Jaisri R. Lingappa

Antiviral Function of APOBEC3G Can Be Dissociated from Cytidine Deaminase Activity

The antiretroviral activity of the cellular enzyme APOBEC3G has been attributed to the excessive deamination of cytidine (C) to uridine (U) in minus strand reverse transcripts, a process resulting in guanosine (G) to adenosine (A) hypermutation of plus strand DNAs. The HIV-1 Vif protein counteracts APOBEC3G by inducing proteasomal degradation and exclusion from virions through recruitment of a cullin5 ECS E3 ubiquitin ligase complex. APOBEC3G belongs to the APOBEC protein family, members of which possess consensus (H/C)-(A/V)-E-(X)24-30-P-C-(X)2-C cytidine deaminase motifs. Earlier analyses of APOBEC-1 have defined specific residues that are important for zinc coordination, proton transfer, and, therefore, catalysis within this motif. Because APOBEC3G contains two such motifs, we used site-directed mutagenesis of conserved residues to assess each regions contribution to anti-HIV-1 activity. Surprisingly, whereas either the N- or C-terminal domain could confer antiviral function in tissue culture-based infectivity assays, only an intact C-terminal motif was essential for DNA mutator activity. These findings reveal the nonequivalency of APOBEC3Gs N- and C-terminal domains and imply that APOBEC3G-mediated DNA editing may not always be necessary for antiviral activity. Accordingly, we propose that APOBEC3G can achieve an anti-HIV-1 effect through an undescribed mechanism that is distinct from cytidine deamination.

Identification of a host protein essential for assembly of immature HIV-1 capsids

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 post-translational events of HIV-1 assembly and reveal a previously unappreciated dimension of host–viral interaction.

Basic Residues in the Nucleocapsid Domain of Gag Are Required for Interaction of HIV-1 Gag with ABCE1 (HP68), a Cellular Protein Important for HIV-1 Capsid Assembly

During human immunodeficiency virus, type 1 (HIV-1) assembly, Gag polypeptides multimerize into immature HIV-1 capsids. The cellular ATP-binding protein ABCE1 (also called HP68 or RNase L inhibitor) appears to be critical for proper assembly of the HIV-1 capsid. In primate cells, ABCE1 associates with Gag polypeptides present in immature capsid assembly intermediates. Here we demonstrate that the NC domain of Gag is critical for interaction with endogenous primate ABCE1, whereas other domains in Gag can be deleted without eliminating the association of Gag with ABCE1. NC contains two Cys-His boxes that form zinc finger motifs and are responsible for encapsidation of HIV-1 genomic RNA. In addition, NC contains basic residues known to play a critical role in nonspecific RNA binding, Gag-Gag interactions, and particle formation. We demonstrate that basic residues in NC are needed for the Gag-ABCE1 interaction, whereas the cysteine and histidine residues in the zinc fingers are dispensable. Constructs that fail to interact with primate ABCE1 or interact poorly also fail to form capsids and are arrested at an early point in the immature capsid assembly pathway. Whereas others have shown that basic residues in NC bind nonspecifically to RNA, which in turn scaffolds or nucleates assembly, our data demonstrate that the same basic residues in NC act either directly or indirectly to recruit a cellular protein that also promotes capsid formation. Thus, in cells, basic residues in NC appear to act by two mechanisms, recruiting both RNA and a cellular ATPase in order to facilitate efficient assembly of HIV-1 capsids.

Innate Immune Signaling Induces High Levels of TC-specific Deaminase Activity in Primary Monocyte-derived Cells through Expression of APOBEC3A Isoforms

In HIV-1-infected individuals, G-to-A hypermutation is found in HIV-1 DNA isolated from peripheral blood mononuclear cells (PBMCs). These mutations are thought to result from editing by one or more host enzymes in the APOBEC3 (A3) family of cytidine deaminases, which act on CC (APOBEC3G) and TC (other A3 proteins) dinucleotide motifs in DNA (edited cytidine underlined). Although many A3 proteins display high levels of deaminase activity in model systems, only low levels of A3 deaminase activity have been found in primary cells examined to date. In contrast, here we report high levels of deaminase activity at TC motifs when whole PBMCs or isolated primary monocyte-derived cells were treated with interferon-α (IFNα) or IFNα-inducing toll-like receptor ligands. Induction of TC-specific deaminase activity required new transcription and translation and correlated with the appearance of two APOBEC3A (A3A) isoforms. Knockdown of A3A in monocytes with siRNA abolished TC-specific deaminase activity, confirming that A3A isoforms are responsible for all TC-specific deaminase activity observed. Both A3A isoforms appear to be enzymatically active; moreover, our mutational studies raise the possibility that the smaller isoform results from internal translational initiation. In contrast to the high levels of TC-specific activity observed in IFNα-treated monocytes, CC-specific activity remained low in PBMCs, suggesting that A3G deaminase activity is relatively inhibited, unlike that of A3A. Together, these findings suggest that deaminase activity of A3A isoforms in monocytes and macrophages may play an important role in host defense against viruses.

