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Sharon Wahl of NIH: PS & HIV...




http://www.jem.org/cgi/content/full/200/10/1337

Secretory Leukocyte Protease Inhibitor Binds to Annexin II, a Cofactor for Macrophage HIV-1 Infection

snips from this NIH work -


"PS is not encoded by HIV-1, but rather is acquired from its host cell membrane as it exits the cells (26). During viral assembly at the cell surface or within cytoplasmic vesicles, cell membrane components become incorporated into the new viral coat along with virally encoded gp120/gp41. The resulting mosaic HIV-1 envelope represents a lipid bilayer with a unique cholesterol/phospholipid composition, embracing viral and host molecules, including PS."

......

"Our data favor a model in which PS in the viral coat interacts with annexin II on the surface of macrophages subsequent to recognition-specific gp120 interactions with CD4 and the chemokine coreceptors that instigate coiled coil exposure of gp41 fusion domains to interact with the lipid bilayer of the target cell (10, 30), and then engage an annexin II–dependent fusion/entry pathway leading to productive infection"

......

"Annexin II on macrophages may preferentially facilitate entry of virions expressing PS acquired during exit from prior macrophage hosts or from T cells undergoing apoptosis.."

......

" It is conceivable that PS–annexin II interactions in the cytoplasmic vesicles and late endosomes of macrophages, where structural assembly of virions occurs (12, 43) and annexin II is found (28), not only serve as a construction scaffolding, but also as a tether to retain virions intracellularly in a covert maneuver to avoid detection at the cell surface. Because the S100A10 component of the annexin II complex has been shown to facilitate arbovirus exocytosis (44), such a role in HIV-1 egress might also be considered. Annexin II/PS may also contribute to the host cell–derived cloak of the hypothetical Trojan exosomes that subversively deliver retroviral particles to nearby cells (45), thus a co-conspirator with HIV-1 both going in and coming out."

.......

"The persistence of HIV-1 infection, coupled with its incredible mutation rate and insular reservoirs, focuses attention on host cell constituents usurped by the virus as potential intervention targets"

......

"Clearly, viral pathogens other than HIV-1, including CMV and respiratory syncytial virus (19, 20), also take advantage of target cell annexin II to enhance their infectivity and/or dissemination, and furthermore, bacteria trigger annexin II recruitment to their attachment sites (27), all suggesting its broader involvement in microbial entrance and pathogenesis"






longer excerpts:




Abstract
The distribution of secretory leukocyte protease inhibitor (SLPI) at entry portals indicates its involvement in defending the host from pathogens, consistent with the ability of SLPI to inhibit human immunodeficiency virus (HIV)-1 infection by an unknown mechanism. We now demonstrate that SLPI binds to the membrane of human macrophages through the phospholipid-binding protein, annexin II. Based on the recent identification of human cell membrane phosphatidylserine (PS) in the outer coat of HIV-1, we define a novel role for annexin II, a PS-binding moiety, as a cellular cofactor supporting macrophage HIV-1 infection. Moreover, this HIV-1 PS interaction with annexin II can be disrupted by SLPI or other annexin II–specific inhibitors. The PS–annexin II connection may represent a new target to prevent HIV-1 infection.


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Annexin II Interacts with PS.
How annexin II might support HIV-1 infection of macrophages was a mystery, but a recent study reporting that HIV-1 membranes contain PS (14) provided a potential clue. Although no binding partner for HIV-PS had been identified, we surmised that annexin II, a phospholipid-binding protein, might be a candidate. In this regard, direct binding of HIV-1 to plate-bound annexin II, but not irrelevant proteins, was demonstrated (Fig. 5 A). Moreover, to verify an HIV-PS–macrophage annexin II connection, we exposed HIV-1 to excess soluble annexin II to bind/coat viral PS before addition to macrophages, and this markedly suppressed subsequent infection (Fig. 5 B), compatible with inhibition of infection by soluble annexin V and/or PS vesicles.


