Review
Structural Determinants and Mechanism of HIV-1 Genome Packaging

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Abstract

Like all retroviruses, the human immunodeficiency virus selectively packages two copies of its unspliced RNA genome, both of which are utilized for strand-transfer-mediated recombination during reverse transcription—a process that enables rapid evolution under environmental and chemotherapeutic pressures. The viral RNA appears to be selected for packaging as a dimer, and there is evidence that dimerization and packaging are mechanistically coupled. Both processes are mediated by interactions between the nucleocapsid domains of a small number of assembling viral Gag polyproteins and RNA elements within the 5′-untranslated region of the genome. A number of secondary structures have been predicted for regions of the genome that are responsible for packaging, and high-resolution structures have been determined for a few small RNA fragments and protein–RNA complexes. However, major questions regarding the RNA structures (and potentially the structural changes) that are responsible for dimeric genome selection remain unanswered. Here, we review efforts that have been made to identify the molecular determinants and mechanism of human immunodeficiency virus type 1 genome packaging.

Introduction

During the late phase of the viral replication cycle, the human immunodeficiency virus type 1 (HIV-1) selectively and efficiently packages two copies of its positive strand, unspliced, 5′-capped, and 3′-polyadenylated RNA genome by a mechanism that has been extensively studied (for previous reviews, see Refs. 1–9) but remains only partially understood. The packaging mechanism efficiently discriminates against the monomeric genome, the spliced viral mRNAs that encode for viral accessory envelope proteins, and the more highly abundant cellular mRNAs.2 Packaging is mediated by the retroviral Gag proteins, which can efficiently assemble in the absence of their native genomes by incorporating an equivalent amount of cellular RNAs.10, 11, 12, 13, 14 Although retroviruses can package essentially any RNA (some mutants even package ribosomes14), RNAs containing the appropriate viral packaging signals are efficiently enriched in assembling virions. The Gag protein contains three independently folded domains (from N- to C-terminus): matrix (MA), capsid (CA), and nucleocapsid (NC), as well as three other unstructured but functionally important segments (Fig. 1). Genome selection appears to proceed via the direct binding of NC to conserved RNA packaging signals, called Ψ-sites, that are generally located near the 5′-end of the viral RNA. It now seems likely that a ribonucleoprotein complex comprising a relatively small number of Gag molecules and two copies of the genome is trafficked to plasma membrane (PM) assembly sites, where several thousand additional Gag molecules localize and assemble to form an immature virus particle.15, 16 During or shortly after budding, the Gag proteins are cleaved by the viral protease to produce the mature MA, CA, and NC proteins, which rearrange to form the mature and infectious virus particle (Fig. 1).

The requirement for two genome molecules is intriguing since all other viruses contain only a single copy of their genetic material.17 Both RNA molecules are utilized for strand-transfer-mediated recombination during reverse transcription,18, 19 but only one DNA allele is generated, and retroviruses are therefore considered “pseudodiploid.” Recombination appears to enhance viral fitness in several ways. It enables strand-transfer-mediated readthrough at sites of RNA damage,20, 21 which could serve as a mechanism for dealing with what appears to be a relatively fragile genome22 and as a defense against restriction nucleases.23 In addition, although cells containing a single integrated provirus can only produce homozygous virions, cells from infected individuals have sometimes been observed to contain several integrated proviruses,24, 25, 26 which probably explains the very high number of circulating recombinant forms of HIV-1.27 Thus, strand-transfer-mediated recombination from heterozygotes likely serves as a primary pathway for the rapid evolution of viruses that are resistant to antiretroviral therapies.28 Retroviral genomes exist as weak, non-covalently linked dimers in immature and young virus particles, and the stability of the RNA dimer29, 30, 31, 32, 33, 34 increases with virus age, which might be important for subsequent reverse transcription events. The genome also appears to play a structural role in virus assembly, although this function can also be achieved by cellular RNAs.13

As for most other retroviruses, the nucleotides that participate in HIV-1 genome selection appear to reside near the 5′-end of the genome and primarily within the 5′-untranslated region (5′-UTR).1, 2, 3, 4, 5, 6, 7, 8, 9 Relatively short elements within the 5′-UTR that are independently capable of directing heterologous RNAs into assembling virus-like particles (VLPs) have been identified for some retroviruses [e.g., the Rous sarcoma virus (RSV)35, 36, 37, 38, 39, 40 and Moloney murine leukemia virus (MoMuLV)41, 42], but HIV-1 appears to require most of its 5′-UTR43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 as well as downstream nucleotides within the gag coding region59, 60, 61 for optimal packaging efficiency. The 5′-UTR is the most conserved region of the HIV-1 genome†,62, 63 and in addition to promoting packaging, it also helps regulate or promote transcriptional activation, splicing, primer binding during reverse transcription, and dimerization. For some retroviruses, elements known to be important for packaging reside downstream of the major 5′ splice donor (SD) site, providing a potential mechanism for selecting the full-length genome and ignoring spliced viral mRNAs.64 RNA elements that are believed to facilitate HIV-1 genome packaging reside near elements that promote RNA dimerization,3, 4, 6, 8 and since both processes are promoted by the NC domain of Gag, it is likely that genome dimerization and packaging are intimately coupled.1, 2, 65 This now certainly seems to be true for the evolutionarily distant MoMuLV.66, 67, 68 Efforts to elucidate the structural determinants and mechanisms that regulate these activities have been made using a variety of biochemical, in vivo packaging, and computational approaches, and there is general consensus that some activities are controlled by well-defined hairpin structures within the HIV-1 5′-UTR.48, 49, 50, 54, 57, 69, 70, 71, 72, 73, 74, 75 However, there is less agreement regarding the structures that regulate genome packaging. Here, we review efforts that have been made to understand the mechanism of HIV-1 genome selection, with emphasis on the RNA and protein structures that appear to play important roles.