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

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.

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

The RNA helicase DDX6 promotes HIV-1 assembly in a co-opted cellular complex containing P body proteins and ABCE1.

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

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.

Host ABCE1 is at plasma membrane HIV assembly sites and its dissociation from Gag is linked to subsequent events of virus production.

In primate cells, assembly of a single HIV‐1 capsid involves multimerization of thousands of Gag polypeptides, typically at the plasma membrane. Although studies support a model in which HIV‐1 assembly proceeds through complexes containing Gag and the cellular adenosine triphosphatase ABCE1 (also termed HP68 or ribonuclease L inhibitor), whether these complexes constitute true assembly intermediates remains controversial. Here we demonstrate by pulse labeling in primate cells that a population of Gag associates with endogenous ABCE1 within minutes of translation. In the next ∼2 h, Gag–ABCE1 complexes increase in size to approximately that of immature capsids. Dissociation of ABCE1 from Gag correlates closely with Gag processing during virion maturation and occurs much less efficiently when the HIV‐1 protease is inactivated. Finally, quantitative double‐label immunogold electron microscopy reveals that ABCE1 is recruited to sites of assembling wild‐type Gag at the plasma membrane but not to sites of an assembly‐defective Gag mutant at the plasma membrane. Together these findings demonstrate that a population of Gag present at plasma membrane sites of assembly associates with ABCE1 throughout capsid formation until the onset of virus maturation, which is then followed by virus release. Moreover, the data suggest a linkage between Gag–ABCE1 dissociation and subsequent events of virion production.

Conservation of a Stepwise, Energy-Sensitive Pathway Involving HP68 for Assembly of Primate Lentivirus Capsids in Cells

ABSTRACT Previously we have described a stepwise, energy-dependent pathway for human immunodeficiency virus type 1 (HIV-1) capsid assembly in a cell-free system. In this pathway, Gag polypeptides utilize the cellular factor HP68 and assemble into immature capsids by way of assembly intermediates that have defined biochemical characteristics. Here we address whether this pathway is universally conserved among primate lentiviruses and can be observed in mammalian cells. We demonstrate that HIV-2 Gag associates with human HP68 in a cell-free system and that Gag proteins of HIV-2, simian immunodeficiency virus SIVmac239, and SIVagm associate with endogenous HP68 in primate cells, as is seen for HIV-1. Analysis of primate cells expressing lentivirus Gag proteins revealed Gag-containing complexes with the same sedimentation values as seen for previously described HIV-1 assembly intermediates in the cell-free system (10S, 80-150S, and 500S). These complexes fit criteria for assembly intermediates as judged by energy sensitivity, pattern of HP68 association, and the failure of specific complexes to be formed by assembly-incompetent Gag mutants. We also demonstrate that virus-like particles released from cells do not appear to contain HP68, suggesting that HP68 is released from Gag upon completion of capsid assembly in cells, as was observed previously in the cell-free system. Together these findings support a model in which all primate lentivirus capsids assemble by a conserved pathway of HP68-containing, energy-dependent assembly intermediates that have specific biochemical features.

How HIV-1 Gag assembles in cells: Putting together pieces of the puzzle

During the late stage of the viral life cycle, HIV-1 Gag assembles into a spherical immature capsid, and undergoes budding, release, and maturation. Here we review events involved in immature capsid assembly from the perspective of five different approaches used to study this process: mutational analysis, structural studies, assembly of purified recombinant Gag, assembly of newly translated Gag in a cell-free system, and studies in cells using biochemical and imaging techniques. We summarize key findings obtained using each approach, point out where there is consensus, and highlight unanswered questions. Particular emphasis is placed on reconciling data suggesting that Gag assembles by two different paths, depending on the assembly environment. Specifically, in assembly systems that lack cellular proteins, high concentrations of Gag can spontaneously assemble using purified nucleic acid as a scaffold. However, in the more complex intracellular environment, barriers that limit self-assembly are present in the form of cellular proteins, organelles, host defenses, and the absence of free nucleic acid. To overcome these barriers and promote efficient immature capsid formation in an unfavorable environment, Gag appears to utilize an energy-dependent, host-catalyzed, pathway of assembly intermediates in cells. Overall, we show how data obtained using a variety of techniques has led to our current understanding of HIV assembly.