Is HIV-1 RNA dimerization a prerequisite for packaging? Yes, no, probably?

During virus assembly, all retroviruses specifically encapsidate two copies of full-length viral genomic RNA in the form of a non-covalently linked RNA dimer. The absolute conservation of this unique genome structure within the Retroviridae family is strong evidence that a dimerized genome is of critical importance to the viral life cycle. An obvious hypothesis is that retroviruses have evolved to preferentially package two copies of genomic RNA, and that dimerization ensures the proper packaging specificity for such a genome. However, this implies that dimerization must be a prerequisite for genome encapsidation, a notion that has been debated for many years. In this article, we review retroviral RNA dimerization and packaging, highlighting the research that has attempted to dissect the intricate relationship between these two processes in the context of HIV-1, and discuss the therapeutic potential of these putative antiretroviral targets

The retroviral RNA dimer linkage: different structures may reflect different roles.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Retroviruses are unique among virus families in having dimeric genomes. The RNA sequences and structures that link the two RNA molecules vary, and these differences provide clues as to the role of this feature in the viral lifecycles. This review draws upon examples from different retroviral families. Differences and similarities in both secondary and tertiary structure are discussed. The implication of varying roles for the dimer linkage in related viruses is considered

6th International Symposium on Retroviral Nucleocapsid

Retroviruses and LTR-retrotransposons are widespread in all living organisms and, in some instances such as for HIV, can be a serious threat to the human health. The retroviral nucleocapsid is the inner structure of the virus where several hundred nucleocapsid protein (NC) molecules coat the dimeric, genomic RNA. During the past twenty years, NC was found to play multiple roles in the viral life cycle (Fig. 1), notably during the copying of the genomic RNA into the proviral DNA by viral reverse transcriptase and integrase, and is therefore considered to be a prime target for anti-HIV therapy. The 6th NC symposium was held in the beautiful city of Amsterdam, the Netherlands, on the 20th and 21st of September 2007. All aspects of NC biology, from structure to function and to anti-HIV vaccination, were covered during this meeting

Analysis of the contribution of reverse transcriptase and integrase proteins to retroviral RNA dimer conformation

All retroviruses contain two copies of genomic RNA that are linked noncovalently. The dimeric RNA of human immunodeficiency virus type 1 (HIV-1) undergoes rearrangement during virion maturation, whereby the dimeric RNA genome assumes a more stable conformation. Previously, we have shown that the packaging of the HIV-1 polymerase (Pol) proteins reverse transcriptase (RT) and integrase (IN) is essential for the generation of the mature RNA dimer conformation. Analysis of HIV-1 mutants that are defective in processing of Pol showed that these mutant virions contained altered dimeric RNA conformation, indicating that the mature RNA dimer conformation in HIV-1 requires the correct proteolytic processing of Pol. The HIV-1 Pol proteins are multimeric in their mature enzymatically active forms; RT forms a heterodimer, and IN appears to form a homotetramer. Using RT and IN multimerization defective mutants, we have found that dimeric RNA from these mutant virions has the same stability and conformation as wild-type RNA dimers, showing that the mature enzymatically active RT and IN proteins are dispensable for the generation of mature RNA dimer conformation. This also indicated that formation of the mature RNA dimer structure occurs prior to RT or IN maturation. We have also investigated the requirement of Pol for RNA dimerization in both Mason-Pfizer monkey virus (M-PMV) and Moloney murine leukemia virus (MoMuLV) and found that in contrast to HIV-1, Pol is dispensable for RNA dimer maturation in M-PMV and MoMuLV, demonstrating that the requirement of Pol in retroviral RNA dimer maturation is not conserved among all retroviruses. <br /

Wrapping up the bad news: HIV assembly and release.

