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ABSTRACT: The inner structural Gag proteins and the envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) traffic independently to the plasma membrane, where they assemble the nascent virion. HIV-1 carries a relatively low number of glycoproteins in its membrane, and the mechanism of Env recruitment and virus incorporation is incompletely understood. We employed dual-color super-resolution microscopy visualizing Gag assembly sites and HIV-1 Env proteins in virus-producing and in Env expressing cells. Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site. Formation of these Env clusters depended on the presence of other HIV-1 proteins and on the long cytoplasmic tail (CT) of Env. CT deletion, a matrix mutation affecting Env incorporation or Env expression in the absence of other HIV-1 proteins led to much smaller Env clusters, which were not enriched at viral assembly sites. These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env(ΔCT) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread . Keeping Env molecules on the nascent virus low may be important for escape from the humoral immune response, while cell-cell contacts mediated by surrounding Env molecules could promote HIV-1 transmission through the virological synapse.
PLoS Pathogens 02/2013; 9(2):e1003198. · 9.13 Impact Factor
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Cellular Microbiology 02/2013; 15(2):159-60. · 5.46 Impact Factor
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ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that obtains its lipid envelope by budding through the plasma membrane of infected host cells. Various studies indicated that the HIV-1 membrane differs from the producer cell plasma membrane suggesting virus budding from pre-existing subdomains or virus-mediated induction of a specialised budding membrane. To perform a comparative lipidomics analysis by quantitative mass spectrometry, we first evaluated two independent methods to isolate the cellular plasma membrane. Subsequent lipid analysis of plasma membranes and HIV-1 purified from two different cell lines revealed a significantly different lipid composition of the viral membrane compared to the host cell plasma membrane, independent of the cell type investigated. Virus particles were significantly enriched in phosphatidylserine, sphingomyelin, hexosylceramide and saturated phosphatidylcholine species when compared with the host cell plasma membrane of the producer cells; they showed reduced levels of unsaturated phosphatidylcholine species, phosphatidylethanolamine and phosphatidylinositol. Cell type-specific differences in the lipid composition of HIV-1 and donor plasma membranes were observed for plasmalogen-phosphatidylethanolamine and phosphatidylglycerol, which were strongly enriched only in HIV-1 derived from MT-4 cells. MT-4 cell-derived HIV-1 also contained dihydrosphingomyelin as reported previously, but this lipid class was also enriched in the host cell membrane. Taken together, these data strongly support the hypothesis that HIV-1 selects a specific lipid environment for its morphogenesis.
Cellular Microbiology 12/2012; · 5.46 Impact Factor
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ABSTRACT: The structural polyprotein Gag of human immunodeficiency virus (HIV-1) is necessary and sufficient for formation of virus like particles. Its C-terminal p6 domain harbors short peptide motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. p6 has been shown to be the major viral phosphoprotein in HIV-1 infected cells and virions, but the sites and functional relevance of p6 phosphorylation are not clear. Here, we identified phosphorylation of several serine and threonine residues in p6 in purified virus preparations using mass spectrometry. Mutation of individual candidate phosphoacceptor residues had no detectable effect on virus assembly, release and infectivity, however, suggesting that phosphorylation of single residues may not be functionally relevant. Therefore, a comprehensive mutational analysis was conducted exchanging all potentially phosphorylatable amino acids in p6, except for a threonine, which is part of an essential peptide motif. To avoid confounding changes in the overlapping pol reading frame, mutagenesis was performed in a provirus with genetically uncoupled gag and pol reading frames. An HIV-1 derivative carrying 12 amino acid exchanges in its p6 region, abolishing all but one potential phosphoacceptor sites, showed no impairment of Gag assembly and virus release and displayed only very subtle deficiencies in viral infectivity in T-cell lines and primary lymphocytes. All mutations were stable over two weeks of culture in primary cells. Based on these findings, we conclude that phosphorylation of p6 is dispensable for HIV-1 assembly, release and infectivity in tissue culture.
