Ubiquitination of HIV-1 and MuLV Gag

AbstractOur previous biochemical studies of HIV-1 and MuLV virions isolated and identified mature Gag products, HIV-1 p6Gag and MuLV p12Gag, that were conjugated to a single ubiquitin. To study the importance of the monoubiquitination of Gag, a series of lysine to arginine mutants were constructed that eliminated ubiquitination at one or both of the lysines in HIV-1NL4-3 p6Gag and both lysines in Moloney MuLV p12Gag. HPLC and immunoblot analysis of the HIV-1 mutants demonstrated that either of the lysines in p6Gag, K27 or K33, could be monoubiquitinated. However, infectivity assays showed that monoubiquitination of HIV-1 p6Gag or MuLV p12Gag is not required for viral replication in vitro. Pulse-chase radiolabeling of HIV-1-producing cells revealed that monoubiquitination of p6Gag does not affect the short-term release of virus from the cell, the maturation of Pr55Gag, or the sensitivity of these processes to proteasome inhibitors. Experiments with protease-deficient HIV-1 showed that Pr55Gag can be monoubiquitinated, suggesting that p6Gag is first modified as a domain within Gag. Examination of the proteins inside an HIV-1 mutant found that free ubiquitin was incorporated into the virions in the absence of the lysines in p6Gag, showing that the ubiquitin inside the virus is not initially brought in as a p6Gag conjugate. Although our results establish that monoubiquitination of p6Gag and p12Gag is not required for viral replication in vitro, this modification may be a by-product of interactions between Gag and cellular proteins during assembly and budding

Mapping the complete glycoproteome of virion-derived HIV-1 gp120 provides insights into broadly neutralizing antibody binding

The surface envelope glycoprotein (SU) of Human immunodeficiency virus type 1 (HIV-1), gp120SU plays an essential role in virus binding to target CD4+ T-cells and is a major vaccine target. Gp120 has remarkably high levels of N-linked glycosylation and there is considerable evidence that this “glycan shield” can help protect the virus from antibody-mediated neutralization. In recent years, however, it has become clear that gp120 glycosylation can also be included in the targets of recognition by some of the most potent broadly neutralizing antibodies. Knowing the site-specific glycosylation of gp120 can facilitate the rational design of glycopeptide antigens for HIV vaccine development. While most prior studies have focused on glycan analysis of recombinant forms of gp120, here we report the first systematic glycosylation site analysis of gp120 derived from virions produced by infected T lymphoid cells and show that a single site is exclusively substituted with complex glycans. These results should help guide the design of vaccine immunogens

Cryoelectron Tomography of HIV-1 Envelope Spikes: Further Evidence for Tripod-Like Legs

A detailed understanding of the morphology of the HIV-1 envelope (Env) spike is key to understanding viral pathogenesis and for informed vaccine design. We have previously presented a cryoelectron microscopic tomogram (cryoET) of the Env spikes on SIV virions. Several structural features were noted in the gp120 head and gp41 stalk regions. Perhaps most notable was the presence of three splayed legs projecting obliquely from the base of the spike head toward the viral membrane. Subsequently, a second 3D image of SIV spikes, also obtained by cryoET, was published by another group which featured a compact vertical stalk. We now report the cryoET analysis of HIV-1 virion-associated Env spikes using enhanced analytical cryoET procedures. More than 2,000 Env spike volumes were initially selected, aligned, and sorted into structural classes using algorithms that compensate for the “missing wedge” and do not impose any symmetry. The results show varying morphologies between structural classes: some classes showed trimers in the head domains; nearly all showed two or three legs, though unambiguous three-fold symmetry was not observed either in the heads or the legs. Subsequently, clearer evidence of trimeric head domains and three splayed legs emerged when head and leg volumes were independently aligned and classified. These data show that HIV-1, like SIV, also displays the tripod-like leg configuration, and, unexpectedly, shows considerable gp41 leg flexibility/heteromorphology. The tripod-like model for gp41 is consistent with, and helps explain, many of the unique biophysical and immunological features of this region

Молекулярно-биологические методы контроля качества субстанций биологических лекарственных препаратов, полученных с использованием технологии рекомбинантной ДНК

