Highly conserved serine residue 40 in HIV-1 p6 regulates capsid processing and virus core assembly

Background: The HIV-1 p6 Gag protein regulates the final abscission step of nascent virions from the cell membrane by the action of two late assembly (L-) domains. Although p6 is located within one of the most polymorphic regions of the HIV-1 gag gene, the 52 amino acid peptide binds at least to two cellular budding factors (Tsg101 and ALIX), is a substrate for phosphorylation, ubiquitination, and sumoylation, and mediates the incorporation of the HIV-1 accessory protein Vpr into viral particles. As expected, known functional domains mostly overlap with several conserved residues in p6. In this study, we investigated the importance of the highly conserved serine residue at position 40, which until now has not been assigned to any known function of p6. Results: Consistently with previous data, we found that mutation of Ser-40 has no effect on ALIX mediated rescue of HIV-1 L-domain mutants. However, the only feasible S40F mutation that preserves the overlapping pol open reading frame (ORF) reduces virus replication in T-cell lines and in human lymphocyte tissue cultivated ex vivo. Most intriguingly, L-domain mediated virus release is not dependent on the integrity of Ser-40. However, the S40F mutation significantly reduces the specific infectivity of released virions. Further, it was observed that mutation of Ser-40 selectively interferes with the cleavage between capsid (CA) and the spacer peptide SP1 in Gag, without affecting cleavage of other Gag products. This deficiency in processing of CA, in consequence, led to an irregular morphology of the virus core and the formation of an electron dense extra core structure. Moreover, the defects induced by the S40F mutation in p6 can be rescued by the A1V mutation in SP1 that generally enhances processing of the CA-SP1 cleavage site. Conclusions: Overall, these data support a so far unrecognized function of p6 mediated by Ser-40 that occurs independently of the L-domain function, but selectively affects CA maturation and virus core formation, and consequently the infectivity of released virions

An Evolutionary Framework for Culture: Selectionism versus Communal Exchange

Dawkins' replicator-based conception of evolution has led to widespread mis-application selectionism across the social sciences because it does not address the paradox that inspired the theory of natural selection in the first place: how do organisms accumulate change when traits acquired over their lifetime are obliterated? This is addressed by von Neumann's concept of a self-replicating automaton (SRA). A SRA consists of a self-assembly code that is used in two distinct ways: (1) actively deciphered during development to construct a self-similar replicant, and (2) passively copied to the replicant to ensure that it can reproduce. Information that is acquired over a lifetime is not transmitted to offspring, whereas information that is inherited during copying is transmitted. In cultural evolution there is no mechanism for discarding acquired change. Acquired change can accumulate orders of magnitude faster than, and quickly overwhelm, inherited change due to differential replication of variants in response to selection. This prohibits a selectionist but not an evolutionary framework for culture. Recent work on the origin of life suggests that early life evolved through a non-Darwinian process referred to as communal exchange that does not involve a self-assembly code, and that natural selection emerged from this more haphazard, ancestral evolutionary process. It is proposed that communal exchange provides a more appropriate evolutionary framework for culture than selectionism. This is supported by a computational model of cultural evolution and a network-based program for documenting material cultural history, and it is consistent with high levels of human cooperation.Comment: 18 pages; 2 tables and 11 figures embedded in tex

