The gag proteins of human immunodeficiency virus type 1: mechanisms of virus assembly and possibilities for interference

Modrow, Susanne; Kattenbeck, Bernhard; Poblotzki, A von; Niedrig, Matthias; Wagner, Ralf; Wolf, Hans

doi:10.1007/bf00194171

Cited by 9 publications

(6 citation statements)

References 67 publications

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“…The exposure of the myristoyl group through interaction with PI(4,5)P 2 is thought to allow anchoring of the protein to the membrane. , To test this hypothesis, Ins(1,4,5)P 3 , a water-soluble analogue of the PI(4,5)P 2 lipid headgroup, was used to examine whether binding of mGag was enhanced by exposing the myristoyl group prior to association with a bilayer by incubating mGag with 0.1 mg/mL Ins(1,4,5)P 3 . The membrane-associated mGag was quantified for a single mGag concentration of 400 nM.…”

Section: Results and Discussionmentioning

confidence: 99%

“…SHG Theory. The SHG intensity produced from the surface at 2ω is proportional to the square of the second-order susceptibility tensor χ ( ) ijk (2) , composed of nonresonant (NR)…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…HIV-1 group-specific antigen (Gag) is a 56 kDa protein, which is integral in the assembly of the HIV-1 virus particle and the only viral protein required to initiate and complete the budding process. , Gag is an N-myristoylated polyprotein comprising, from the N- to C-terminus, the MA domain (which is cleaved and forms the matrix assembly in the mature virus), the CA domain (forms the capsid assembly in the mature virus), the NC domain (forms the nucleocapsid assembly in the mature virus), and the p6 domain . The MA domain also contains a highly basic region (HBR), which is believed to facilitate interaction with lipid membranes.…”

Section: Introductionmentioning

confidence: 99%

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Mechanistic Investigation of HIV-1 Gag Association with Lipid Membranes

et al. 2019

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An extensive investigation into the initial association of HIV-1 Gag with lipid membranes was conducted with second harmonic generation. The roles of the lipid phase, phospholipid 1,2-dioleoyl-sn-glycero-3-phospho-(1-myo-inositol-4,5-bisphosphate) [PI(4,5)P2], the presence of the myristoyl group on Gag, the C-terminus of Gag, and the presence of transfer ribonucleic acid (tRNA) in Gag–membrane association were examined using the physiologically most relevant full-length Gag protein studied thus far. The tighter packing of a bilayer composed of gel-phase lipids was found to have a lower relative amount of membrane-bound Gag in comparison to its fluid-phase counterpart. Rather than driving membrane association of Gag, the presence of PI(4,5)P2 and the myristoyl group were found to anchor Gag at the membrane by decreasing the rate of desorption. Specifically, the interaction with PI(4,5)P2 allows Gag to overcome electrostatic repulsion with negatively charged lipids at the membrane surface. This behavior was verified by measuring the binding properties of Gag mutants in the matrix domain of Gag, which prevented anchoring to the membrane either by blocking interaction with PI(4,5)P2 or by preventing exposure of the myristoyl group. The presence of tRNA was found to inhibit Gag association with the membrane by specifically blocking the PI(4,5)P2 binding region, thereby preventing exposure of the myristoyl group and precluding subsequent anchoring of Gag to the membrane. While Gag likely samples all membranes, only the anchoring provided by the myristoyl group and PI(4,5)P2 allows Gag to accumulate at the membrane. These quantitative results on the kinetics and thermodynamics of Gag association with lipid membranes provide important new information about the mechanism of Gag–membrane association.

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Section: Results and Discussionmentioning

confidence: 99%

“…SHG Theory. The SHG intensity produced from the surface at 2ω is proportional to the square of the second-order susceptibility tensor χ ( ) ijk (2) , composed of nonresonant (NR)…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Mechanistic Investigation of HIV-1 Gag Association with Lipid Membranes

et al. 2019

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“…By shortening the respective active peptides down to the overlapping por tions we were able to define residues 47-59 (air /, NPGLLETSEGCRQ) in pl7MA and 339-349 (air 2, PAATLEEMMTA) in p24CA as active parts [36,38]. When peptide-treated cells were analyzed in the elec tron microscope, particulate structures were found to be released from the cell surface which did not contain the conical core structure typical for mature lentivirus parti cles.…”

Section: Synthetic Peptides Derived From the Matrix And Capsid Proteimentioning

confidence: 96%

Defined Amino Acids in the Gag Proteins of Human Immunodeficiency Virus Type 1 Are Functionally Active during Virus Assembly

et al. 1996

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A structurally highly ordered arrangement of the polyprotein precursor Pr55gag is a necessary prerequisite for assembly, budding and maturation of the human immunodeficiency virus type 1 (HIV-1). In particular, distinct regions of the matrix protein (pi7) and the capsid protein (p24) contained within Pr55gag are functionally active during these processes. In order to determine such regions we exchanged amino acid triplets within pi7 (amino acids 46-61) and p24 (amino acids 341-352) for alanine residues and deleted the whole regions. Synthetic peptides derived from these regions had been shown previously to inhibit the production of infectious virus. The effect of the mutations on the release of viral particles was investigated by using recombinant baculoviruses for the expression of mutated Pr55gag as virus-like particles and by use of the respective HI proviruses for monitoring the production of infectious particles.

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“…However, as expected, in comparison to wild-type Gag VLP yields, the chimeric VLP yields in this study were almost 10-fold lower. 24 The yields were dependent on the position of the fused protein on Gag as well as the length of the fused protein.…”

Section: Optimization Conditions and Vlp Yieldsmentioning

confidence: 99%

Optimization of chimeric HIV‐1 virus‐like particle production in a baculovirus‐insect cell expression system

Pillay

Meyers

Williamson

et al. 2009

Biotechnology Progress

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A baculovirus-insect cell expression system potentially provides the means to produce prophylactic HIV-1 virus-like particle (VLP) vaccines inexpensively and in large quantities. However, the system must be optimized to maximize yields and increase process efficiency. In this study, we optimized the production of two novel, chimeric HIV-1 VLP vaccine candidates (GagRT and GagTN) in insect cells. This was done by monitoring the effects of four specific factors on VLP expression: these were insect cell line, cell density, multiplicity of infection (MOI), and infection time. The use of western blots, Gag p24 ELISA, and four-factorial ANOVA allowed the determination of the most favorable conditions for chimeric VLP production, as well as which factors affected VLP expression most significantly. Both VLP vaccine candidates favored similar optimal conditions, demonstrating higher yields of VLPs when produced in the Trichoplusia ni Pro insect cell line, at a cell density of 1 x 10(6) cells/mL, and an infection time of 96 h post infection. It was found that cell density and infection time were major influencing factors, but that MOI did not affect VLP expression significantly. This work provides a potentially valuable guideline for HIV-1 protein vaccine optimization, as well as for general optimization of a baculovirus-based expression system to produce complex recombinant proteins.

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The gag proteins of human immunodeficiency virus type 1: mechanisms of virus assembly and possibilities for interference

Cited by 9 publications

References 67 publications

Mechanistic Investigation of HIV-1 Gag Association with Lipid Membranes

Mechanistic Investigation of HIV-1 Gag Association with Lipid Membranes

Defined Amino Acids in the Gag Proteins of Human Immunodeficiency Virus Type 1 Are Functionally Active during Virus Assembly

Optimization of chimeric HIV‐1 virus‐like particle production in a baculovirus‐insect cell expression system

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