Journal of Virology 10/2012; · 5.40 Impact Factor
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ABSTRACT: The structural polyprotein Gag of human immunodeficiency virus (HIV-1) is necessary and sufficient for formation of virus like particles. Its C-terminal p6 domain harbors short peptide motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. p6 has been shown to be the major viral phosphoprotein in HIV-1 infected cells and virions, but the sites and functional relevance of p6 phosphorylation are not clear. Here, we identified phosphorylation of several serine and threonine residues in p6 in purified virus preparations using mass spectrometry. Mutation of individual candidate phosphoacceptor residues had no detectable effect on virus assembly, release and infectivity, however, suggesting that phosphorylation of single residues may not be functionally relevant. Therefore, a comprehensive mutational analysis was conducted exchanging all potentially phosphorylatable amino acids in p6, except for a threonine, which is part of an essential peptide motif. To avoid confounding changes in the overlapping pol reading frame, mutagenesis was performed in a provirus with genetically uncoupled gag and pol reading frames. An HIV-1 derivative carrying 12 amino acid exchanges in its p6 region, abolishing all but one potential phosphoacceptor sites, showed no impairment of Gag assembly and virus release and displayed only very subtle deficiencies in viral infectivity in T-cell lines and primary lymphocytes. All mutations were stable over two weeks of culture in primary cells. Based on these findings, we conclude that phosphorylation of p6 is dispensable for HIV-1 assembly, release and infectivity in tissue culture.
Journal of Virology 10/2012; · 5.40 Impact Factor
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ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) buds from the cell as an immature particle requiring subsequent proteolysis of the main structural polyprotein Gag for morphological maturation and infectivity. Visualization of the viral envelope (Env) glycoprotein distribution on the surface of individual HIV-1 particles with stimulated emission depletion (STED) superresolution fluorescence microscopy revealed maturation-induced clustering of Env proteins that depended on the Gag-interacting Env tail. Correlation of Env surface clustering with the viral entry efficiency revealed coupling between the viral interior and exterior: Rearrangements of the inner protein lattice facilitated the alteration of the virus surface in preparation for productive entry. We propose that Gag proteolysis-dependent clustering of the sparse Env trimers on the viral surface may be an essential aspect of HIV-1 maturation.
Science 10/2012; 338(6106):524-8. · 31.20 Impact Factor
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ABSTRACT: GS-8374 is a potent HIV protease inhibitor (PI) with a unique diethyl-phosphonate moiety. Due to a balanced contribution of enthalpic and entropic components to its interaction with the protease (PR) active site, the compound retains activity against HIV mutants with high-level multi-PI resistance. Here we report the in vitro selection and characterization of HIV variants resistant to GS-8374. While highly resistant viruses with multiple mutations in PR were isolated in the presence of control PIs, an HIV variant displaying moderate (14-fold) resistance to GS-8374 was generated only after prolonged passaging for >300 days . The isolate showed low-level cross-resistance to darunavir, atazanavir, lopinavir, and saquinavir, but not other PIs, and contained a single R41K mutation in PR combined with multiple genotypic changes in the Gag matrix, capsid, nucleocapsid, and SP2 domains. Mutations also occured in the trans-frame peptide and p6* domain of the Gag-Pol polyprotein. Analysis of recombinant HIV variants indicated that mutations in Gag, but not the R41K in PR, conferred reduced susceptibility to GS-8374. The Gag mutations acted in concert, as they did not affect susceptibility when introduced individually. Analysis of viral particles revealed that the mutations rendered Gag more susceptible to PR-mediated cleavage in the presence of GS-8374. In summary, the emergence of resistance to GS-8374 involved a combination of substrate mutations without typical resistance mutations in PR. These substrate changes were distributed throughout Gag and acted in an additive manner. Thus, they are classified as primary resistance mutations indicating a unique mechanism and pathway of resistance development for GS-8374.