Structure and function of the HIV-1 gag-protein p6

Das HIV-1-Protein p6 ist im C-terminalen Bereich von Gag lokalisiert und besitzt trotz seiner geringen Größe von nur 52 Aminosäuren eine komplexe strukturelle und funktionelle Organisation. Es bindet mindestens zwei zelluläre Buddingfaktoren (Tsg101 und ALIX) sowie zwei virale Proteine (Vpr und Env) und übt eine Vielzahl biologischer Funktionen aus. Außerdem besitzt p6 sowohl C- als auch N-terminal jeweils eine L-Domäne, welche unter anderem den letzten Schritt in der Abschnürung des naszierenden Virions von der Zellmembran, in einem bis heute noch nicht vollständig geklärten Mechanismus, regulieren. Neben den Modifikationen der Ubiquitinylierung und Sumoylierung fungiert p6 außerdem als Substrat für verschiedene Kinasen: Im Jahr 2002 wurde p6 als Hauptphosphoprotein in HIV-1-Partikeln identifiziert und es konnte gezeigt werden, dass p6 durch die MAP-Kinase ERK-2 an der Position Thr-23 phosphoryliert wird. Weiter gibt es Hinweise, dass die genannte Kinase durch die Phosphorylierung an Thr-23 von p6 den Viruszusammenbau sowie die Virusfreisetzung reguliert. Zur Identifikation weiterer potentieller Kinasen wurde zu Beginn dieser Arbeit eine in silico Analyse durchgeführt und schließlich mittels in vitro Experimenten mit synthetischem und viralem p6-Peptid die Proteinkinase C, welche spezifisch Ser-Reste an Position 14, 25 und 40 in p6 phosphoryliert, identifiziert. Vergleicht man die Konsensussequenzen verschiedener HIV-1-Isolate der M-Hauptgruppe, so ist eine absolute Konservierung von Ser-40 festzustellen, was auf eine mögliche biologische Funktion dieser Stelle hindeutet. Zur Untersuchung der biologischen Funktion der Phosphorylierungsstellen in p6 wurden Konstrukte kloniert, die an den identifizierten Ser-Resten mutiert wurden. Die Substitution aller Ser-Reste einzeln zeigte in Infektionsstudien, dass nur die Mutation von Ser-40 zu einer verminderten Replikationskapazität in T-Zellen sowie in primären humanen Zellen führte. Die Mutation von Ser-14 und Ser-25 zeigte keinen Effekt auf die Replikation von HIV-1. Die Mutation aller drei PKC-Stellen zusammen führte hingegen zu einem kompletten Verlust der Replikationsfähigkeit. Obwohl die Mutation der Phosphoakzeptorstellen die Freisetzung der Viruspartikel nicht beeinflusste, sind die produzierten Virionen der Ser-40-Mutante weniger infektiös und die Dreifachmutation führte sogar zu einem kompletten Verlust der Infektiösität. Als molekularer Mechanismus hinter diesem Phänomen konnte in Pulse-Chase Experimenten beobachtet werden, dass die CA-Prozessierung von p25 zu p24 im Fall der Ser-40-Mutante gestört ist. Elektronenmikroskopische Aufnahmen zeigten, dass der Defekt in der CA-Prozessierung zu einer irregulären Core-Morphologie und zur Bildung einer elektronendichten Struktur außerhalb des Cores führt. Die erhaltenen Ergebnisse belegen eine neue, bis heute unbekannte Funktion von p6, die nicht die Virusfreisetzung, sondern die Gag-Prozessierung betrifft. Das konservierte Ser-40 reguliert auf noch unbekannte Weise, womöglich durch PKC-Phosphorylierung, die CA-Reifung und somit die Infektiösität von HIV-1. Neben den verschiedenen Bindungsstellen für virale und zelluläre Proteine sowie den Modifikationsstellen besitzt p6 die inhärente Fähigkeit zur Ausbildung oligomerer Formen. Diese wurden durch die Behandlung von sp6 und vp6 mit chemischen Crosslinkern und anschließender Western Blot Analyse untersucht. Es zeigte sich, dass das C-terminale Fragment von p6 oligomere Strukturen ausbildet. Somit konnte der in silico Befund, dass p6 eine im C-terminalen Bereich lokalisierte Oligomerisierungsdomäne besitzt, bestätigt werden. Ob diese Oligomerisierungsdomäne in p6 zur Gag-Gag-Multimerisierung, welcher eine wichtige Rolle in der Gag-Assemblierung und der Produktion von Viruspartikeln zukommt, beiträgt, sie eine ganz andere funktionelle Relevanz hat oder gar funktionslos ist, ist bislang nicht bekannt.The HIV-1 protein is located at the C-terminus of Gag and despite its small size of 52 amino acids it comprises a quite complex structural and functional organization: it binds at least two cellular budding factors (Tsg101 and ALIX) as well as two viral proteins (Vpr and Env) and it exerts a variety of biological functions. Among others, it regulates the final abscission step of the nascent virion from the cell membrane by the action of two different l domains, one located at the C- and one located at the N-terminus of the peptide, in a yet not completely elucidated mechanism. Besides the ubiquitinylation and sumoylation p6 acts as a substrate for distinct kinases: 2002 p6 was identified as the major phosphoprotein found in HIV-1 particles and it could be shown that phosphorylation of p6 by MAP-kinase ERK-2 at position Thr-23 regulates the assembly and release of progeny virions. To identify further potential kinases an in silico analysis was performed and finally in vitro experiments with synthetic and viral p6 identified PKC to specifically phosphorylate Ser residues in position 14, 25 and 40 in p6. When the consensus sequences derived from various HIV-1 group M isolates were compared, one can see that Ser-40 is absolutely conserved. Therefore, the biological function of this site has to be identified. To analyze the biological function of the identified phosphorylation sites, different constructs carrying mutations of the identified Ser residues were generated. Individual substitutions of the Ser residues revealed that only mutation of Ser-40 reduced virus replication in T-cell lines and in primary human cells, while mutation of Ser-14 and Ser-25 had no effect on the replication. In contrast, mutation of all three PKC-sites completely abrogated replication. Although mutation of PKC sites had no influence on virus release, the infectivity was reduced for the Ser-40 mutant and completely lost for the triple mutant. As molecular mechanism behind this phenomenon pulse-chase experiments illustrated that the efficiency in final processing of CA from p25 to p24 was dependent on the integrity of Ser-40. Electron micrographs showed that the defect in CA processing led to an irregular morphology of the virus core and the formation of an electron dense extra core structure. Thus, the obtained results support a novel, till this day unknown function of p6, that does not affect virus release but concerns Gag processing. The conserved Ser-40 regulates in a yet unknown manner CA maturation and thereby infectivity of HIV-1 in a process that might involve PKC phosphorylation. In addition to the different binding sites for viral and cellular proteins and the modification sites p6 displays the inherent ability to undergo oligomerization. This observation was analyzed by incubating sp6 and vp6 with chemical crosslinkers and western blotting. It turned out that the C-terminal fragment of p6 has the propensity to form oligomeric structures. This is consistent with in silico analysis, which predicts an oligomerization domain in the C-terminus of p6. Whether this oligomerization domain in p6 contributes to the Gag-Gag multimerization, which has an important role in Gag assembly and virus particle production, or whether it has a complete different functional relevance or it is totally out of any function is previously not known

