor standard cell culture systems support significant assembly of HCV capsids, leaving a large gap in our knowledge of HCV virion formation. Recently, we established a cell-free system in which over 60% of full-length HCV core protein synthesized de novo in cell extracts assembles into HCV capsids by biochemical and morphological criteria. Here we used mutational analysis to identify residues in HCV core that are important for capsid assembly in this highly reproducible cell-free system. We found that basic residues present in two clusters within the N-terminal 68 amino acids of HCV core played a critical role, while the uncharged linker domain between them was not. Furthermore, the aspartate at position 111, the region spanning amino acids 82 to 102, and three serines that are thought to be sites of phosphorylation do not appear to be critical for HCV capsid formation in this system. Mutation of prolines important for targeting of core to lipid droplets also failed to alter HCV capsid assembly in the cell-free system. In addition, wild-type HCV core did not rescue assembly-defective mutants. These data constitute the first systematic and quantitative analysis of the roles of specific residues and domains of HCV core in capsid formation.


Recent Insights into Biological Regulation from Cell-Free Protein-Synthesizing Systems

The Mount Sinai Journal of Medicine, Volume 72: 141-160, 2005
Vishwanath R. Lingappa and Jaisri R. Lingappa

ABSTRACT: We review the important role that cell-free protein-synthesizing systems (CFPSS) have played in the history of modern biology, and highlight two recent applications that illustrate their continued utility for the exploration of otherwise intractable aspects of gene expression and its regulation. Viral capsid assembly recreated in CFPSS reveals a catalyzed biochemical pathway involving transient, energy-dependent action of host proteins and discrete assembly intermediates, rather than the classical notion of self-assembly that was expected for capsid formation. Study of prion protein biogenesis reveals a new conformation critical for disease pathogenesis and advances the paradigm of protein bioconformatics, by which cells may productively regulate the folding of various proteins. In each example, the CFPSS made it easier to analyze biochemical mechanism than is possible in other currently available whole cell systems, illustrating why this approach is likely to be a continuing source of insight into important features of biological regulation. Key Words: Protein synthesis, in vitro translation, biogenesis, protein folding, viral capsid, prion, wheat germ, reticulocyte lysate, translocation, endoplasmic reticulum, translocon.


Cell-Free Systems for Capsid Assembly of Primate Lentiviruses from Three Different Lineages

The Journal of Medical Primatology, Volume 33: 272-280, 2004
Julia E. Dooher, Jaisri R. Lingappa

ABSTRACT: We recently demonstrated that capsids from three main primate lentiviral lineages appear to form via a pathway of assembly intermediates in primate cells. Retroviral capsid assembly intermediates were initially identified and characterized using a cell-free system for assembly of immature HIV-1 capsids. Because cell-free capsid assembly systems are useful tools, we are interested in developing such systems for other primate lentiviruses besides HIV-1. Here we extend previous cell-free studies by showing that Gag proteins of HIV-2, from a second primate lentiviral lineage, progress from early intermediates to late intermediates and completed capsids over time. Additionally, we demonstrate that Gag proteins of SIVagm, from a third primate lentiviral lineage, associate with the cellular factor HP68 and complete assembly in this system. Therefore, cell-free systems reproduce assembly of Gag from three main primate lentiviral lineages, and can be used to compare mechanistic features of capsid assembly of genetically divergent primate lentiviruses.


Unique Features of Hepatitis C Virus Capsid Formation Revealed by De Novo Cell-Free Assembly

Journal of Virology, Volume 78: 9257-9269, 2004
Kevin C. Klein, Stephen J. Polyak, and Jaisri R. Lingappa

ABSTRACT: The assembly of hepatitis C virus (HCV) is poorly understood, largely due to the lack of mammalian cell culture systems that are easily manipulated and produce high titers of virus. This problem is highlighted by the inability of the recently established HCV replicon systems to support HCV capsid assembly despite high levels of structural protein synthesis. Here we demonstrate that up to 80% of HCV core protein synthesized de novo in cell-free systems containing rabbit reticulocyte lysate or wheat germ extracts assembles into HCV capsids. This contrasts with standard primate cell culture systems, in which almost no core assembles into capsids. Cell-free HCV capsids, which have a sedimentation value of 100S, have a buoyant density (1.28 g/ml) on cesium chloride similar to that of HCV capsids from other systems. Capsids produced in cell-free systems are also indistinguishable from capsids isolated from HCV-infected patient serum when analyzed by transmission electron microscopy. Using these cell-free systems, we show that HCV capsid assembly is independent of signal sequence cleavage, is dependent on the N terminus but not the C terminus of HCV core, proceeds at very low nascent chain concentrations, is independent of intact membrane surfaces, and is partially inhibited by cultured liver cell lysates. By allowing reproducible and quantitative assessment of viral and cellular requirements for capsid formation, these cell-free systems make a mechanistic dissection of HCV capsid assembly possible.


