This was subsequently demonstrated with recombinant HIV Gag protein; for technical reasons, this protein differed from authentic Gag in lacking the fatty acid moiety at its N-terminus and the p6 domain at its C-terminus [ 12 ].
Also, in the HIV case, the particles are morphologically abnormal, but their structural defect was corrected if there was a sizable deletion within the MA domain, or if an additional cofactor, inositol pentakisphosphate, was added to the assembly reaction [ 13 ].
Thus, both within the cell and in vitro, particle assembly seems to require nucleic acid, but there is no obvious specificity in this requirement. Ultimately, one would hope to understand the mechanisms underlying selective packaging of viral RNA at a detailed molecular level. Thus, many investigators have devoted years of effort to structural characterization of Gag, viral RNA, and their interactions.
These studies have been very challenging, but important progress has been made in recent years. As noted above, Gag is a multi-domain protein with flexible linkers between the independently folded domains. Thus, while there is extensive, detailed structural information about the individual domains, the flexibility of the connections between them means that there is no unique structure for the full-length protein.
The NC protein has been exhaustively analyzed from many points of view and performs a remarkable array of functions during retroviral replication. It is also an essential cofactor during the synthesis of DNA in the initial stages of infection.
This stems largely from its nucleic acid chaperone activity, i. In fact, during virus assembly the Gag protein, using its NC domain, catalyzes the unwinding of a specific cellular tRNA and its annealing to a complementary base sequence on the viral genomic RNA: this is essential for replication as the annealed tRNA will serve as the primer for DNA synthesis when the virus infects a new host cell [ 15 ].
It is highly basic pI 9. In fact all retroviral NC proteins except those of the spumaretroviruses, an outlier group of viruses not considered here contain one or two zinc fingers, and this spacing of the cysteines and histidines is absolutely conserved among them. The basic residues are found particularly in the N-terminal portion of the protein and between the two fingers.
The fingers, as well as the basic residues, are of great importance in interactions of NC and therefore of Gag with nucleic acids. As electrostatic interaction is nearly independent of the nucleotide sequence of the target nucleic acid, it is the fingers that are largely responsible for specificity in nucleic acidbinding. Thus, viruses whose zinc fingers are disrupted by single point mutations in the conserved cysteines or histidines can successfully assemble into virus-like particles, but they fail to selectively package genomic RNA [ 5 , 6 ].
No 3-dimensional structure has been determined for free NC protein. However, NMR studies indicate that both N-terminal and C-terminal regions of the protein are flexible, while the zinc fingers are rigid.
It is striking that the conformation of NC is different in the two complexes: in other words, the overall structure of NC is not fixed, but can adapt to optimize binding to RNA. Specifically, the highly basic N-terminal tail of the protein forms a 3 10 helix in both structures, but the position of this helix is different in the two structures.
The relative orientations of the two zinc fingers are also different in the structures. While this discussion focuses on the NC domain, it has recently become clear that the MA domain can also bind RNAs, and the biological significance of these interactions is under active investigation [ 18 - 20 ]. As noted above, retroviral genomic RNAs are always packaged as dimers. Computer predictions suggest that nucleotides of the RNA, i. In this structure, the initiation codon for Gag nt is at the base of SL4, and thus might be in a poor context for translational initiation.
However, as first pointed out by Berkhout and colleagues, the same sequence can be folded into a different secondary structure. In this alternate conformation, the DIS palindrome is occluded by intramolecular base-pairing and thus unavailable for intermolecular interactions [ 21 , 22 ], while the Gag initiation codon is partially exposed.
They proposed that the two structures correspond to the two functions of this RNA species: a packaging into virions, which evidently requires dimerization, and b translation into Gag and the Gag-Pol fusion protein.
It has recently been found that HIV RNA molecules in the infected cell differ with respect to the exact transcriptional start site, and that this in turn influences the proportions of the two conformations and the probability that a given RNA molecule will be packaged [ 23 , 24 ].
While the details of the two proposed structures have evolved with time, the basic idea is now widely accepted: the RNA can either assume a conformation in which the DIS is exposed, permitting dimerization as required for packaging, while the Gag AUG is base-paired with upstream sequences; or alternatively, a conformation in which the DIS is buried but the Gag AUG is exposed see Fig. In a major step forward in , Keane et al.
The model is based on NMR analysis of smaller RNAs designed to mimic the relevant portions of the real structure; in addition, these investigators constructed the RNA from fragments, some of which were deuterated and thus invisible to the NMR. Even with the judicious removal of unwanted sequences, the structure encompasses bases, and is thus considerably larger than any RNA structure previously determined by NMR.
Adapted from [ 25 , 35 ]. The structure is shown in Fig. This RNA is designed to represent one of the monomers in the dimeric RNA which will be packaged: in this model structure, the DIS stem-loop and the U5:AUG stem-loop are both intramolecular, but in the authentic dimer, the stems are apparently formed by intermolecular base-pairing [ 26 ]. As mentioned above, structures were previously determined for complexes of NC protein with individual RNA stem-loops [ 16 , 17 ]. A striking feature of these structures was the intimate interaction between the NC zinc fingers and unpaired or mispaired guanosine residues in the RNA, and it has often been suggested that these bases might be important in the recognition of genomic RNA by the NC domain of Gag.
In fact, yet another attractive feature of the new structure [ 25 ] is that it contains a number of unpaired G residues. Interestingly, independent studies of RNA packaging in murine leukemia virus from a distinct retroviral genus have arrived at analogous conclusions [ 28 ].
The nature of this advantage is evidently the key to selective packaging. It is technically challenging to measure the affinity of Gag for an RNA, since under many experimental conditions Gag-RNA complexes assemble into virus-like particles, precluding the determination of an equilibrium constant for the binding. This difficulty can be overcome, however, by making measurements at extremely low concentrations.