Section snippets

Genome Selection Is Mediated by the NC Domain of Gag

There is now considerable evidence that genome selection is mediated primarily by the NC domain of the HIV-1 Gag polyprotein. Substitution of the HIV-1 NC domain by that of MoMuLV leads to the preferential packaging of the MoMuLV RNA76 into HIV-1-derived chimeric virions, and conversely, substitution of the MoMuLV NC domain by that of HIV-1 results in preferential packaging of the HIV-1 genome.77 Similar results have been observed for other retroviruses.78 Retroviruses belonging to a given

Potential Roles for the MA Domain in Genome Packaging

The MA domain of Gag plays important roles in intracellular trafficking and membrane targeting142, 143, 144, 145 and by this means may also be important for genome packaging. In 1993, Nash et al. showed that approximately 18% of the cellular MoMuLV Gag protein is associated with the nucleus, and a role of nuclear Gag in regulating splicing and/or dimerization activities was proposed.146 Dupont et al. subsequently reported that mutating two basic residues in the HIV-1 MA domain of Gag can lead

Genomes Are Selected for Packaging as Dimers

Early studies by Mangel et al. showed that RNAs extracted from the RSV under mild denaturing conditions were dimeric, as determined by sucrose gradient sedimentation, but formed monomers in the presence of the gene-32 protein that unwinds double-helical nucleic acid structures and preferentially binds to single-stranded RNAs.167 These studies showed that the RNA in virions exists as non-covalently linked dimers, but they did not rule out the possibility that the RNAs might be recognized by Gag

Structural Studies of the HIV-1 5′-UTR

Over the past 20 years, considerable effort to identify the RNA residues and structures that are responsible for genome selection and packaging has been made by many research groups. Most studies indicate that the primary packaging determinants reside within the 5′-UTR, and most structural studies therefore focused on this region of viral genomes. Deletion mutagenesis studies have shown that ∼ 120 nt located upstream of the Gag start codon are required for efficient packaging.43, 44, 45, 46, 48,

The 5′-UTR Can Adopt Multiple Conformations—A Regulatory Role?

There is considerable evidence that the HIV-1 genome, and its 5′-UTR, can adopt multiple conformations. Even the earliest nucleotide-probing experiments by Baudin et al. suggested that the dimeric form of the 5′-end of the viral genome (residues 1–500) likely adopts multiple distinct and mutually exclusive structures that could modulate the function of the 5′-UTR.73 The isolated, recombinant HIV-1 5′-UTR (residues 1–373) appears to form a mixture of monomers and dimers, and native gel

Overview and Future Directions

A number of findings made over the past 5 years or so have significantly advanced our understanding of the mechanism of HIV-1 genome packaging. Fluorescence imaging studies have enabled real-time visualization in living cells of RNA trafficking, the assembly of Gag proteins, and the recruitment of cellular proteins during virus assembly.15, 16, 273, 274, 275 These studies have shown that genome trafficking to virus assembly sites is dependent on Gag, that virions do not assemble repeatedly at

Acknowledgement

Support from the National Institutes of Health (R01 GM42561 and AI30917) is gratefully acknowledged.

References (277)

  • BaudinF. et al.

    Functional sites in the 5′ region of human immunodeficiency virus type 1 RNA form defined structural domains

    J. Mol. Biol.

    (1993)
  • HendersonL.E. et al.

    Primary structure of the low molecular weight nucleic acid-binding proteins of murine leukemia viruses

    J. Biol. Chem.

    (1981)
  • ReinA.

    Retroviral RNA packaging: a review

    Arch. Virol.

    (1994)
  • BerkowitzR. et al.

    RNA packaging

    Curr. Top. Microbiol. Immunol.

    (1996)
  • GreatorexJ. et al.

    Retroviral RNA dimer linkage

    J. Gen. Virol.

    (1998)
  • JewellN.A. et al.

    In the beginning: genome recognition, RNA encapsidation and the initiation of complex retrovirus assembly

    J. Gen. Virol.

    (2000)
  • PaillartJ.C. et al.

    Dimerization of retroviral RNA genomes: an inseparable pair

    Nat. Rev., Microbiol.