Identification of a Host Protein Essential for Assembly of Immature HIV-1 Capsids

Nature, Volume 415: 88-92, 2002
Concepcion Zimmerman, Kevin C. Klein, Patti K. Kiser, Aalok R. Singh, Bonnie L. Firestein, Shannyn C. Riba, and Jaisri R. Lingappa

ABSTRACT: To form an immature HIV-1 capsid, 1,500 HIV-1 Gag (p55) polypeptides must assemble properly along the host cell plasma membrane. Insect cells and many higher eukaryotic cell types support efficient capsid assembly, but yeast and murine cells do not, indicating that host machinery is required for immature HIV-1 capsid formation. Additionally, in a cell-free system that reconstitutes HIV-1 capsid formation, post-translational assembly events require ATP and a subcellular fraction, suggesting a requirement for a cellular ATP-binding protein. Here we identify such a protein (HP68), described previously as an RNase L inhibitor, and demonstrate that it associates post-translationally with HIV-1 Gag in a cell-free system and human T cells infected with HIV-1. Using a dominant negative mutant of HP68 in mammalian cells and depletion-reconstitution experiments in the cell-free system, we demonstrate that HP68 is essential for post-translational events in immature HIV-1 capsid assembly. Furthermore, in cells the HP68-Gag complex is associated with HIV-1 Vif, which is involved in virion morphogenesis and infectivity. These findings support a critical role for HP68 in posttranslational events of HIV-1 assembly and reveal a previously unappreciated dimension of host-viral interaction.


Effect of Mutations in Gag on Assembly of Immature Human Immunodeficiency Virus Type 1 Capsids in a Cell-Free System

Virology, Volume 279: 257-270, 2001
Aalok R. Singh, Rebecca L. Hill, and Jaisri R. Lingappa

ABSTRACT: Studies of HIV-1 capsid formation in a cell-free system revealed that capsid assembly occurs via an ordered series of assembly intermediates and requires host machinery. Here we use this system to examine 12 mutations in HIV-1 Gag that others studied previously in intact cells. With respect to capsid formation, these mutations generally produced the same phenotype in the cell-free system as in cells, indicating the cell-free system’s high degree of fidelity. Analysis of assembly intermediates reveals that a mutation in the distal region of CA (322 LΔS) and truncations proximal to the second cys-his box in NC block multimerization of Gag at early stages in the cell-free capsid assembly pathway. In contrast, mutations in the region of amino acids 56–68 (located in the proximal portion of MA) inhibit assembly at a later point in the pathway. Other mutations, including truncations distal to the first cys-his box in NC and mutations in the distal half of MA (88HΔG, 85YΔG, Δ104–115, and Δ115–129), do not affect formation of immature capsids in the cell-free system. These data provide new information on the role of different domains in Gag during the early events of capsid assembly.


A Multistep, ATP-Dependent Pathway for Assembly of Human Immunodeficiency Virus Capsids in a Cell-Free System

The Journal of Cell Biology, Volume 136: 567-581, 1997
Jaisri R. Lingappa, Rebecca L. Hill, Mei Lie Wong, and Ramanujan S. Hegde

ABSTRACT: To understand the mechanism by which human immunodeficiency virus type 1 (HIV) capsids are formed, we have reconstituted the assembly of immature HIV capsids de novo in a cell-free system. Capsid authenticity is established by multiple biochemical and morphologic criteria. Known features of the assembly process are closely reproduced, indicating the fidelity of the cell-free reaction. Assembly is separated into co- and posttranslational phases, and three independent posttranslational requirements are demonstrated: (a) ATP, (b) a detergent-sensitive host factor, and (c) a detergent-insensitive host subcellular fraction that can be depleted and reconstituted. Assembly appears to proceed by way of multiple intermediates whose conversion to completed capsids can be blocked by either ATP depletion or treatment with nondenaturing detergent. Specific subsets of these intermediates accumulate upon expression of various assembly-defective Gag mutants in the cell-free system, suggesting that each mutant is blocked at a particular step in assembly. Furthermore, the accumulation of complexes of similar sizes in cells expressing the corresponding mutants suggests that comparable intermediates may exist in vivo. From these data, we propose a multi-step pathway for the biogenesis of HIV capsids, in which the assembly process can be disrupted at a number of discrete points.


A Eukaryotic Cytosolic Chaperonin Is Associated with a High Molecular Weight Intermediate in the Assembly of Hepatitis B Virus Capsid, a Multimeric Particle

The Journal of Cell Biology, Volume 125: 99-111, 1994
Jaisri R. Lingappa, Robert L. Martin, Mei Lie Wong, Don Ganem, William J. Welch, and Vishwanath R. Lingappa

ABSTRACT: We have established a system for assembly of hepatitis B virus capsid, a homomultimer of the viral core polypeptide, using cell-free transcriptionlinked translation. The mature particles that are produced are indistinguishable from authentic viral capsids by four criteria: velocity sedimentation, buoyant density, protease resistance, and electron microscopic appearance. Production of unassembled core polypeptides can be uncoupled from production of capsid particles by decreasing core mRNA concentration. Addition of excess unlabeled core polypeptides allows the chase of the unassembled polypeptides into mature capsids. Using this cell-free system, we demonstrate that assembly of capsids proceeds by way of a novel high molecular weight intermediate. Upon isolation, the high molecular weight intermediate is productive of mature capsids when energy substrates are manipulated. A 60-kD protein related to the chaperonin t-complex polypoptide 1 (TCP-1) is found in association with core polypeptides in two different assembly intermediates, but is not associated with either the initial unassembled polypeptides or with the final mature capsid product. These findings implicate TCP-1 or a related chaperonin in viral assembly and raise the possibility that eukaryotic cytosolic chaperonins may play a distinctive role in multimer assembly apart from their involvement in assisting monomer folding. {PDF}