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Annexin II Is Associated with Entry/Fusion of HIV-1 in Macrophages.
Next, we focused on when in the virus life cycle annexin II cooperates with HIV-1 to promote infection. Consistent with SLPI (2, 3), blockade of annexin II on macrophages did not significantly interrupt HIV-1 binding to the cells (Fig. 5 C), likely dissociating it from a direct interaction with CD4 and/or CCR5. In confirmatory studies, anti–annexin II was incorporated into a fusion assay in which effector cells expressing recombinant Env, but lacking viral PS, were cocultured with target cells expressing recombinant CD4 and coreceptors (23). In the absence of PS, anti–annexin II was ineffective in interrupting this fusion process (not depicted), ruling out a specific interaction with Env, CD4, and/or coreceptors. These data emphasize the potential unique constraints of macrophage–HIV-1 entry events that might be optimized through an annexin II–PS cofactor linkage. Although the role of annexin II in viral entry may involve participation in internalization or structural/functional facilitation of fusion events, it was unclear whether membrane annexin II had intracellular access. Because apoptotic cells also bind through PS to annexin II on macrophages (24, 25), we exposed macrophages to apoptotic Jurkat cells (PS+) as a model of annexin II–dependent internalization. By immunofluorescence, annexin II could be found within the early phagosome membrane, consistent with surface membrane internalization (Fig. 5 D). Whether internalization is essential to viral entry is under investigation, but by multiple criteria, the role of annexin II appears instrumental early in the infection process. To confirm that disconnecting the HIV–annexin II bond inhibits the virus life cycle after HIV-1 binding but before reverse transcription, as shown for SLPI (3), macrophages were infected with or without annexin II inhibitors and the formation of nascent viral DNA was assessed using a nested PCR-based assay. The presence of anti–annexin II during the initial virus inoculation period dramatically inhibited subsequent viral DNA synthesis, as demonstrated by the HIV-1–specific 730-bp PCR product (Fig. 5 E), even though annexin II blockade did not disengage the viral-binding step (Fig. 5 C). Collectively, these data indicate that interference with macrophage membrane annexin II inhibits infectivity after binding, but pre-reverse transcription, consistent with a stranglehold on the viral entry/fusion step.


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DISCUSSION
In this study, we identify annexin II as a novel macrophage membrane–binding protein for the innate host defense protein, SLPI, by multiple parameters including immunoprecipitation, mass spectrometry, peptide sequencing, and binding specificity. Striking was the ability of inhibitors of annexin II to mimic the kinetics and apparent mechanism of HIV-1 suppression by SLPI, denoting a shared site of action. Although not ruling out additional binding targets for SLPI, annexin II appears to be significant in mediating its anti–HIV-1 activity. Subsequent to binding of HIV-1 to the canonical receptors, CD4 and CCR5, HIV-1 fuses with the host cell membrane that might be facilitated by viral envelope PS (14). PS is not encoded by HIV-1, but rather is acquired from its host cell membrane as it exits the cells (26). During viral assembly at the cell surface or within cytoplasmic vesicles, cell membrane components become incorporated into the new viral coat along with virally encoded gp120/gp41. The resulting mosaic HIV-1 envelope represents a lipid bilayer with a unique cholesterol/phospholipid composition, embracing viral and host molecules, including PS. Although PS enhances infection, it does not mediate initial binding of the virus to the target cells (14), consistent with our observations that neither soluble annexin II, RNAi, anti–annexin II, nor the annexin II ligand, SLPI, blocks HIV-1 binding, but rather inhibit postbinding and pre-reverse transcription, a point in the viral life cycle consistent with a proposed role for annexin II as a cellular fusogenic cofactor.

Annexin II reportedly is found in caveolae and lipid rafts in association with cholesterol (27) and mediates interactions between cholesterol-rich membrane domains and the actin cytoskeleton (28, 29), which may navigate HIV-1 through the labyrinth of the cell membrane. Our data favor a model in which PS in the viral coat interacts with annexin II on the surface of macrophages subsequent to recognition-specific gp120 interactions with CD4 and the chemokine coreceptors that instigate coiled coil exposure of gp41 fusion domains to interact with the lipid bilayer of the target cell (10, 30), and then engage an annexin II–dependent fusion/entry pathway leading to productive infection.