A pioneering study was carried out by Webb et al. Comas-Garcia et al. Both studies used recombinant HIV Gag protein lacking the myristate modification at its N-terminus and the p6 domain at its C-terminus. As it has been suggested that p6 influences interactions with RNA [ 31 ], its absence in this work adds one caveat to the conclusions. At the same time, further experimentation showed that this high-affinity binding to RNA represents the sum of both specific and non-specific interactions.
There are several ways that these interactions can be distinguished experimentally. For example, as the non-specific interaction is largely electrostatic, it is ablated by raising the salt concentration in the assay buffer. Webb et al. This hypothesis was recently tested experimentally in an in vitro assembly system [ 32 ], and all of the data were strongly supportive of this proposal.
As shown in Fig. The incorporation of the RNAs into virus-like particles was monitored by sedimenting them through sucrose gradients. Adapted from [ 32 ]. However, particle assembly is accomplished by interactions among a large number of Gag molecules.
Moreover, it seems plausible that there are two stages in particle assembly: a initiation or nucleation, and b polymerization. Types of Funding Opportunities. See Funded Projects. Sample Applications. Determine Eligibility. Prepare Your Application. Additional Application Elements. Research with Special Considerations. Submit an Application. Track Your Application. Understand the Review Process. Respond to Pre-Award Requests. After Award. Contract Solicitations.
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Profiles, Awards and Honors. Laboratory of Immunoregulation. Previous Directors. Office of the Director. Division of AIDS. Division of Allergy, Immunology, and Transplantation. This voluntary measure further reduces the theoretical viral load of plasma pools for the production of plasma derivatives.
The production and purification of individual proteins from plasma is not sufficient to completely rem ove HIV. Therefore additional validated procedures for an effective depletion and inactivation of viruses must be applied [ ].
No transmissions of HIV by plasma derivatives have been reported since the consistent implementation of effective methods for removing and inactivating viruses in the production process. Accordingly, the experimentally determined inactivation capacity of the manufacturing process is supported by epidemiologic data. HIV is sensitive to heat and detergents see 1.
Heat treatment of lyophilized products e. Because of the heat sensitivity of plasma proteins the inactivation procedures must be carried out under appropriate validated conditions [ ]. The product should optimally maintain its biologic activity and native conformation, while potentially contaminating viruses should be inactivated under the production conditions [ , ].
Further methods for inactivation of HIV and other viruses in blood components have been developed. These are chemical e. Clinical trials concerning the potency and tolerability of plasma treated with methylene blue, inactivated with SD or treated with amotosalen [ ] and riboflavin [ ] have been performed only to a limited extent [ ].
A further substance to inactivate HIV in whole blood or packed red blood cells is S [ ]. Validation of the various removal and inactivation steps must be carried out following the actual production processes using HIV [ , , ]. HIV can be propagated to sufficient amounts in cell culture, enabling the spiking of the different source materials under laboratory conditions.
Individual steps have to be investigated mimicking the different production processes with regard to their virus removal or inactivation capacity by determining the infectious titres at the start and the end of each production step. The occurrence of HIV in the blood and plasma donors collectives since has been leading to a continuous increase in the viral safety requirements for the production of blood components and plasma derivatives.
The initially observed diagnostic window period of days is now reduced to about 11 days due to the introduction of HIV NAT. No HIV infection with fresh frozen plasma, which is subject to quarantine storage, has become known since Only products with a high safety margin regarding HIV and hepatitis viruses are approved by the authorities.
After detection of circulating HIV strains that were not detected in NAT assays due to mutation or deletion of primer-binding regions, it has become mandatory to use dual-target NAT in order to increase the safety of blood products. However, HIV is genetically variable, and there is a need for continuous research to sustain the achieved level of safety.
Continuous monitoring of circulating viruses is necessary to enable the detection of emerging new variants of HIV as early as possible and to enable accordingly the adaption and improvement of HIV detection test systems.
National Center for Biotechnology Information , U. Journal List Transfus Med Hemother v. Transfus Med Hemother. Published online May 9. Author information Article notes Copyright and License information Disclaimer.
Received Jan 13; Accepted Feb Karger GmbH, Freiburg. This article has been cited by other articles in PMC. Open in a separate window. Table 1 Overview of HIV-1 proteins and their function.
Table 2 Newly diagnosed HIV infections and modes of transmission in different European countries in www. Confirmatory Test Western blot, Immunoblot Because ELISAs were developed also for the detection of low antibody levels with the highest sensitivity, false-positive results occur, especially when immune complexes are present in the serum, e.
Permanently deferred from donation are the following: — Persons with a confirmed HIV infection. Cost-Benefit Calculation In view of the high costs of NAT and the currently low incidence in blood donors, the financial investment for the elimination of infectious, but still HIV antibody-negative donations is high, but justified.
HIV Vaccination Since , attempts were made to develop a vaccine against HIV with high expenditures regarding both personnel and finances. Table 3 Overview of the drugs available for HIV therapy — see also [ ].
Martin Aepfelbacher Dr. Ursula Bauerfeind PD Dr. Reinhard Burger Prof. Margarethe Heiden Prof. Martin Hildebrandt Prof. Bernd Jansen Dr. Ruth Offergeld Prof. Georg Pauli Dr. Uwe Schlenkrich Dr. Volkmar Schottstedt Prof. Rainer Seitz Dr. Johanna Strobel Dr. Hannelore Willkommen. References 1. Luciw PA. Human immunodeficiency viruses and their replication. In: Fields BN, editor. Philadelphia: Lippincott-Raven; Origin of HIV-1 in the chimpanzee Pan troglodytes troglodytes.
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