Elizabeth R. Wonderlich

To facilitate viral infection and spread, HIV-1 Nef disrupts the surface expression of the viral receptor (CD4) and molecules capable of presenting HIV antigens to the immune system (MHC-I). To accomplish this, Nef binds to the cytoplasmic tails of both molecules and then, by mechanisms that are not well understood, disrupts the trafficking of each molecule in different ways. Specifically, Nef promotes CD4 internalization after it has been transported to the cell surface, whereas Nef uses the clathrin adaptor, AP-1, to disrupt normal transport of MHC-I from the TGN to the cell surface. Despite these differences in initial intracellular trafficking, we demonstrate that MHC-I and CD4 are ultimately found in the same Rab7(+) vesicles and are both targeted for degradation via the activity of the Nef-interacting protein, beta-COP. Moreover, we demonstrate that Nef contains two separable beta-COP binding sites. One site, an arginine (RXR) motif in the N-terminal alpha helical domain of Nef, is necessary for maximal MHC-I degradation. The second site, composed of a di-acidic motif located in the C-terminal loop domain of Nef, is needed for efficient CD4 degradation. The requirement for redundant motifs with distinct roles supports a model in which Nef exists in multiple conformational states that allow access to different motifs, depending upon which cellular target is bound by Nef.

T cells are faster for intact than for defective proviruses, and intact provirus frequencies are similar in mucosal and circulating T cells.

To evade the anti-human immunodeficiency virus (HIV) immune response, the HIV Nef protein disrupts major histocompatibility complex class I (MHC-I) trafficking by recruiting the clathrin adaptor protein 1 (AP-1) to the MHC-I cytoplasmic tail. Under normal conditions AP-1 binds dileucine and tyrosine signals (YXX phi motifs) via physically separate binding sites. In the case of the Nef-MHC-I complex, a tyrosine in the human leukocyte antigen (HLA)-A2 cytoplasmic tail ((320)YSQA) and a methionine in Nef (Met(20)) are absolutely required for AP-1 binding. Also present in Nef is a dileucine motif, which does not normally affect MHC-I trafficking and is not needed to recruit AP-1 to the Nef-MHC-I-complex. However, evidence is presented here that this dileucine motif can be activated by fusing Nef to the HLA-A2 tail in cis. Thus, the inability of this motif to function in trans likely results from a structural change that occurs when Nef binds to the MHC-I cytoplasmic tail. The physiologically relevant tyrosine-dependent recruitment of AP-1 to MHC-I, which occurs whether Nef is present in cis or trans, was stabilized by the acidic and polyproline domains within Nef. Additionally, amino acids Ala(324) and Asp(327) in the cytoplasmic tails of HLA-A and (but not HLA-C and HLA-E) molecules also stabilized AP-1 binding. Finally, mutation of the tyrosine binding pocket in the mu subunit of AP-1 created a dominant negative inhibitor of Nef-induced down-modulation of HLA-A2 that disrupted binding of wild type AP-1 to the Nef-MHC-I complex. Thus, these data provide evidence that Nef binding to the MHC-I cytoplasmic tail stabilizes the interaction of a tyrosine in the MHC-I cytoplasmic tail with the natural tyrosine binding pocket in AP-1.

Human infections with highly pathogenic avian influenza A (H5N1) virus are frequently fatal but the mechanisms of disease remain ill-defined. H5N1 infection is associated with intense production of proinflammatory cytokines, but whether this cytokine storm is the main cause of fatality or is a consequence of extensive virus replication that itself drives disease remains controversial. Conventional intratracheal inoculation of a liquid suspension of H5N1 influenza virus in nonhuman primates likely results in efficient clearance of virus within the upper respiratory tract and rarely produces severe disease. We reasoned that small particle aerosols of virus would penetrate the lower respiratory tract and blanket alveoli where target cells reside. We show that inhalation of aerosolized H5N1 influenza virus in cynomolgus macaques results in fulminant pneumonia that rapidly progresses to acute respiratory distress syndrome with a fatal outcome reminiscent of human disease. Molecular imaging revealed intense lung inflammation coincident with massive increases in proinflammatory proteins and IFN-α in distal airways. Aerosolized H5N1 exposure decimated alveolar macrophages, which were widely infected and caused marked influx of interstitial macrophages and neutrophils. Extensive infection of alveolar epithelial cells caused apoptosis and leakage of albumin into airways, reflecting loss of epithelial barrier function. These data establish inhalation of aerosolized virus as a critical source of exposure for fatal human infection and reveal that direct viral effects in alveoli mediate H5N1 disease. This new nonhuman primate model will advance vaccine and therapeutic approaches to prevent and treat human disease caused by highly pathogenic avian influenza viruses.

Persistent production of type I interferon (IFN) by activated plasmacytoid dendritic cells (pDC) is a leading model to explain chronic immune activation in human immunodeficiency virus (HIV) infection but direct evidence for this is lacking. We used a dual antagonist of Toll-like receptor (TLR) 7 and TLR9 to selectively inhibit responses of pDC but not other mononuclear phagocytes to viral RNA prior to and for 8 weeks following pathogenic simian immunodeficiency virus (SIV) infection of rhesus macaques. We show that pDC are major but not exclusive producers of IFN-α that rapidly become unresponsive to virus stimulation following SIV infection, whereas myeloid DC gain the capacity to produce IFN-α, albeit at low levels. pDC mediate a marked but transient IFN-α response in lymph nodes during the acute phase that is blocked by administration of TLR7 and TLR9 antagonist without impacting pDC recruitment. TLR7 and TLR9 blockade did not impact virus load or the acute IFN-α response in plasma and had minimal effect on expression of IFN-stimulated genes in both blood and lymph node. TLR7 and TLR9 blockade did not prevent activation of memory CD4+ and CD8+ T cells in blood or lymph node but led to significant increases in proliferation of both subsets in blood following SIV infection. Our findings reveal that virus-mediated activation of pDC through TLR7 and TLR9 contributes to substantial but transient IFN-α production following pathogenic SIV infection. However, the data indicate that pDC activation and IFN-α production are unlikely to be major factors in driving immune activation in early infection. Based on these findings therapeutic strategies aimed at blocking pDC function and IFN-α production may not reduce HIV-associated immunopathology.