Structural studies on the RNA-recognition motif of NELF E, a cellular negative transcription elongation factor involved in the regulation of HIV transcription

The elongation of transcription of HIV RNA at the TAR (transactivation-response element) is highly regulated by positive and negative factors. The cellular negative transcription elongation factor NELF (negative elongation factor) was suggested to be involved in transcriptional regulation of HIV-1 (HIV type 1) by binding to the stem of the viral TAR RNA which is synthesized by cellular RNA polymerase II at the viral long terminal repeat. NELF is a heterotetrameric protein consisting of NELF A, B, C or the splice variant D, and E. In the present study, we determined the solution structure of the RRM (RNA-recognition motif) of the RNA-binding subunit NELF E and studied its interaction with the viral TAR RNA. Our results show that the separately expressed recombinant NELF E RRM has α-helical and β-strand elements adopting a βαββαβ fold and is able to bind to TAR RNA. Fluorescence equilibrium titrations with fluorescently labelled double- and single-stranded oligoribonucleotides representing the TAR RNA stem imply that NELF E RRM binds to the single-stranded TAR RNAs with K(d) values in the low-micromolar range

Clinical and genetic characteristics of late-onset Huntington's disease

Background: The frequency of late-onset Huntington's disease (>59 years) is assumed to be low and the clinical course milder. However, previous literature on late-onset disease is scarce and inconclusive. Objective: Our aim is to study clinical characteristics of late-onset compared to common-onset HD patients in a large cohort of HD patients from the Registry database. Methods: Participants with late- and common-onset (30–50 years)were compared for first clinical symptoms, disease progression, CAG repeat size and family history. Participants with a missing CAG repeat size, a repeat size of ≤35 or a UHDRS motor score of ≤5 were excluded. Results: Of 6007 eligible participants, 687 had late-onset (11.4%) and 3216 (53.5%) common-onset HD. Late-onset (n = 577) had significantly more gait and balance problems as first symptom compared to common-onset (n = 2408) (P <.001). Overall motor and cognitive performance (P <.001) were worse, however only disease motor progression was slower (coefficient, −0.58; SE 0.16; P <.001) compared to the common-onset group. Repeat size was significantly lower in the late-onset (n = 40.8; SD 1.6) compared to common-onset (n = 44.4; SD 2.8) (P <.001). Fewer late-onset patients (n = 451) had a positive family history compared to common-onset (n = 2940) (P <.001). Conclusions: Late-onset patients present more frequently with gait and balance problems as first symptom, and disease progression is not milder compared to common-onset HD patients apart from motor progression. The family history is likely to be negative, which might make diagnosing HD more difficult in this population. However, the balance and gait problems might be helpful in diagnosing HD in elderly patients