Journal of Virology 10/2012; · 5.40 Impact Factor
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ABSTRACT: HIV-1 buds as an immature, non-infectious virion. Proteolysis of its main structural component Gag is required for morphological maturation and infectivity, and leads to release of four functional domains and the spacer peptides SP1 and SP2. The N-terminal cleavages of Gag and the separation of SP1 from CA are all essential for viral infectivity, while the roles of the two C-terminal cleavages and the role of SP2, separating the NC and p6 domains, are less well defined. We have analyzed HIV-1 variants with defective cleavage at either or both sites flanking SP2, or largely lacking SP2, regarding virus production, infectivity and structural maturation. Neither the presence nor proteolytic processing of SP2 were required for particle release. Viral infectivity was almost abolished when both cleavage sites were defective and severely reduced when the fast cleavage site between SP2 and p6 was defective. This correlated with an increased proportion of irregular core structures observed by cryo-electron tomography, although processing of CA was unaffected. Mutating the slow cleavage site between NC and SP2 or deleting most of SP2 had only a minor effect on infectivity, and did not induce major alterations in mature core morphology. We speculate that not only separation of NC and p6, but also the processing kinetics in this region are essential for successful maturation, while SP2 itself is dispensable.
Journal of Virology 10/2012; · 5.40 Impact Factor
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ABSTRACT: We apply single-molecule super-resolution microscopy and coordinate-based cluster analysis to extract information on the distribution and on the morphology and size of clusters of the human immunodeficiency virus (HIV-1) Gag polyprotein in fixed cells. Three different patterns of Gag distribution could be distinguished. A major type of assembly observed was in accordance with previous electron microscopy analyses revealing ~140 nm-sized HIV-1 buds at the plasma membrane of virus-producing cells. The distribution of Gag molecules in the 2D projection at these sites was consistent with a semi-spherical 3D assembly. We compared different methods of cluster analysis and demonstrated that we can reliably distinguish different distribution patterns of the Gag polyprotein. These methods were applied to extract information on the properties of the different Gag clusters.
Histochemie 08/2012; · 2.59 Impact Factor
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ABSTRACT: Entry of human immunodeficiency virus type 1 (HIV-1) into the host cell involves interactions between the viral envelope glycoproteins (Env) and the cellular receptor CD4 as well as a coreceptor molecule (most importantly CCR5 or CXCR4). Viral preference for a specific coreceptor (tropism) is in particular determined by the third variable loop (V3) of the Env glycoprotein gp120. The approval and use of a coreceptor antagonist for antiretroviral therapy make detailed understanding of tropism and its accurate prediction from patient derived virus isolates essential. The aim of the present study is the development of an extended description of the HIV entry phenotype reflecting its co-dependence on several key determinants as the basis for a more accurate prediction of HIV-1 entry phenotype from genotypic data.
Here, we established a new protocol of quantitation and computational analysis of the dependence of HIV entry efficiency on receptor and coreceptor cell surface levels as well as viral V3 loop sequence and the presence of two prototypic coreceptor antagonists in varying concentrations. Based on data collected at the single-cell level, we constructed regression models of the HIV-1 entry phenotype integrating the measured determinants. We developed a multivariate phenotype descriptor, termed phenotype vector, which facilitates a more detailed characterization of HIV entry phenotypes than currently used binary tropism classifications. For some of the tested virus variants, the multivariant phenotype vector revealed substantial divergences from existing tropism predictions. We also developed methods for computational prediction of the entry phenotypes based on the V3 sequence and performed an extrapolating calculation of the effectiveness of this computational procedure.
Our study of the HIV cell entry phenotype and the novel multivariate representation developed here contributes to a more detailed understanding of this phenotype and offers potential for future application in the effective administration of entry inhibitors in antiretroviral therapies.
