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Researchers Find Cell Protein That Nips HIV in the Bud: DC-SIGN EFFICIENTLY BLOCKS HIV VIRAL BUDDING
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Released: Sun 13-Jan-2008
Source: University of California, Los Angeles (UCLA), Health Sciences
Pdf of article attached. SEE SELECT TEXT FOLLOWING THIS PRESS RELEASE BELOW
UCLA researchers have found that a key protein in the body's dendritic cells can stop the virus that causes AIDS from "budding" - part of the virus' life cycle that is crucial to its ability to replicate and infect other cells.
Newswise - UCLA researchers have found that a key protein in the body's dendritic cells can stop the virus that causes AIDS from "budding" - part of the virus' life cycle that is crucial to its ability to replicate and infect other cells.
The study, scheduled for publication in the April issue of the Federation of American Societies for Experimental Biology's FASEB Journal, is currently available online at
www.fasebj.org/cgi/rapidpdf/fj.07-9443comv3.pdf
"If we can block virus generation, then we can control the disease," said lead author Shen Pang, associate professor in the division of oral biology and medicine at the UCLA School of Dentistry and a member of the UCLA AIDS Institute.
Dendritic cells are specialized white blood cells in the skin, mucosa and lymph nodes that kick-start a primary immune response to foreign invaders by activating lymphocytes, including the T cells that HIV targets. Though dendritic cells can be infected with HIV - and indeed play a crucial role in transmitting the virus to T cells - studies have shown that viral generation from these cells is nearly a hundred times lower than from infected T cells, indicating that the cells may possess some inhibiting property.
Pang hypothesized that DC-SIGN, a protein expressed in dendritic cells, may be responsible for such inhibition. He and his colleagues found that DC-SIGN and a related protein, DC-SIGNR, both demonstrated 95 percent to 99.5 percent inhibition of viral production from host cells.
Very few cells are infected when HIV first enters the human body, but the virus rapidly creates new copies of itself, which in turn infect more cells. To achieve this, the virus, after infecting a cell, sends envelopes of protein to the cell's membrane. The viral genomes then combine with viral structural proteins and move into these envelopes. The envelopes bubble, or bud, outward, releasing viral particles that will infect more cells and start new viral life cycles.
According to the researchers, DC-SIGN appears to block HIV generation by efficiently neutralizing an HIV glycoprotein on the surface of the HIV envelope known as gp120, a key to viral infection. In such cases, while some viral particles may still be released from the infected dendritic cells, the lack of gp120 in their envelopes means they are not infectious to CD4-positive T-lymphocytes and macrophages. In other words, these viral particles have been rendered uninfectious.
Current methods to interrupt the life cycle of the virus are limited because they generally target HIV at the stages of viral entry, reverse transcription and post-translational protein cleavages. Once the virus passes through these stages, treatment fails. The UCLA researchers, therefore, focused on halting the virus' generation at different stages in its life cycle.
"The strong inhibition of viral production by DC-SIGN suggests the possibility of using this protein for treatment of HIV-infected patients," the researchers write. "Expression of this protein in various CD4-positive cells should inhibit viral production from infected cells. Because it can also enhance the immune response, DC-SIGN is expected to be useful for in vivo studies for developing an HIV vaccine."
Pang's co-author is Qiuwei Wang, a postgraduate researcher in the division of oral biology and medicine at the UCLA School of Dentistry.
This study was supported by the UCLA AIDS Institute, the UCLA School of Dentistry and a grant from the National Institutes of Health.
The UCLA AIDS Institute, established in 1992, is a multidisciplinary think tank drawing on the skills of top-flight researchers in the worldwide fight against HIV and AIDS, the first cases of which were reported in 1981 by UCLA physicians. Institute members include researchers in virology and immunology, genetics, cancer, neurology, ophthalmology, epidemiology, social science, public health, nursing, and disease prevention. Their findings have led to advances in treating HIV, as well as other diseases, such as hepatitis B and C, influenza, and cancer.