    (2004)
  • RussellR.S. et al.

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

    Retrovirology

    (2004)
  • GreatorexJ.

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

    Retrovirology

    (2004)
  • D'SouzaV. et al.

    How retroviruses select their genomes

    Nat. Rev., Microbiol.

    (2005)
  • PoonD.T.K. et al.

    Charged amino acid residues of human immunodeficiency virus type-1 nucleocapsid P7 protein involved in RNA pacakaging and infectivity

    J. Virol.

    (1996)
  • CimarelliA. et al.

    Basic residues in human immunodeficiency virus type 1 nucleocapsid promote virion assembly via interaction with RNA

    J. Virol.

    (2000)
  • WangS.W. et al.

    RNA incorporation is critical for retroviral particle integrity after cell membrane assembly of Gag complexes

    J. Virol.

    (2002)
  • MuriauxD. et al.

    RNA is a structural element in retrovirus particles

    Proc. Natl Acad. Sci. USA

    (2001)
  • MuriauxD. et al.

    Murine leukemia virus nucleocapsid mutant particles lacking viral RNA encapsidate ribosomes

    J. Virol.

    (2002)
  • JouvenetN. et al.

    Imaging the interaction of HIV-1 genomes and Gag during assembly of individual viral particles

    Proc. Natl Acad. Sci. USA

    (2009)
  • JouvenetN. et al.

    Imaging the biogenesis of individual HIV-1 virions in live cells

    Nature

    (2008)
  • VogtV.M.

    Retroviral virions and genomes

  • HuW.S. et al.

    Genetic consequences of packaging two RNA genomes in one retroviral particle: pseudodiploidy and high rate of genetic recombination

    Proc. Natl Acad. Sci. USA

    (1990)
  • HuW.S. et al.

    Retroviral recombination and reverse transcription

    Science

    (1990)
  • KingS.R. et al.

    Pseudodiploid genome organization aids full-length human immunodeficiency virus type 1 DNA synthesis

    J. Virol.

    (2008)
  • CoffinJ.M.

    Structure, replication, and recombination of retrovirus genomes: some unifying hypotheses

    J. Gen. Virol.

    (1979)
  • Onafuwa-NugaA. et al.

    The remarkable frequency of human immunodeficiency virus type 1 genetic recombination

    Microbiol. Mol. Biol. Rev.

    (2009)
  • GrattonS. et al.

    Highly restricted spread of HIV-1 and multiply infected cells within splenic germinal centers

    Proc. Natl Acad. Sci. USA

    (2000)
  • JungA. et al.

    Recombination: multiply infected spleen cells in HIV patients

    Nature

    (2002)
  • Quinones-MateuM.E. et al.

    Recombination in HIV-1: update and Implications

    AIDS Rev.

    (1999)
  • McCutchanF.E.

    Global epidemiology of HIV

    J. Med. Virol.

    (2006)
  • NoraT. et al.

    Contribution of recombination to the evolution of human immunodeficiency viruses expressing resistance to antiretroviral treatment

    J. Virol.

    (2007)
  • BenderW. et al.

    High-molecular-weight RNAs of AKR, NZB and wild mouse viruses and avian reticuloendotheliosis virus all have similar dimer structures

    J. Virol.

    (1978)
  • MaiselJ. et al.

    Structure of 50 to 70S RNA from Moloney sarcoma viruses

    J. Virol.

    (1978)
  • MurtiK.G. et al.

    Secondary structural features in the 70S RNAs of Moloney murine leukemia and Rous sarcoma viruses as observed by electron microscopy

    J. Virol.

    (1981)
  • FuW. et al.

    Maturation of dimeric viral RNA of Moloney murine leukemia virus

    J. Virol.

    (1993)
  • Oritz-CondeB.A. et al.

    Studies of the genomic RNA of leukosis viruses: implications for RNA dimerization

    J. Virol.

    (1999)
  • ArnoffR. et al.

    Specificity of retroviral RNA packaging

    J. Virol.

    (1991)
  • ArnoffR. et al.

    Avian retroviral RNA encapsidation: reexamination of functional 5′ RNA sequences and the role of nucleocapsid Cys-His motifs

    J. Virol.

    (1993)
  • BanksJ.D. et al.

    A minimal avian retroviral packaging sequence has a complex structure

    J. Virol.

    (1998)
  • Doria-RoseN.A. et al.

    In vivo selection of Rous sarcoma virus mutants with randomized sequences in packaging signals

    J. Virol.

    (1998)
  • BanksJ.D. et al.

    An MΨ containing heterologous RNA, but not env mRNA, is efficiently packaged into avian retroviral particles

    J. Virol.

    (1999)
  • BanksJ.D. et al.

    Secondary structure analysis of a minimal avian leukosis-sarcoma virus packaging signal

    J. Virol.

    (2000)
  • AdamM.A. et al.

    Identification of a signal in a murine retrovirus that is sufficient for packaging of nonretroviral RNA into virions

    J. Virol.

    (1988)
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