The late Nobel Laureate Sir Peter Medawar once memorably described viruses as 'bad news wrapped in protein'. Virus assembly in HIV is a remarkably well coordinated process in which the virus achieves extracellular budding using primarily intracellular budding machinery and also the unusual phenomenon of export from the cell of an RNA. Recruitment of the ESCRT system by HIV is one of the best documented examples of the comprehensive way in which a virus hijacks a normal cellular process. This review is a summary of our current understanding of the budding process of HIV, from genomic RNA capture through budding and on to viral maturation, but centering on the proteins of the ESCRT pathway and highlighting some recent advances in our understanding of the cellular components involved and the complex interplay between the Gag protein and the genomic RNA.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Investigation of the sequences and structural elements required for HIV-2 infectivity and genome dimerisation

A unique feature of retroviruses is that two copies of the genomic RNA are packaged in each particle. The selective encapsidation of viral genomes is ensured by the binding of the Nucleocapsid to a specific motif on the RNA genome, the packaging signal (Psi). The Psi regions of many retroviruses overlap with sequences that promote the dimerisation of the genome, the dimerisation initiation site (DIS), and it has been suggested that the two mechanisms are closely linked. The aim of the research presented herein was to identify the sequences and structural elements required for the dimerisation of HIV-2 genomic RNA and to investigate the relationship between HIV-2 genome dimerisation and encapsidation, infectivity and particle morphogenesis. Mutations of two palindromic sequences, introduced in an infectious molecular clone of the HIV-2rod isolate, revealed that a palindrome within HIV-2 Psi was important for genome dimerisation. In contrast with previous studies, the palindrome termed DIS is not required for genome dimerisation and viral replication. Viruses bearing mutations within the Psi region failed to dimerise and to replicate in T-cells, a defect that could not be rescued by targeting more genomes to the cells. Psi-deleted viruses also displayed a defect in particle morphogenesis. A reduced packaging efficiency, combined with the presence of RNA monomers or unstable dimers in these virions, resulted in the production of fewer mature particles. However an increase in the number of particles containing two cores was observed. Further characterisation of the sequences and structural elements required for RNA dimerisation, packaging and viral replication showed that the formation of stem B is not critical for viral replication. However, a GGAG purine-rich motif at position 392-395 of the HIV-2rod genome is absolutely essential for genome dimerisation and viral infectivity, and a correlation was observed between dimer formation and viral replication

Virus assembly, allostery, and antivirals

Assembly of virus capsids and surface proteins must be regulated to ensure that the resulting complex is an infectious virion. In this review, we examine assembly of virus capsids, focusing on hepatitis B virus and bacteriophage MS2, and formation of glycoproteins in the alphaviruses. These systems are structurally and biochemically well-characterized and are simplest-case paradigms of self-assembly. Published data suggest that capsid and glycoprotein assembly is subject to allosteric regulation, that is regulation at the level of conformational change. The hypothesis that allostery is a common theme in viruses suggests that deregulation of capsid and glycoprotein assembly by small molecule effectors will be an attractive antiviral strategy, as has been demonstrated with hepatitis B virus

