Current HIV Research - Volume 9, Issue 7, 2011

Ever since the protein received its inappropriate acronym “negative facor”, the primate lentiviral Nef protein has opened a field of research marked by controversy. Two decades after the discovery of its importance for pathogenic infection of rhesus macaques followed by similar observations in HIV infected humans, the mechanism of action of this protein remains notoriously enigmatic, a puzzle that is solved only piece by piece. Nef is thought to be exclusively involved in virus-host interactions. It has no catalytical nor structural function, but serves as an adaptor protein. It significantly enhances virus infection and spread in primary T cells or in ex vivo infected lymphoid tissues. It affects T cell activation, T cell development and trafficking of several cellular proteins like CD4, CD8, CD28, CXCR4, CCR5, MHC class I and II. The identification of new cellular interaction partners and the understanding of the interaction of Nef with these and already known partners will not only increase our basic understanding of HIV replication but will also provide new therapeutic avenues to interfere with HIV replication. In this themed issue, several research groups active in Nef research review our current knowledge. The enhancement of primate lentivirus infectivity by Nef is reviewed by Vermeire et al. They discuss the mechanisms proposed to explain this property of the protein, affecting the virion composition and the early events post-infection of the target cell. Margherita Doria reviews the controversy on the importance of CD4 downregulation by Nef in both the producer and target cell for enhanced virion infectivity by Nef. The downregulation of cell surface molecules like CD4 by Nef and the possible advantageous consequences for viral infection, replication, and evasion from immunity is the focus of the review by Landi et al. While most knowledge on Nef is gathered from the study of the protein in lymphocytes and derived cell lines, it is clear that expression of the protein affects both the viral life cycle as well as cellular functions in macrophages, as reviewed by Yanina Ghiglione and Gabriela Turk. The challenges primate lentiviruses have overcome during inter-species transmission reveal the plasticity of the viral proteins, needed to interact with different species-specific host protein domains. The impairment of the tetherin restriction factor function by Nef illustrates this plasticity, reviewed by Daniel Sauter and Frank Kirchhoff. Despite species differences, the expression of Nef driven by the regulatory sequences of the human CD4 gene in transgenic mice causes disease, reminiscent to AIDS in humans. Paul Jolicoeur reviews what this model can learn us on the contribution of Nef to morbidity of HIV infection. In the search for the molecular mechanism behind the Nef functions reviewed in this issue, most progress was made in the study of the interaction of Nef with Src homology-3 (SH3) domains of cellular host proteins. The biological and therapeutic relevance of this interaction is reviewed by Kalle Saksela. As the molecular details of this and other Nef - host protein interaction are being unveiled, targeted therapies can be developed. Lulf et al. discuss recent progress in this field. While the enigma has not been solved, a lot progress has been made in our understanding of the function of lentiviral Nef. By these revelations, both the beauty of the viruses as a possible Achilles' heel to target therapeutically are exposed.

The Nef protein is an essential factor for lentiviral pathogenesis in humans and other simians. Despite a multitude of functions attributed to this protein, the exact role of Nef in disease progression remains unclear. One of its most intriguing functions is the ability of Nef to enhance the infectivity of viral particles. In this review we will discuss current insights in the mechanism of this well-known, yet poorly understood Nef effect. We will elaborate on effects of Nef, on both virion biogenesis and the early stage of the cellular infection, that might be involved in infectivity enhancement. In addition, we provide an overview of different HIV-1 Nef domains important for optimal infectivity and briefly discuss some possible sources of the frequent discrepancies in the field. Hereby we aim to contribute to a better understanding of this highly conserved and therapeutically attractive Nef function.

The regulatory Nef protein of HIV-1/2 and SIV is required for high viral replication and disease progression, thus represents a very attractive therapeutic target. Because of the multi-functional nature of the Nef protein, it is unclear which of the several Nef activities are most crucial in vivo for the outcome of viral infection. Some findings indicate that the CD4 down-regulation activity of Nef is critical for viral infectivity as well as for progression to immunodeficiency. On the other hand, more recent evidences suggest that CD4 targeting and stimulation of infectivity are two separate functions of Nef. This controversial issue will be discussed here in the light of the latest findings.

The HIV-1, HIV-2 and SIV Nef protein are known to modulate the expression of several cell surface receptors and molecules to escape the immune system, to alter T cell activation, to enhance viral replication, infectivity and transmission and overall to ensure the optimal environment for infection outcome. Consistent and continuous efforts have been made over the years to characterize the modulation of expression of each of these molecules, in the hope that a better understanding of these processes essential for HIV infection and/or pathogenesis will eventually highlight new therapeutic targets. In this article we provide an extensive review of the knowledge gained so far on this important and evolving topic.