An intercellular adhesion molecule-3 (ICAM-3) - grabbing nonintegrin (DC-SIGN) efficiently blocks HIV viral budding
The FASEB Journal
Vol. 22 April 2008
Qiuwei Wang and Shen Pang1
University of California, Los Angeles, School of Dentistry, Los Angeles, California, USA
ABSTRACT
Efficient inhibition of the HIV infection life cycle at the stages of viral infection, reverse transcription, and post-translational processing has been
extensively studied. However, efficient inhibition of HIV assembly and budding has not been reported. Here, we report that dendritic cell-specific intercellular adhesion molecule-3 (ICAM-3) -grabbing nonintegrin (DC-SIGN) and its related protein, DC-SIGNR, effectively block HIV budding from infected cells. Cotransfection of DC-SIGN or DC-SIGNR with HIV demonstrated 95-99.5% inhibition of viral production from host cells. DC-SIGN or DC-SIGNR can also effectively
inhibit 90-95% of HIV generation from infected cells. DC-SIGN efficiently reduces the amount of gp120 present on the cell plasma membrane, and completely strips off gp120 from the virions produced by the host cells, suggesting that blockage of HIV budding is due to internalization of gp120 by DC-SIGN.-Wang, Q., Pang, S. An intercellular adhesion molecule-3 (ICAM-3) -grabbing nonintegrin (DC-SIGN) efficiently blocks HIV viral budding. FASEB J. 22, 000-000 (2008)
Key Words: gp120 viral production
Methods to interrupt the HIV life cycle at the stages of viral entry, reverse transcription, integration, and post-translational processing have been well documented. Anti-HIV drugs designed to attack viral entry (e.g., T-20, enfuvirtide, Fuzeon), reverse transcription (AZT), and post-translational protein modification (protease inhibitors such as indinavir, ritonavir, and nelfinavir) have been used clinically. However, blockage at these stages has limitations, in that once the virus has passed through these stages, treatment fails. Therefore, approaches are necessary to target other stages in the viral life cycle, including viral assembly and budding.
Our approach was to find a protein to interrupt viral assembly and/or budding. In this study, we focused on a protein that is expressed in dendritic cells (DCs). The normal function of DCs is to present pathogen-derived antigens to T lymphocytes. DCs scavenge pathogens before migrating to the lymph nodes, where they present processed antigens to resting T-cells and initiate an adaptive immune response (1-2). It is proposed that HIV can be internalized into DCs by either DC engulfment or receptor-mediated viral fusion, because many DCs express low levels of CD4 and CCR5 or CXCR4 coreceptors (3-7) and several lectins (8). The presence of a C-type (calcium-dependent) lectin, dendritic
cell-specific intercellular adhesion molecule-3 (ICAM-3) -grabbing nonintegrin (DC-SIGN), in some subsets of DCs may also help the virus to attach to and
fuse into DCs (9). However, there are studies demonstrating that although DCs can be infected, viral generation from DCs is much lower than from infected CD4-positive T-lymphocytes (10-17). HIV production from infected DCs in vivo is often 10- to 100-fold lower (17) than from CD4-positive T-lymphocytes, suggesting that there is a specific mechanism of DCs that can efficiently block viral release from the infected cells.
DC-SIGN, which is specifically expressed on certain DC subsets located in mucosal tissues, has been proposed to play an essential role in transmission of HIV from DCs to T-cells (9, 18, 19). DC-SIGN binds the HIV glycoprotein, gp120, with a high affinity, and transmits the virus to T-cells (9, 18, 19). In addition to playing roles in viral transmission, this molecule may also play other roles. On the basis of the low production of HIV from DCs (10-17), we hypothesized that DC-SIGN might block viral generation in DCs. To investigate the role of DC-SIGN in viral production, gene transfection was used to express this protein in 293T and HeLa-CD4 cells, into which X4 or R5 HIV clones were introduced by either infection or transfection. To accurately quantify viral infection in this study, we used HIV clones that express the enhanced green fluorescent protein (EGFP) (20-22).
DISCUSSION
Our results propose a role for DCs in human immunity. As a type of antigen-presenting cell, the surface lectins of DCs are believed to interact with pathogens before engulfing them. Our results suggest that DCs can engulf certain pathogens, including HIV, and permit certain types of pathogens to continue their life cycles so that more pathogen-derived peptides can be generated (29). Certain lectins such as DC-SIGN can block these pathogens at the stages of assembly and budding, so that only very limited numbers of viral particles can be generated from DCs. Using such a mechanism, DCs are able to efficiently present pathogen-derived peptides on their surfaces without sacrificing themselves to the process of pathogen replication.