Biotechnological products manufactured by recombinant DNA technology are widely used nowadays. According to the national and  international requirements the amount of residual host cell DNA in  such products should not exceed 10 ng per dose. However, for  products intended for frequent or long-term use, this amount must  not exceed 100 pg per dose. This article describes methods most  frequently used for quantification of residual host cell DNA in  biological active substances contained in biotechnological products:  molecular hybridization with biotin- or digoxigenin-labelled DNA- probes (semiquantitative method), Threshold system, real-time PCR, method based on the use of a fluorescent reagent (assays). If  a method based on the use of a fluorescent reagent or real-time PCR are used to replace the current procedure, it is necessary to  demonstrate their validity, e.g. by comparing the results of residual DNA quantification obtained by the two methods — the new one and the current one. The article dwells upon the advantages and  disadvantages of the methods and potential sources of uncertainty.  It highlights the importance of using appropriately certified reference standards and retention samples. The biological active substances  included into the State Register of Medicinal Products conform to the international requirements in terms of the amount of residual host cell DNA.Биотехнологические препараты, полученные с применением технологии рекомбинантных ДНК, широко используются в настоящее время. В соответствии с международными и  отечественными требованиями содержание остаточной ДНК штамма-продуцента в подобных  препаратах не должно превышать 10 нг на 1 дозу, для препаратов, предназначенных для  частого или длительного введения, эта величина не должна превышать 100 пг на 1 дозу. В  статье представлено описание наиболее часто используемых методов определения  содержания остаточной ДНК штамма-продуцента в субстанциях биотехнологических препаратов: молекулярная гибридизация с биотиновой или  дигоксигениновой меткой ДНК-зонда (полуколичественный метод), система Threshold, ПЦР в  режиме реального времени, метод с флуоресцентным реагентом (количественные  методы). В случае использования методов с флуоресцентным реагентом или ПЦР в режиме  реального времени для замены ранее использовавшегося метода требуется подтверждение  их правильности, например с помощью сравнительной оценки содержания остаточной ДНК  двумя методами — вновь вводимым и ранее использовавшимся методом. Отмечены  преимущества и недостатки методов, источники неопределенности результатов при  проведении испытаний, указана необходимость использования стандартных и контрольных  образцов, аттестованных в установленном порядке. В субстанциях, внесенных в  Государственный реестр лекарственных средств, содержание остаточной ДНК штамма-продуцента соответствует международным рекомендациям

HIV-1 is budded from CD4+ T lymphocytes independently of exosomes

The convergence of HIV-1 budding and exosome biogenesis at late endosomal compartments called multivesicular bodies has fueled the debate on whether HIV-1 is budded from its target cells and transmitted in the form of exosomes. The point of contention appears to primarily derive from the types of target cells in question and lack of a well-defined protocol to separate exosomes from HIV-1. In this study, we adapted and established a simplified protocol to define the relationship between HIV-1 production and exosome biogenesis. Importantly, we took advantage of the newly established protocol to unequivocally show that HIV-1 was produced from CD4+ T lymphocytes Jurkat cells independently of exosomes. Thus, this study not only presents a simplified way to obtain highly purified HIV-1 virions for identification of host proteins packaged into virions, but also provides a technical platform that can be employed to define the relationship between exosome biogenesis and budding of HIV-1 or other viruses and its contributions to viral pathogenesis

Tsunami hazards in the Catalan Coast, a low-intensity seismic activity area

The final publication is available at Springer via http://dx.doi.org/10.1007/s11069-017-2918-zThe potential impacts of tsunamis along the Catalan Coast (NW Mediterranean) are analysed using numerical modelling. The region is characterized by moderate to low seismic activity and by moderate- to low-magnitude earthquakes. However, the occurrence of historical strong earthquakes and the location of several active offshore faults in front of the coast suggest that the possibility of an earthquake-triggered tsunami is not negligible although of low probability. Up to five faults have been identified to generate tsunamis, being the highest associated possible seismic magnitudes of up to 7.6. Coastal flooding and port agitation are characterized using the Worst-case Credible Tsunami Scenario Analysis approach. The results show a multiple fault source contribution to tsunami hazard. The shelf dimensions and the existence of submerged canyons control the tsunami propagation. In wide shelves, waves travelling offshore may become trapped by refraction causing the wave energy to reach the coastline at some distance from the origin. The free surface water elevation increases at the head of the canyons due to the sharp depth gradients. The effects of potential tsunamis would be very harmful in low-lying coastal stretches, such as deltas, with a high population concentration, assets and infrastructures. The Ebro delta appears to be the most exposed coast, and about the 20% of the delta surface is prone to flooding due to its extremely low-lying nature. The activity at Barcelona port will be severely affected by inflow backflow current at the entrance of up to 2 m/s.Peer ReviewedPostprint (author's final draft

Inhibition of HIV virus by neutralizing Vhh attached to dual functional liposomes encapsulating dapivirine