Retrovirology 07/2012; 9:60. · 6.47 Impact Factor
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ABSTRACT: A defining property of retroviruses is their ability to assemble into particles that can leave producer cells and spread infection to susceptible cells and hosts. Virion morphogenesis can be divided into three stages: assembly, wherein the virion is created and essential components are packaged; budding, wherein the virion crosses the plasma membrane and obtains its lipid envelope; and maturation, wherein the virion changes structure and becomes infectious. All of these stages are coordinated by the Gag polyprotein and its proteolytic maturation products, which function as the major structural proteins of the virus. Here, we review our current understanding of the mechanisms of HIV-1 assembly, budding, and maturation, starting with a general overview and then providing detailed descriptions of each of the different stages of virion morphogenesis.
Cold Spring Harbor perspectives in medicine. 07/2012; 2(7):a006924.
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Milan Kozísek,
Sandra Henke,
Klára Grantz Sasková,
Graeme Brendon Jacobs,
Anita Schuch,
Bernd Buchholz,
Viktor Müller, Hans-Georg Kräusslich,
Pavlína Rezácová,
Jan Konvalinka,
Jochen Bodem
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ABSTRACT: During the last few decades, the treatment of HIV-infected patients by highly active antiretroviral therapy, including protease inhibitors (PIs), has become standard. Here, we present results of analysis of a patient-derived, multiresistant HIV-1 CRF02_AG recombinant strain with a highly mutated protease (PR) coding sequence, where up to 19 coding mutations have accumulated in the PR. The results of biochemical analysis in vitro showed that the patient-derived PR is highly resistant to most of the currently used PIs and that it also exhibits very poor catalytic activity. Determination of the crystal structure revealed prominent changes in the flap elbow region and S1/S1' active site subsites. While viral loads in the patient were found to be high, the insertion of the patient-derived PR into a HIV-1 subtype B backbone resulted in reduction of infectivity by 3 orders of magnitude. Fitness compensation was not achieved by elevated polymerase (Pol) expression, but the introduction of patient-derived gag and pol sequences in a CRF02_AG backbone rescued viral infectivity to near wild-type (wt) levels. The mutations that accumulated in the vicinity of the processing sites spanning the p2/NC, NC/p1, and p6pol/PR proteins lead to much more efficient hydrolysis of corresponding peptides by patient-derived PR in comparison to the wt enzyme. This indicates a very efficient coevolution of enzyme and substrate maintaining high viral loads in vivo under constant drug pressure.
Antimicrobial Agents and Chemotherapy 05/2012; 56(8):4320-30. · 4.84 Impact Factor
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ABSTRACT: There are no data on the outcome of highly active antiretroviral therapy (HAART) in HIV-infected adults in rural Burkina Faso. We therefore assessed CD4(+) T-cell counts and HIV-1 plasma viral load (VL), the proportion of naive T-cells (co-expressing CCR7 and CD45RA) and T-cell activation (expression of CD95 or CD38) in 61 previously untreated adult patients from Nouna, Burkina Faso, at baseline and 2 weeks, 1, 3, 6, 9 and 12 months after starting therapy. Median CD4(+) T-cell counts increased from 174 (10(th)-90(th) percentile: 33-314) cells/µl at baseline to 300 (114- 505) cells/µl after 3 months and 360 (169-562) cells/µl after 12 months of HAART. Median VL decreased from 5.8 (4.6- 6.6) log10 copies/ml at baseline to 1.6 (1.6-2.3) log10 copies/ml after 12 months. Early CD4(+) T-cell recovery was accompanied by a reduction of the expression levels of CD95 and CD38 on T-cells. Out of 42 patients with complete virological follow-up under HAART, 19 (45%) achieved concordant good immunological (gain of ≥100 CD4(+) T-cells/µl above baseline) and virological (undetectable VL) responses after 12 months of treatment (intention-to-treat analysis). Neither a decreased expression of the T-cell activation markers CD38 and CD95, nor an increase in the percentage of naive T-cells reliably predicted good virological treatment responses in patients with good CD4(+) T-cell reconstitution. Repeated measurement of CD4(+) T-cell counts during HAART remains the most important parameter for immunologic monitoring. Substitution of repeated VL testing by determination of T-cell activation levels (e.g., CD38 expression on CD8(+) T-cells) should be applied with caution.
