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: 42.35). 08/2008; 454(7201):236-40. DOI: 10.1038/nature06998
Source: PubMed

ABSTRACT 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, Aug 26, 2015
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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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    • "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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    • "It has been known for decades that retroviruses can tolerate the incorporation of heterologous proteins (Jones et al., 1990; Weldon et al., 1990). Lentiviral particles (LPs) have been engineered to carry foreign proteins for the purpose of visualizing the intracellular behavior of the virus during infection (McDonald et al., 2002; Jouvenet et al., 2008) and altering the viral integration profile (Bushman, 1994; Goulaouic and Chow, 1996; Bushman and Miller, 1997), as well as for ferrying antiviral (Okui et al., 2000; Ao et al., 2008) and antitumor (Link et al., 2006; Miyauchi et al., 2012) protein therapeutics . As the delicate structural composition of HIV-1-derived lentiviral particles is easily disturbed by an inappropriate load of nonviral proteins, leading to suboptimal vector yields and/or reduced transduction capability, various strategies for transducing heterologous protein cargo have been scrutinized. "
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