Although highly active antiretroviral therapy (HAART) has greatly improved the life expectancy of HIV/AIDS patients, the treatment is not curative. It is a global challenge which fosters an urgent need to develop an effective drug or neutralizing antibody delivery approach for the prevention and treatment of this disease. Due to the low density of envelope spikes with restricted mobility present on the surface of HIV virus, which limit the antibody potency and allow virus mutation and escape from the immune system, it is important for a neutralizing antibody to form bivalent or multivalent bonds with the virus. Liposome constructs could fulfil this need due to the flexible mobility of the membrane with its attached antibodies and the capacity for drug encapsulation. In this study, we evaluated the neutralization activity of a range of liposome formulations in different sizes coated with anti-gp120 llama antibody fragments (Vhhs) conjugated via either non-covalent metal chelation or a covalent linkage. The non-covalent construct demonstrated identical binding affinity to HIV-1 envelope glycoprotein gp120 and neutralizing ability for HIV virus as free Vhh. Although covalently linked Vhh showed significant binding affinity to gp120, it unexpectedly had a lower neutralization potency. This may be due to the comparability in size of the viral and liposome particles restricting the number which can be bound to the liposome surface so involving only a fraction of the antibodies, whereas non-covalently attached antibodies dissociate from the surface after acting with gp120 and free the remainder to bind further viruses. Covalently conjugated Vhh might also trigger the cellular uptake of a liposome-virion complex. To explore the possible ability of the antibody-coated liposomes to have a further function, we encapsulated the hydrophobic antiviral drug dapivirine into both of the non-covalently and covalently conjugated liposome formulations, both of which revealed high efficacy in reducing viral replication in vitro. Thus, dual function liposomes may lead to a novel strategy for the prophylaxis of HIV/AIDS by combining the neutralizing activity of Vhh with antiviral effects of high drug concentrations

Potent Inhibition of HIV-1 Replication by a Tat Mutant

Herein we describe a mutant of the two-exon HIV-1 Tat protein, termed Nullbasic, that potently inhibits multiple steps of the HIV-1 replication cycle. Nullbasic was created by replacing the entire arginine-rich basic domain of wild type Tat with glycine/alanine residues. Like similarly mutated one-exon Tat mutants, Nullbasic exhibited transdominant negative effects on Tat-dependent transactivation. However, unlike previously reported mutants, we discovered that Nullbasic also strongly suppressed the expression of unspliced and singly-spliced viral mRNA, an activity likely caused by redistribution and thus functional inhibition of HIV-1 Rev. Furthermore, HIV-1 virion particles produced by cells expressing Nullbasic had severely reduced infectivity, a defect attributable to a reduced ability of the virions to undergo reverse transcription. Combination of these inhibitory effects on transactivation, Rev-dependent mRNA transport and reverse transcription meant that permissive cells constitutively expressing Nullbasic were highly resistant to a spreading infection by HIV-1. Nullbasic and its activities thus provide potential insights into the development of potent antiviral therapeutics that target multiple stages of HIV-1 infection

Clathrin Facilitates the Morphogenesis of Retrovirus Particles

The morphogenesis of retroviral particles is driven by Gag and GagPol proteins that provide the major structural component and enzymatic activities required for particle assembly and maturation. In addition, a number of cellular proteins are found in retrovirus particles; some of these are important for viral replication, but many lack a known functional role. One such protein is clathrin, which is assumed to be passively incorporated into virions due to its abundance at the plasma membrane. We found that clathrin is not only exceptionally abundant in highly purified HIV-1 particles but is recruited with high specificity. In particular, the HIV-1 Pol protein was absolutely required for clathrin incorporation and point mutations in reverse transcriptase or integrase domains of Pol could abolish incorporation. Clathrin was also specifically incorporated into other retrovirus particles, including members of the lentivirus (simian immunodeficiency virus, SIVmac), gammaretrovirus (murine leukemia virus, MLV) and betaretrovirus (Mason-Pfizer monkey virus, M-PMV) genera. However, unlike HIV-1, these other retroviruses recruited clathrin primarily using peptide motifs in their respective Gag proteins that mimicked motifs found in cellular clathrin adaptors. Perturbation of clathrin incorporation into these retroviruses, via mutagenesis of viral proteins, siRNA based clathrin depletion or adaptor protein (AP180) induced clathrin sequestration, had a range of effects on the accuracy of particle morphogenesis. These effects varied according to which retrovirus was examined, and included Gag and/or Pol protein destabilization, inhibition of particle assembly and reduction in virion infectivity. For each retrovirus examined, clathrin incorporation appeared to be important for optimal replication. These data indicate that a number of retroviruses employ clathrin to facilitate the accurate morphogenesis of infectious particles. We propose a model in which clathrin contributes to the spatial organization of Gag and Pol proteins, and thereby regulates proteolytic processing of virion components during particle assembly