The Open AIDS Journal 01/2012; 6:16-25.
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ABSTRACT: Pairs of amino acid positions that evolve in a correlated manner are proposed to play important roles in protein structure or function. Methods to detect them might fare better with families for which sequences of thousands of closely related homologs are available than families with only a few distant relatives. We applied co-evolution analysis to thousands of sequences of HIV Gag, finding that the most significantly co-evolving positions are proximal in the quaternary structures of the viral capsid. A reduction in infectivity caused by mutating one member of a significant pair could be rescued by a compensatory mutation of the other.
PLoS ONE 01/2012; 7(8):e42468. · 4.09 Impact Factor
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ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) Gag is the main structural protein driving assembly and release of virions from infected cells. Gag alone is capable of self-assembly in vitro, but host factors have been shown to play a role in efficient viral replication and particle morphogenesis within the living cell. In a series of affinity purification experiments, we identified the cellular protein Lyric to be an HIV-1 Gag-interacting protein. Lyric was previously described to be an HIV-inducible gene and is involved in various signaling pathways. Gag interacts with endogenous Lyric via its matrix (MA) and nucleocapsid (NC) domains. This interaction requires Gag multimerization and Lyric amino acids 101 to 289. Endogenous Lyric is incorporated into HIV-1 virions and is cleaved by the viral protease. Gag-Lyric interaction was also observed for murine leukemia virus and equine infectious anemia virus, suggesting that it represents a conserved feature among retroviruses. Expression of the Gag binding domain of Lyric increased Gag expression levels and viral infectivity, whereas expression of a Lyric mutant lacking the Gag binding site resulted in lower Gag expression and decreased viral infectivity. The results of the current study identify Lyric to be a cellular interaction partner of HIV-1 Gag and hint at a potential role in regulating infectivity. Further experiments are needed to elucidate the precise role of this interaction.
Journal of Virology 09/2011; 85(24):13322-32. · 5.40 Impact Factor
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ABSTRACT: Assembly of human immunodeficiency virus type 1 is driven by oligomerization of the Gag polyprotein at the plasma membrane of an infected cell, leading to membrane envelopment and budding of an immature virus particle. Proteolytic cleavage of Gag at five positions subsequently causes a dramatic rearrangement of the interior virion organization to form an infectious particle. Within the mature virus, the genome is encased within a conical capsid core. Here, we describe the molecular architecture of the virus assembly site, the immature virus, the maturation intermediates and the mature virus core and highlight recent advances in our understanding of these processes from electron microscopy and X-ray crystallography studies.
Journal of Molecular Biology 07/2011; 410(4):491-500. · 4.00 Impact Factor
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ABSTRACT: Viruses intricately interact with and modulate cellular membranes at several stages of their replication, but much less is known about the role of viral lipids compared to proteins and nucleic acids. All animal viruses have to cross membranes for cell entry and exit, which occurs by membrane fusion (in enveloped viruses), by transient local disruption of membrane integrity, or by cell lysis. Furthermore, many viruses interact with cellular membrane compartments during their replication and often induce cytoplasmic membrane structures, in which genome replication and assembly occurs. Recent studies revealed details of membrane interaction, membrane bending, fission, and fusion for a number of viruses and unraveled the lipid composition of raft-dependent and -independent viruses. Alterations of membrane lipid composition can block viral release and entry, and certain lipids act as fusion inhibitors, suggesting a potential as antiviral drugs. Here, we review viral interactions with cellular membranes important for virus entry, cytoplasmic genome replication, and virus egress.
