Review
HIV-1 Vif versus the APOBEC3 cytidine deaminases: An intracellular duel between pathogen and host restriction factors

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Abstract

The Vif protein of HIV is essential for the effective propagation of this pathogenic retrovirus in vivo. Vif acts by preventing virion encapsidation of two potent antiviral factors, the APOBEC3G and APOBEC3F cytidine deaminases. Decreased encapsidation in part involves Vif-mediated recruitment of a ubiquitin E3 ligase complex that promotes polyubiquitylation and proteasome-mediated degradation of APOBEC3G/F. The resultant decline in intracellular levels of these enzymes leads to decreased encapsidation of APOBECG/F into budding virions. This review discusses recent advances in our understanding of the dynamic interplay of Vif with the antiviral APOBEC3 enzymes.

Introduction

Human immunodeficiency virus-1 (HIV-1) encodes four accessory proteins—Vif, Vpu, Vpr, and Nef. Early studies indicated that these accessory proteins are not always required for viral replication in cell cultures, but each is important for the success of natural infections. These accessory proteins often function by modulating host immune responses, including countering the intrinsic antiviral effects of host restriction factors (Bieniasz, 2004, Malim and Emerman, 2008). Vif, a cytoplasmic, 23-kDa basic phosphoprotein encoded during late stages of the HIV-1 life cycle, is a notable example. Vif is conserved among all lentiviruses except equine infectious anemia virus, suggesting a prominent role in the life cycle of these retroviruses. Remarkably, its precise function remained mysterious for many years.

Section snippets

Early Vif observations

Soon after the discovery of HIV-1, it became apparent that viral infectivity factor (Vif) is required for HIV replication in some, but not all cell types (Fisher et al., 1987, Gallo et al., 1988, Sodroski et al., 1986, Strebel et al., 1987). Specifically, HIV-1 virions lacking Vif (ΔVif HIV-1) can only spread in so-called “permissive” adherent cell cultures (e.g., HeLa and 293T) and various leukemic T-cell lines (e.g., CEM-SS and SupT1); they fail to spread in “nonpermissive” cells, which

Discovery of A3G

Using a subtractive hybridization approach between the nonpermissive CEM cell line and the closely related but permissive cell line CEM-SS, Sheehy and colleagues identified this inhibitor as CEM-15 (Sheehy et al., 2002), now known as APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G, or A3G); its expression in permissive cells is sufficient to render these cells nonpermissive. This finding showed that A3G can block HIV-1 replication in the absence of Vif (Fig. 1).

A3G

Intravirion packaging of A3G/F

In nonpermissive cells, inhibition of ΔVif HIV-1 by A3G only occurs during the next round of viral infection, indicating that A3G exerts its antiviral activity in the target cell. A3G is effectively incorporated into budding ΔVif virions, providing a potential mechanism for how A3G influences the infectivity of progeny virions in the next target cell (Gaddis et al., 2003, Sheehy et al., 2002, Suspene et al., 2004) (Fig. 1). The amount of A3G molecules incorporated into the budding virus is

Cytidine deaminase activity of A3G and A3F and inhibition of viral growth

Once incorporated into the virion, A3G is introduced into the next target cell as a result of virion fusion. Within the cell, the enzyme triggers massive deamination, converting specific dC residues to dU during synthesis of the minus-strand viral DNA (Suspene et al., 2004, Yu et al., 2004a) (Fig. 1, point 2). During synthesis of the DNA plus-strand, adenosines are incorporated instead of the original guanines, resulting in G-to-A mutation (Harris et al., 2003, Mangeat et al., 2003, Mariani et

A3G and A3F exert antiviral effects independent of their deaminase activity

Although the antiviral activity of A3 proteins is clearly linked to their cytidine deaminase activity (Mangeat et al., 2003, Shindo et al., 2003, Zhang et al., 2003) (Fig. 2, point 2), increasing evidence suggests that the A3G and A3F proteins also exert antiviral activity independently of cytidine deamination (Fig. 1, point 1). Specifically, mutagenesis of key amino acids at the center of the enzymatically active CDA2 prevents deaminase activity (Navarro et al., 2005, Newman et al., 2005), yet

Vif-mediated degradation of A3G

A3G can effectively inhibit the spread of ΔVif HIV-1 but not wildtype HIV-1. A principal function of the Vif protein is to circumvent the action of A3G (Fig. 2). After the discovery of A3G, it was observed that Vif reduces A3G incorporation into the budding virion by ∼99% (Kao et al., 2003, Mariani et al., 2003, Marin et al., 2003, Mehle et al., 2004b, Sheehy et al., 2003, Stopak et al., 2003), suggesting that Vif inhibits the packaging of A3G into virions. To achieve this, Vif could directly

Vif facilitates HIV-1 in addition to promoting the degradation of APOBEC3

Several mechanisms have been proposed to explain how Vif increases the viral infectivity of HIV-1 besides degrading A3 proteins. For instance, Vif can deplete intracellular A3G by impairing the translation of its mRNA (Kao et al., 2003, Mariani et al., 2003, Stopak et al., 2003). Vif achieves this by binding to the 3′UTR and the 5′UTR of the A3G mRNA (Mercenne et al., 2010) (Fig. 2, point 2).

