Mechanisms of viral capsid assembly around a polymer

Capsids of many viruses assemble around nucleic acids or other polymers. Understanding how the properties of the packaged polymer affect the assembly process could promote biomedical efforts to prevent viral assembly or nanomaterials applications that exploit assembly. To this end, we simulate on a lattice the dynamical assembly of closed, hollow shells composed of several hundred to 1000 subunits, around a flexible polymer. We find that assembly is most efficient at an optimum polymer length that scales with the surface area of the capsid; significantly longer than optimal polymers often lead to partial-capsids with unpackaged polymer `tails' or a competition between multiple partial-capsids attached to a single polymer. These predictions can be tested with bulk experiments in which capsid proteins assemble around homopolymeric RNA or synthetic polyelectrolytes. We also find that the polymer can increase the net rate of subunit accretion to a growing capsid both by stabilizing the addition of new subunits and by enhancing the incoming flux of subunits; the effects of these processes may be distinguishable with experiments that monitor the assembly of individual capsids.Comment: 7 figure

HIV-1 Vpu Promotes Release and Prevents Endocytosis of Nascent Retrovirus Particles from the Plasma Membrane

The human immunodeficiency virus (HIV) type-1 viral protein U (Vpu) protein enhances the release of diverse retroviruses from human, but not monkey, cells and is thought to do so by ablating a dominant restriction to particle release. Here, we determined how Vpu expression affects the subcellular distribution of HIV-1 and murine leukemia virus (MLV) Gag proteins in human cells where Vpu is, or is not, required for efficient particle release. In HeLa cells, where Vpu enhances HIV-1 and MLV release approximately 10-fold, concentrations of HIV-1 Gag and MLV Gag fused to cyan fluorescent protein (CFP) were initially detected at the plasma membrane, but then accumulated over time in early and late endosomes. Endosomal accumulation of Gag-CFP was prevented by Vpu expression and, importantly, inhibition of plasma membrane to early endosome transport by dominant negative mutants of Rab5a, dynamin, and EPS-15. Additionally, accumulation of both HIV and MLV Gag in endosomes required a functional late-budding domain. In human HOS cells, where HIV-1 and MLV release was efficient even in the absence of Vpu, Gag proteins were localized predominantly at the plasma membrane, irrespective of Vpu expression or manipulation of endocytic transport. While these data indicated that Vpu inhibits nascent virion endocytosis, Vpu did not affect transferrin endocytosis. Moreover, inhibition of endocytosis did not restore Vpu-defective HIV-1 release in HeLa cells, but instead resulted in accumulation of mature virions that could be released from the cell surface by protease treatment. Thus, these findings suggest that a specific activity that is present in HeLa cells, but not in HOS cells, and is counteracted by Vpu, traps assembled retrovirus particles at the cell surface. This entrapment leads to subsequent endocytosis by a Rab5a- and clathrin-dependent mechanism and intracellular sequestration of virions in endosomes

A facile quantitative assay for viral particle genesis reveals cooperativity in virion assembly and saturation of an antiviral protein

Conventional assays of viral particle assembly and release are time consuming and laborious. We have developed an enzymatic virus-like particle (VLP) genesis assay that rapid and quantitative and is also versatile and applicable to diverse viruses including HIV-1 and Ebola virus. Using this assay, which has a dynamic range of several orders of magnitude, we show that the efficiency of VLP assembly and release, i.e., the fraction of the expressed protein that is assembled into extracellular particles, is dependent on the absolute level of expression of either HIV-1 Gag or Ebola virus VP40. We also demonstrate that the activity of the antiviral factor tetherin is dependent on the level of HIV-1 Gag expression and the numbers of VLPs generated, and appears to become saturated as these parameters are increased

Understanding the Concentration Dependence of Viral Capsid Assembly Kinetics - the Origin of the Lag Time and Identifying the Critical Nucleus Size

The kinetics for the assembly of viral proteins into a population of capsids can be measured in vitro with size exclusion chromatography or dynamic light scattering, but extracting mechanistic information from these studies is challenging. For example, it is not straightforward to determine the critical nucleus size or the elongation time (the time required for a nucleated partial capsid to grow completion). We show that, for two theoretical models of capsid assembly, the critical nucleus size can be determined from the concentration dependence of the assembly reaction half-life and the elongation time is revealed by the length of the lag phase. Furthermore, we find that the system becomes kinetically trapped when nucleation becomes fast compared to elongation. Implications of this constraint for determining elongation mechanisms from experimental assembly data are discussed.Comment: Submitted to Biophysical Journa

A genome-wide data assessment of the African lion (Panthera leo) population genetic structure and diversity in Tanzania

The African lion (Panthera leo), listed as a vulnerable species on the IUCN Red List of Threatened Species (Appendix II of CITES), is mainly impacted by indiscriminate killing and prey base depletion. Additionally, habitat loss by land degradation and conversion has led to the isolation of some subpopulations, potentially decreasing gene flow and increasing inbreeding depression risks. Genetic drift resulting from weakened connectivity between strongholds can affect the genetic health of the species. In the present study, we investigated the evolutionary history of the species at different spatiotemporal scales. Therefore, the mitochondrial cytochrome b gene (N = 128), 11 microsatellites (N = 103) and 9,103 SNPs (N = 66) were investigated in the present study, including a large sampling from Tanzania, which hosts the largest lion population among all African lion range countries. Our results add support that the species is structured into two lineages at the continental scale (West-Central vs East-Southern), underlining the importance of reviewing the taxonomic status of the African lion. Moreover, SNPs led to the identification of three lion clusters in Tanzania, whose geographical distributions are in the northern, southern and western regions. Furthermore, Tanzanian lion populations were shown to display good levels of genetic diversity with limited signs of inbreeding. However, their population sizes seem to have gradually decreased in recent decades. The highlighted Tanzanian African lion population genetic differentiation appears to have resulted from the combined effects of anthropogenic pressure and environmental/climatic factors, as further discussed