Cold Spring Harbor perspectives in biology 05/2011; 3(10):a004820. · 9.40 Impact Factor
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ABSTRACT: Productive infection of macrophages is central to HIV-1 pathogenesis. Newly formed virions bud into a tubular membranous compartment that is contiguous with the plasma membrane. However, little is known about the structure of this compartment and its potential regulation by infection. Here we characterized this compartment in macrophages using electron tomography and electron microscopy with stereology. We found an intricate, interconnected membrane network that constitutes a preexisting physiologic structure in macrophages but which expands in size upon HIV-1 infection. Membranes required for this expansion were apparently derived from preexisting pools of plasma membrane. Physical connections between this compartment and the extracellular milieu were frequently made by tube-like structures of insufficient diameter for virion passage. We conclude that HIV-1 induces the expansion of a complex membranous labyrinth in macrophages in which the virus buds and can be retained, with potential consequences for transmission and immune evasion.
Journal of Virology 05/2011; 85(15):7922-7. · 5.40 Impact Factor
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ABSTRACT: HIV (human immunodeficiency virus) diverts the cellular ESCRT (endosomal sorting complex required for transport) machinery to promote virion release from infected cells. The ESCRT consists of four heteromeric complexes (ESCRT-0 to ESCRT-III), which mediate different membrane abscission processes, most importantly formation of intralumenal vesicles at multivesicular bodies. The ATPase VPS4 (vacuolar protein sorting 4) acts at a late stage of ESCRT function, providing energy for ESCRT dissociation. Recruitment of ESCRT by late-domain motifs in the viral Gag polyprotein and a role of ESCRT in HIV release are firmly established, but the order of events, their kinetics and the mechanism of action of individual ESCRT components in HIV budding are unclear at present. Using live-cell imaging, we show late-domain-dependent recruitment of VPS4A to nascent HIV particles at the host cell plasma membrane. Recruitment of VPS4A was transient, resulting in a single or a few bursts of at least two to five VPS4 dodecamers assembling at HIV budding sites. Bursts lasted for ∼35 s and appeared with variable delay before particle release. These results indicate that VPS4A has a direct role in membrane scission leading to HIV-1 release.
Nature Cell Biology 03/2011; 13(4):469-74. · 19.49 Impact Factor
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Alex de Marco,
Norman E Davey,
Pavel Ulbrich,
Judith M Phillips,
Vanda Lux,
James D Riches,
Tibor Fuzik,
Tomas Ruml, Hans-Georg Kräusslich,
Volker M Vogt,
John A G Briggs
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ABSTRACT: The assembly of retroviruses is driven by oligomerization of the Gag polyprotein. We have used cryo-electron tomography together with subtomogram averaging to describe the three-dimensional structure of in vitro-assembled Gag particles from human immunodeficiency virus, Mason-Pfizer monkey virus, and Rous sarcoma virus. These represent three different retroviral genera: the lentiviruses, betaretroviruses and alpharetroviruses. Comparison of the three structures reveals the features of the supramolecular organization of Gag that are conserved between genera and therefore reflect general principles of Gag-Gag interactions and the features that are specific to certain genera. All three Gag proteins assemble to form approximately spherical hexameric lattices with irregular defects. In all three genera, the N-terminal domain of CA is arranged in hexameric rings around large holes. Where the rings meet, 2-fold densities, assigned to the C-terminal domain of CA, extend between adjacent rings, and link together at the 6-fold symmetry axis with a density, which extends toward the center of the particle into the nucleic acid layer. Although this general arrangement is conserved, differences can be seen throughout the CA and spacer peptide regions. These differences can be related to sequence differences among the genera. We conclude that the arrangement of the structural domains of CA is well conserved across genera, whereas the relationship between CA, the spacer peptide region, and the nucleic acid is more specific to each genus.
Journal of Virology 11/2010; 84(22):11729-36. · 5.40 Impact Factor