Proteolytic cleavage events in the maturation of HIV-1 reverse transcriptase

Each of the HIV-1 pol-encoded enzymes, protease (PR), reverse transcriptase (RT) and integrase (IN) are released during virion maturation and are active only as dimers. Of the three, only RT comprises subunits of different mass. RT in mature infectious virions is a heterodimer of 66 kDa and 51 kDa subunits, even though its gene encodes a 66 kDa protein. The RT p51 subunit is formed by HIV-1 PR-catalyzed cleavage of RT p66, resulting in the removal of a ribonuclease H (RNH) domain. Given the existence of completely active recombinant p66/66 RT homodimers and alternative RT oligomers in other retroviruses, the apparent need for p66/51 RT heterodimers in the HIV-1 virion is unclear. To determine why the generation of active viral RT requires three processing events, we introduced mutations in the p51-RNH and RT-IN protease cleavage sites of an infectious HIV-1 molecular clone. Mutation of the RT-IN cleavage site had no effect on the activity or proteolytic stability of the p98/51 RT product, although infectivity was severely attenuated. This result was similar to findings previously reported for the PR-RT cleavage site. Surprisingly, mutation of the internal p51-RNH cleavage site did not increase RT p66 content, but resulted in attenuated virus containing greatly decreased levels of RT that was primarily RT p51. We further identified a compensatory second-site mutation T477A, found to restore RT activity and processing to p66/51 RT when introduced in the background of p51-RNH cleavage site mutations. These studies demonstrate that cleavage of the internal p51-RNH junction, not the flanking N-terminal or C-terminal junctions is essential for proteolytic stability of functional RT during virion maturation. These findings further emphasize the importance of the RNH domain in regulating proteolytic generation of p66/51 RT. The overall need for the RT heterodimer is attributable to the generation of its subunits. Formation of the 51 kDa subunit or cleavage of the p51-RNH junction is essential for RT stability in the virion, whereas formation of the 66 kDa subunit is important for efficient viral replication

INSIGHTS INTO THE PACKAGING OF MOUSE MAMMARY TUMOR VIRUS (MMTV) GENOMIC RNA BY IDENTIFYING PR77ᴳᴬᴳ BINDING SITES INVOLVED DURING ITS SELECTIVE ENCAPSIDATION

Selective encapsidation and/or packaging of retroviral genomic RNA (gRNA) by Gag during retrovirus assembly is a crucial step for generating infectious virus particles. Despite having been studied extensively, the mechanism by which the retroviral Gag precursor selects and packages the retroviral genome remains largely unclear. Therefore, to understand the molecular mechanism(s) of mouse mammary tumor virus (MMTV) gRNA packaging, as a first step, expression of full-length recombinant Pr77Gag -His6-tag fusion protein in bacteria was done. The recombinant Gag protein was then purified from the soluble fractions of bacterial cultures using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). The purified recombinant Pr77Gag -His6-tag protein retained the ability to assemble in vitro into virus-like particles (VLPs). In parallel, the VLPs made in vivo following expression of the recombinant Pr77Gag -His6-tag fusion protein in eukaryotic cells could package MMTV subgenomic RNAs. Next, RNA binding and footprinting assays using the purified protein and in cell gRNA packaging experiments identified two critical, non-redundant Pr77Gag binding sites. These binding sites include: i) a stretch of purines in a hairpin loop immediately adjacent to the dimerization initiation site (DIS) hairpin, thus forming a bifurcated stem-loop structure and ii) the primer binding site (PBS). Despite the presence of the packaging signals on both unspliced and spliced RNAs, Pr77Gag specifically bound to unspliced RNA, which is the only one that can adopt the native bifurcated stem-loop structure. Together this study demonstrates the minimal packaging elements at both sequence and structural levels required to initiate MMTV gRNA packaging. Unlike purine rich regions, the direct involvement of PBS in retroviral gRNA packaging has not been documented in retroviruses. These findings add to the knowledge of retroviral gRNA packaging and assembly, making it a potential target for novel therapeutic approaches as well as the development of safer gene therapy vectors

Implications of the Nucleocapsid and the Microenvironment in Retroviral Reverse Transcription

This mini-review summarizes the process of reverse-transcription, an obligatory step in retrovirus replication during which the retroviral RNA/DNA-dependent DNA polymerase (RT) copies the single-stranded genomic RNA to generate the double-stranded viral DNA while degrading the genomic RNA via its associated RNase H activity. The hybridization of complementary viral sequences by the nucleocapsid protein (NC) receives a special focus, since it acts to chaperone the strand transfers obligatory for synthesis of the complete viral DNA and flanking long terminal repeats (LTR). Since the physiological microenvironment can impact on reverse-transcription, this mini-review also focuses on factors present in the intra-cellular or extra-cellular milieu that can drastically influence both the timing and the activity of reverse-transcription and hence virus infectivity