Following transmission of human immunodeficiency virus (HIV) into a new host, cells of the monocyte/macrophage lineage play a central role in host invasion and viral replication. In particular, macrophages survive infection and support long-standing viral replication, contributing to viral persistence within the host and representing a viral reservoir in vivo. On the other hand, HIV Nef protein is a small though versatile molecule that plays an unquestioned key role in viral pathogenesis. In macrophages, Nef is able to modulate cell surface receptor expression, to intersect intracellular signaling pathways and to augment the release of pro-inflammatory and chemotactic molecules. In addition, Nef can alter macrophage phagocytic capacity, autophagy machinery and metabolism. Altogether, these Nef activities support viral replication and persistence in this cell type while at the same time favor viral dissemination. Here, we will review the newest findings describing how monocytes/macrophages natural pathways are altered by Nef protein, highlighting how viral and host biology are perturbed in consequence.

The multifunctional Nef protein of primate lentiviruses is commonly considered an early viral factor that downmodulates various receptors from the cell surface and modulates several signaling pathways to facilitate viral immune evasion and to render the cell conducive for viral replication. However, Nef also acts during the late stages of infection, e.g. by increasing the infectivity of progeny virions. Just recently, it has become clear that many primate lentiviruses that have been detected in about 40 different monkey and ape species also use Nef to antagonize tetherin (BST2/CD317), a cellular factor that inhibits virus release by tethering nascent viral particles to the cell surface. Exceptions are some simian immunodeficiency viruses (SIVs) infecting Cercopithecus monkeys that employ their accessory Vpu protein to counteract the restriction by tetherin. Furthermore, pandemic HIV-1 group M strains switched from Nef to Vpu and HIV-2 group A isolates from Nef to Env after zoonotic transmission from chimpanzees and sooty mangabeys, respectively, to antagonize the tetherin restriction in humans. These evolutionary switches were most likely enforced by a deletion in the cytoplasmic domain of the human tetherin orthologue that confers resistance to Nef. Here, we summarize some of our current knowledge about Nef-mediated tetherin antagonism.

CD4C/HIVNef Tg mice express HIV-1 Nef in relevant target cells through the regulatory sequences of the human CD4 gene. These mice develop a severe disease showing many characteristics of human AIDS. A summary of the results obtained with these Tg mice is presented.

A bewildering number of host cell proteins associated with Nef can be found in the literature and in the public protein interaction databases. However, only in a few of these cases, including binding of Nef to certain Src homology-3 (SH3) domain proteins, is the interaction understood in any molecular detail or even known to be direct. Indeed, SH3 binding capacity by Nef is required for many of the other protein interactions to take place, suggesting that a large proportion of the latter is indirectly coupled to Nef via an SH3 protein. Accordingly, the proline-rich SH3 binding site, the “PxxP motif”, is one of the key functional determinants of Nef. It is highly conserved among the lentiviral Nef proteins, and mutations disrupting it abrogate the majority of the known effects of Nef on host cell physiology. This review summarizes the current understanding as well as the outstanding gaps in our knowledge regarding the relevant SH3 protein partners and SH3-dependent cellular functions of Nef. The roles of these interactions in the pathogenesis of AIDS and their potential as targets for antiviral drug development are also discussed.

The HIV-1 Nef protein is an accessory protein of 24-27 kDa mass that mediates a multitude of effector functions in infected cells. Although not essentially required for viral replication, HIV-1 Nef exhibits stimulating potential towards disease progression to AIDS and is therefore considered a pathogenic factor in retroviridae. Here we correlate sequence conservation in HIV-1 Nef with surface hydrophobicity and functionality in protein-protein interaction to identify accessible substructures on the surface of Nef that might be suitable as pharmacological target sites. Recent advances in targeting of Nef by small molecular compounds that interfere with SH3 domain binding or MHC class I down-regulation are discussed. Similarly, approaches for the use of larger molecules are introduced, such as tailored fusion proteins that simultaneously interact with multiple highly conserved sequence motifs of Nef. In addition, the design of a single domain antibody from llama that interferes with CD4 down-regulation activity and PAK2 binding is discussed. The flexibility in binding recognition is exemplarily shown for the modulation of RT-loop binding using engineered SH3 domains. The various considerations corroborate the potential of HIV-1 Nef as a promising target for the development of potent Nef inhibitors.