Vif also directly prevents the encapsidation of A3G, as shown by Vif’s ability to inhibit the packaging

Interaction between Vif, A3G, and A3F

Because it is difficult to express soluble full-length A3G or Vif at high levels in prokaryotic cells or insect cells, data on the three-dimensional structure of the A3G–Vif interaction are not yet available (Auclair et al., 2007, Iwatani et al., 2006, Reingewertz et al., 2009, Stanley et al., 2008). However, considerable information has emerged from the analysis of mutants in Vif–A3G interaction assays (Fig. 3).

The interaction between A3G and Vif is critically dependent on amino acids 128–130

Other A3 proteins

As noted above, the A3 family is comprised of seven members, A3A–H (Conticello et al., 2005). A3G and A3F have been the most intensively studied members due to their ability to restrict HIV spread. These proteins are over 50% identical, and both use the same conserved amino acids to bind RNA and to facilitate cytidine deamination. Like A3G, A3F acts on the minus-strand DNA, resulting in G-to-A mutation on the plus-strand. However, unlike A3G, whose target sequence is CC, the target sequence of

Intravirion encapsidation of Vif

Vif is an RNA-binding protein, but it binds RNA in a relatively nonspecific manner. This is illustrated by its effective binding to homopolymeric RNA (Zhang et al., 2000). Importantly, Vif interacts with HIV-1 genomic RNA in the cytoplasm of infected cells (Dettenhofer and Yu, 1999, Khan et al., 2001, Zhang et al., 2000). In this case, Vif binds in a cooperative manner to the 5′-untranslated region of HIV-1 RNA and Gag (Bernacchi et al., 2007). More precisely, it binds with strong affinity to

Naturally occurring variations of Vif, Cullin5, and A3G/F affect the fitness of HIV-1

The finding that ΔVif HIV-1 cannot effectively spread in cultures of CD4 T-cells—a natural target of HIV-1 in vivo—has been principally attributed to the antiviral action of A3G and A3F proteins. Overexpression of A3G can modestly suppress wildtype HIV-1 (Mangeat et al., 2003, Sheehy et al., 2002, Zhang et al., 2003), probably by overwhelming the intracellular Vif block. These facts make the Vif–APOBEC3 interaction a worthwhile pharmacological target. Although current antiretroviral drugs

The Vif–A3G interaction as a pharmacological target

Recently, increased efforts were made to block the A3G interaction with Vif. For example, inhibition of the Vif–Vif interaction with peptides that interfere with the PPLP motif of Vif restored A3G encapsidation in nonpermissive cells and reduced HIV-1 infectivity (Miller et al., 2007, Yang et al., 2003). While peptides will not emerge as viable Vif antagonists, these studies support the notion that true Vif antagonists could be an exciting new class of antiviral drugs. The fact that the

Acknowledgments

We thank John Carroll for assistance with the graphics, Stephen Ordway for editorial assistance, and Robin Givens and Sue Cammack for administrative support. The research in our laboratory is supported by funding from the National Institutes of Health (1P01 AI083050-01 and R01 AI065329 to W.C.G.) and California Institute of Regenerative Medicine (TRI-01227 to W.C.G. and TG2/01160 to S.W.).

Warner C. Greene, M.D., Ph.D. is Director of the Gladstone Institute of Virology and Immunology and the Nick and Sue Hellmann Distinguished Professor of Translational Medicine. In addition, he is Professor of Medicine, Microbiology and Immunology at the University of California, San Francisco. He also serves as President of the Accordia Global Health Foundation and Co-Director of the UCSF-GIVI Center for AIDS Research. The author of more than 330 publications, his research focuses on human

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    Warner C. Greene, M.D., Ph.D. is Director of the Gladstone Institute of Virology and Immunology and the Nick and Sue Hellmann Distinguished Professor of Translational Medicine. In addition, he is Professor of Medicine, Microbiology and Immunology at the University of California, San Francisco. He also serves as President of the Accordia Global Health Foundation and Co-Director of the UCSF-GIVI Center for AIDS Research. The author of more than 330 publications, his research focuses on human retroviral pathogenesis.

    Silke Wissing, Ph.D., was born in Emsdetten, Germany. She attended the University of Tuebingen, Germany, where she obtained a Diploma degree in Biochemistry in 2001. She then joined the group of Frank Madeo at the University of Tuebingen, where she worked on the molecular mechanisms of apoptosis in Saccharomyces cerevisiae and earned her PhD in 2005. She then joined the laboratory of Warner C. Greene at the Gladstone Institute of Virology and Immunology in San Francisco, USA, working as a postdoctoral researcher. Her current research focuses on the protective role of APOBEC3 proteins against exogenous and endogenous retroviruses.

    Nicole L.K. Galloway, B.A., was born in Portland, Oregon. She attended Bowdoin College in Maine, where she earned her B.A. diploma with Honors in Biochemistry in 2005. Following graduation, she worked in the laboratory of Tom J. Hope at Northwestern University in Chicago, where she researched the early events of HIV-1 transmission. She currently is in graduate school at the University of California, San Francisco and joined Warner C. Greene’s Lab at the Gladstone Institute of Virology and Immunology. Here she focuses on the two HIV-1 non-structural proteins, Vif and Vpr.

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