It was also noted that the levels of DC-SIGN in DCs may not be as high as in transfected 293T cells or in DC-SIGN lentiviral vector-transduced CEM or Raji cells. Therefore, although it is very likely that DC-SIGN can block HIV production in DCs, the real effect of such blockage in DC-SIGN-positive DCs may still need to be quantified. Previous studies demonstrated that although DCs can be infected, much fewer virions can be generated from DCs than from infected CD4-positive T-lymphocytes (10-17), suggesting that there are certain proteins that can efficiently block viral release from the infected cells. Whether DC-SIGN is the only critical protein in such inhibition merits further exploration, since there are other lectins expressed in DCs and some of them can also bind to gp120 (30).
Although the expression levels of DC-SIGN in DCSIGN- positive DCs may not be high, because the copy numbers of HIV in infected DCs may also not be high, it is still possible that low expression of DC-SIGN can efficiently block HIV budding in infected cells. If so, decreases in DC-SIGN expression when DCs become mature may be a mechanism of DC-mediated transinfection.
Previous studies demonstrated that DC-SIGN plays an essential role in transmission of HIV from DCs to T-cells (9, 18, 19). These results, combined with ours, suggest that DC-SIGN plays dual roles in HIV infection. DC-SIGN-mediated viral transmission requires a clathrin involved in endocytosis, with the cytoplasmic domain of DC-SIGN being essential (19). Since DC-SIGN-mediated
inhibition of HIV budding uses a different mechanism, the cytoplasmic domain of DC-SIGN is not required (Fig. 3). The different requirements of these two mechanisms may be important in development of new therapies for HIV-infected patients. We may use C-terminal-truncated DC-SIGN to transduce cells. Truncation of the cytoplasmic domain still retains blockage of DC-SIGN, which abrogates the capacity of DC-SIGN in viral transmission.
The gp160 precursor for the two envelope glycoproteins, gp120 and gp41, is synthesized on ribosomes associated with the endoplasmic reticulum (ER), and forms a trimeric structure (31). This is transported from the ER to the Golgi complex, where the mannoserich oligosaccharide side chains are added and the precursor is cleaved to gp120 and gp41. These two viral proteins are noncovalently associated following cleavage of the precursor, and move to the plasma membrane. For this reason, together with our results, we propose a model for this process (Fig. 5D), in which most gp160 is processed through pathway A (Fig. 5D). The gp120 and gp41 complex is targeted by DC-SIGN, resulting in no infectious viral particles being generated. In pathway B (Fig. 5D), some gp160 may not be correctly folded and cleaved. Membrane-bound gp120 can be degraded by DC-SIGN, but the gp120-free gp41 molecules would not be affected. The virus generated from the DC-SIGN-expressing cells contain gp41 but not gp120, as shown in Fig. 5A. It is expected that only a small fraction of gp160 enters pathway B, so that the amount of virus generated from DC-SIGN-expressing cells is significantly less than from cells that do not express DC-SIGN.
In humans, DC-SIGN has been reported to be expressed in vivo by DCs, macrophages, activated B-cells, the dermis of the skin, the placenta, the intestinal and genital mucosa, and lymphoid tissues (8). DC-SIGNR is expressed in several types of endothelial cells (32). We have studied several epithelial cell lines and found low expression levels of DC-SIGN or DC-SIGNR (Fig. 5C). In addition to binding HIV gp120, DC-SIGN(R) was also able to bind the hepatitis C virus (HCV) E2 glycoprotein (33) and CMV envelope glycoprotein B (34). It will be of interest to determine whether DCSIGN can also block production of HCV and CMV in host cells.
The strong inhibition of viral production by DCSIGN suggests the possibility of using this protein for treatment of HIV-infected patients. Expression of this protein in various CD4-positive cells should inhibit viral production from infected cells. Because it can also enhance the immune response (29), DC-SIGN is expected to be useful for in vivo studies for developing an HIV vaccine. We also noted that DC-SIGN(R) is expressed
in the CD4-positive T cell line, CEM, and certain CD4-negative cell lines (Fig. 5C). However, most of these proteins may lack part of the DC-SIGN(R) sequence. The full-length DC-SIGN is at least 45,774 Da (if not glycosylated), and DC-SIGNR is at least 45,350. The band of about 40 kDa may either lack the TM or a part of the CRD (35). A minor band of about 45 kDa in CEM cell preparations has been detected (Fig. 5C). It is not clear whether this band represents a functional isoform of DC-SIGN, since the soluble DC-SIGN form that lacks the TM domain is only 0.6 kDa smaller than that containing the full-length TM domain (35). Characterizing the expression and splicing of DC-SIGN in CD4-positive T-lymphocytes and macrophages will be necessary to find an approach to use endogenous DC-SIGN to block HIV production in patients.
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