Annexin II may represent a molecular pathway exploited by HIV-1 unique to macrophage hosts and thus, a potential target to block their virus susceptibility. Both laboratory-adapted and clinical M tropic isolates appear to coopt this host cell bridge into the cell's interior. Whether annexin II selectively boosts the viral entry/fusion process or possibly also influences pathways involved in HIV-1–mediated macrophage signaling, viral DNA transport, or subsequent virion construction, budding and release remains to be deciphered. Annexin II has the potential to traverse into intracellular compartments and interaction of annexin II with the actin cytoskeleton may not only facilitate internalization, but also the trafficking of HIV-1 within the cell and/or represent the scaffolding for viral transcription (30). Nonetheless, our data support a dominant role of annexin II to be in the early steps of the infection process, preceding reverse transcription. Annexin II may represent one of multiple potential cofactors, such as syndecan and human neutrophil elastase (31, 32), which independently or collaboratively might be usurped by HIV-1 to facilitate the infectious process. Because annexin II is a membrane-associated protein, best known as a docking station for tissue plasminogen activator/plasminogen (16), it is unclear if it transduces a signal because it lacks a hydrophobic signal sequence, but conceivably, may serve as an adaptor in a signaling cascade. Annexin II can be phosphorylated at key residues by several kinases, including the src oncogene (17) and Pyk-2 (15), a tyrosine kinase activated by HIV-1 (33, 34), but such a pathway awaits delineation. Although less persuasive due to the temporal association of SLPI/anti–annexin II inhibition with preintegration events, SLPI may also influence NF-{kappa}B activation and/or proteasome inhibition (6, 8), both of which are requisite in an optimal infection process (35–37).

Whether the relative lack of abundance of annexin II on the perimeter of immature blood monocytes compared with mature macrophages influences their differential permissiveness to HIV-1 infection is of interest. A related, intriguing question is whether the viral tropism characteristic of T cell and macrophage targets bears any association with their divergent annexin II expression. Annexin II on macrophages may preferentially facilitate entry of virions expressing PS acquired during exit from prior macrophage hosts or from T cells undergoing apoptosis that only then express PS on their outer membrane leaflet (38, 39), as compared with virions budded from PS-less viable T cells. R5 viruses mediate both mucosal and blood-borne transmission of HIV-1 infection, whereas the X4 (T tropic) viruses typically abound in the later stages of disease during clinical progression to AIDS (11, 40, 41). Moreover, when infected T cells succumb to apoptosis, recognition of their newly exposed PS will promote clearance by annexin II–bearing macrophages with the potential for HIV-1 transfer (42). It is conceivable that PS–annexin II interactions in the cytoplasmic vesicles and late endosomes of macrophages, where structural assembly of virions occurs (12, 43) and annexin II is found (28), not only serve as a construction scaffolding, but also as a tether to retain virions intracellularly in a covert maneuver to avoid detection at the cell surface. Because the S100A10 component of the annexin II complex has been shown to facilitate arbovirus exocytosis (44), such a role in HIV-1 egress might also be considered. Annexin II/PS may also contribute to the host cell–derived cloak of the hypothetical Trojan exosomes that subversively deliver retroviral particles to nearby cells (45), thus a co-conspirator with HIV-1 both going in and coming out.

The identification of a novel role for annexin II as a cellular cofactor in HIV-1 entry/fusion has implications for specific antiviral strategies, albeit primarily targeting macrophage infection. Nonetheless, as macrophages may contribute to initial viral selection, dissemination, and transmission of virus to CD4+ T cells, and serve as long-term covert reservoirs of HIV-1 (11, 40, 46), this would be an enviable goal. Particularly evident is the enormous viral burden in macrophages in later stage HIV-1/AIDS during opportunistic infections (12, 21, 22). Further unraveling of the complex interplay between viral envelope and macrophage membrane constituents remains crucial to the development of antiviral agents active before permanent viral integration into the host cell genome when the virus is most vulnerable. The persistence of HIV-1 infection, coupled with its incredible mutation rate and insular reservoirs, focuses attention on host cell constituents usurped by the virus as potential intervention targets. In this regard, annexin II, a host cell molecule that the virus has appropriated for easing its entrance into the host cell, represents a likely candidate, and SLPI, an endogenous ligand for annexin II, or other annexin II–specific blockades, may represent a therapeutic impediment to the infection process. Clearly, viral pathogens other than HIV-1, including CMV and respiratory syncytial virus (19, 20), also take advantage of target cell annexin II to enhance their infectivity and/or dissemination, and furthermore, bacteria trigger annexin II recruitment to their attachment sites (27), all suggesting its broader involvement in microbial entrance and pathogenesis.



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