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

Aaron Diamond AIDS Research Center, The Rockefeller University, New York, New York 10065, USA.
Nature (Impact Factor: 41.46). 08/2008; 454(7201):236-40. DOI: 10.1038/nature06998
Source: PubMed


Observations of individual virions in live cells have led to the characterization of their attachment, entry and intracellular transport. However, the assembly of individual virions has never been observed in real time. Insights into this process have come primarily from biochemical analyses of populations of virions or from microscopic studies of fixed infected cells. Thus, some assembly properties, such as kinetics and location, are either unknown or controversial. Here we describe quantitatively the genesis of individual virions in real time, from initiation of assembly to budding and release. We studied fluorescently tagged derivatives of Gag, the major structural component of HIV-1-which is sufficient to drive the assembly of virus-like particles-with the use of fluorescence resonance energy transfer, fluorescence recovery after photobleaching and total-internal-reflection fluorescent microscopy in living cells. Virions appeared individually at the plasma membrane, their assembly rate accelerated as Gag protein accumulated in cells, and typically 5-6 min was required to complete the assembly of a single virion. These approaches allow a previously unobserved view of the genesis of individual virions and the determination of parameters of viral assembly that are inaccessible with conventional techniques.

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Available from: Nolwenn Jouvenet, Oct 04, 2015
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    • "The HIV-1 Gag polyprotein is a key player in virus assembly due to its ability to form oligomers in the cytoplasm [5] [16] [22] [23] and at the PM [11] [12] [41] [50]. "
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    ABSTRACT: The Pr55 Gag of HIV-1 orchestrates viral particle assembly in producer cells, which requires the genomic RNA and a lipid membrane as scaffolding platforms. The nucleocapsid (NC) domain with its two invariant CCHC zinc fingers flanked by unfolded basic sequences is thought to direct genomic RNA selection, dimerization and packaging during virus assembly. To further investigate the role of NC domain, we analyzed the assembly of Gag with deletions in the NC domain in parallel with that of wild-type Gag using fluorescence lifetime imaging microscopy (FLIM) combined with Förster resonance energy transfer (FRET) in HeLa cells. We found that upon binding to nucleic acids, the NC domain promotes the formation of compact Gag oligomers in the cytoplasm. Moreover, the intracellular distribution of the population of oligomers further suggests that oligomers progressively assemble during their trafficking towards the plasma membrane (PM), but with no dramatic changes in their compact arrangement. This ultimately results in the accumulation at the PM of closely packed Gag oligomers that likely arrange in hexameric lattices, as revealed by the perfect match between the experimental FRET value with the one calculated from the structural model of Gag in immature viruses. The distal finger and flanking basic sequences, but not the proximal finger appear to be essential for Gag oligomer compaction and membrane binding. Moreover, the full NC domain was found to be instrumental in the kinetics of Gag oligomerization and intracellular trafficking. These findings further highlight the key roles played by the NC domain in virus assembly.
    Journal of Molecular Biology 01/2015; 427(6). DOI:10.1016/j.jmb.2015.01.015 · 4.33 Impact Factor
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    • "In subsequent sections we illustrate how different experimental systems have yielded complementary pieces of the HIV-1 assembly puzzle, while highlighting important questions that remain unanswered and concepts that could reconcile contrasting assembly models. Other topics related to HIV-1 assembly have been reviewed elsewhere and will only be mentioned here in passing, including gRNA trafficking and packaging (reviewed in Kuzembayeva et al., 2014; Lu et al., 2011), HIV-1 budding and release (reviewed in Votteler and Sundquist, 2013), HIV-1 maturation (reviewed in Sundquist and Krausslich, 2012), and the subcellular localization of HIV-1 assembly (reviewed in Jouvenet et al., 2008; Klein et al., 2007). "
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    ABSTRACT: During the late stage of the viral life cycle, HIV-1 Gag assembles into a spherical immature capsid, and undergoes budding, release, and maturation. Here we review events involved in immature capsid assembly from the perspective of five different approaches used to study this process: mutational analysis, structural studies, assembly of purified recombinant Gag, assembly of newly-translated Gag in a cell-free system, and studies in cells using biochemical and imaging techniques. We summarize key findings obtained using each approach, point out where there is consensus, and highlight unanswered questions. Particular emphasis is placed on reconciling data suggesting that Gag assembles by two different paths, depending on the assembly environment. Specifically, in assembly systems that lack cellular proteins, high concentrations of Gag can spontaneously assemble using purified nucleic acid as a scaffold. However, in the more complex intracellular environment, barriers that limit self-assembly are present in the form of cellular proteins, organelles, host defenses, and the absence of free nucleic acid. To overcome these barriers and promote efficient immature capsid formation in an unfavorable environment, Gag appears to utilize an energy-dependent, host-catalyzed, pathway of assembly intermediates in cells. Overall, we show how data obtained using a variety of techniques has led to our current understanding of HIV assembly.
    Virus Research 11/2014; 193. DOI:10.1016/j.virusres.2014.07.001 · 2.32 Impact Factor
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    • "During maturation, HIV also packages its reverse transcriptase and integrase enzymes (encoded by pol gene), which are required for infection (Seibert et al., 1995) (Fig. 2C). Complete assembly takes about 5–6 min (Jouvenet et al., 2008, 2009). The genomes of plant viruses are either monopartite or multipartite (segmented) (Rao, 2006). "
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    ABSTRACT: Genome encapsidation is an essential step in the life cycle of viruses. Viruses either use some of the most powerful ATP-dependent motors to compel the genetic material into the preformed capsid or make use of the positively charged proteins to bind and condense the negatively charged genome in an energy-independent manner. While the former is a hallmark of large DNA viruses, the latter is commonly seen in small DNA and RNA viruses. Discoveries of many complex giant viruses such as mimivirus, megavirus, pandoravirus, etc., belonging to the nucleo-cytoplasmic large DNA virus (NCLDV) superfamily have changed the perception of genome packaging in viruses. From what little we have understood so far, it seems that the genome packaging mechanism in NCLDVs has nothing in common with other well-characterized viral packaging systems such as the portal-terminase system or the energy-independent system. Recent findings suggest that in giant viruses, the genome segregation and packaging processes are more intricately coupled than those of other viral systems. Interestingly, giant viral packaging systems also seem to possess features that are analogous to bacterial and archaeal chromosome segregation. Although there is a lot of diversity in terms of host range, type of genome, and genome size among viruses, they all seem to use three major types of independent innovations to accomplish genome encapsidation. Here, we have made an attempt to comprehensively review all the known viral genome packaging systems, including the one that is operative in giant viruses, by proposing a simple and expanded classification system that divides the viral packaging systems into three large groups (types I–III) on the basis of the mechanism employed and the relatedness of the major packaging proteins. Known variants within each group have been further classified into subgroups to reflect their unique adaptations.
    Virology 10/2014; 466. DOI:10.1016/j.virol.2014.06.022 · 3.32 Impact Factor
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