Quantitative Live-Cell Imaging of Human Immunodeficiency Virus (HIV-1) Assembly

Advances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site

HIV-1 Infection of T cells and macrophages are differentially modulated by virion-associated Hck: a nef-dependent phenomenon

The proline repeat motif (PxxP) of Nef is required for interaction with the SH3 domains of macrophage-specific Src kinase Hck. However, the implication of this interaction for viral replication and infectivity in macrophages and T lymphocytes remains unclear. Experiments in HIV-1 infected macrophages confirmed the presence of a Nef:Hck complex which was dependent on the Nef proline repeat motif. The proline repeat motif of Nef also enhanced both HIV-1 infection and replication in macrophages, and was required for incorporation of Hck into viral particles. Unexpectedly, wild-type Hck inhibited infection of macrophages, but Hck was shown to enhance infection of primary T lymphocytes. These results indicate that the interaction between Nef and Hck is important for Nef-dependent modulation of viral infectivity. Hck-dependent enhancement of HIV-1 infection of T cells suggests that Nef-Hck interaction may contribute to the spread of HIV-1 infection from macrophages to T cells by modulating events in the producer cell, virion and target cell

The RING-CH ligase K5 antagonizes restriction of KSHV and HIV-1 particle release by mediating ubiquitin-dependent endosomal degradation of tetherin

Tetherin (CD317/BST2) is an interferon-induced membrane protein that inhibits the release of diverse enveloped viral particles. Several mammalian viruses have evolved countermeasures that inactivate tetherin, with the prototype being the HIV-1 Vpu protein. Here we show that the human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) is sensitive to tetherin restriction and its activity is counteracted by the KSHV encoded RING-CH E3 ubiquitin ligase K5. Tetherin expression in KSHV-infected cells inhibits viral particle release, as does depletion of K5 protein using RNA interference. K5 induces a species-specific downregulation of human tetherin from the cell surface followed by its endosomal degradation. We show that K5 targets a single lysine (K18) in the cytoplasmic tail of tetherin for ubiquitination, leading to relocalization of tetherin to CD63-positive endosomal compartments. Tetherin degradation is dependent on ESCRT-mediated endosomal sorting, but does not require a tyrosine-based sorting signal in the tetherin cytoplasmic tail. Importantly, we also show that the ability of K5 to substitute for Vpu in HIV-1 release is entirely dependent on K18 and the RING-CH domain of K5. By contrast, while Vpu induces ubiquitination of tetherin cytoplasmic tail lysine residues, mutation of these positions has no effect on its antagonism of tetherin function, and residual tetherin is associated with the trans-Golgi network (TGN) in Vpu-expressing cells. Taken together our results demonstrate that K5 is a mechanistically distinct viral countermeasure to tetherin-mediated restriction, and that herpesvirus particle release is sensitive to this mode of antiviral inhibition

Dimerization of Tetherin Is Not Essential for Its Antiviral Activity against Lassa and Marburg Viruses

Tetherin (also known as BST2, CD317 or HM1.24) has recently been reported to inhibit a wide range of viruses. However, the antiviral mechanism of action of tetherin has not been determined. Both ends of the tetherin molecule are associated with the plasma membrane and it forms a homodimer. Therefore, a model in which progeny virions are retained on the cell surface by dimer formation between tetherin molecules on the viral envelope and plasma membrane has been proposed as the antiviral mechanism of action of this molecule. To investigate this possibility, we examined the correlation between dimerization and antiviral activity of tetherin in Lassa and Marburg virus-like particle production systems using tetherin mutants deficient in dimer formation. However, the tetherin mutant with complete loss of dimerization activity still showed apparent antiviral activity, indicating that dimerization of tetherin is not essential for its antiviral activity. This suggests that tetherin retains progeny virions on the cell surface by a mechanism other than dimerization

Analysis of the Initiating Events in HIV-1 Particle Assembly and Genome Packaging

HIV-1 Gag drives a number of events during the genesis of virions and is the only viral protein required for the assembly of virus-like particles in vitro and in cells. Although a reasonable understanding of the processes that accompany the later stages of HIV-1 assembly has accrued, events that occur at the initiation of assembly are less well defined. In this regard, important uncertainties include where in the cell Gag first multimerizes and interacts with the viral RNA, and whether Gag-RNA interaction requires or induces Gag multimerization in a living cell. To address these questions, we developed assays in which protein crosslinking and RNA/protein co-immunoprecipitation were coupled with membrane flotation analyses in transfected or infected cells. We found that interaction between Gag and viral RNA occurred in the cytoplasm and was independent of the ability of Gag to localize to the plasma membrane. However, Gag:RNA binding was stabilized by the C-terminal domain (CTD) of capsid (CA), which participates in Gag-Gag interactions. We also found that Gag was present as monomers and low-order multimers (e.g. dimers) but did not form higher-order multimers in the cytoplasm. Rather, high-order multimers formed only at the plasma membrane and required the presence of a membrane-binding signal, but not a Gag domain (the CA-CTD) that is essential for complete particle assembly. Finally, sequential RNA-immunoprecipitation assays indicated that at least a fraction of Gag molecules can form multimers on viral genomes in the cytoplasm. Taken together, our results suggest that HIV-1 particle assembly is initiated by the interaction between Gag and viral RNA in the cytoplasm and that this initial Gag-RNA encounter involves Gag monomers or low order multimers. These interactions per se do not induce or require high-order Gag multimerization in the cytoplasm. Instead, membrane interactions are necessary for higher order Gag multimerization and subsequent particle assembly in cells