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Project

Exploration of the role of HIV envelope glycans on virus entry and the antiviral activity of carbohydrate-binding agents (CBAs)

The human immunodeficiency virus (HIV) is the causative agent of the acquired immunodeficiency syndrome (AIDS) and, although extensive research has resulted in the development of over 25 anti-HIV drugs, the pandemic continues to be a major public health threat worldwide. Annually, about 2.1 million people get newly infected with the virus and approximately 1.5 million people are subject of an AIDS-related death. These facts clearly demonstrate the ongoing need to invest in additional highly efficient strategies to treat HIV-infected individuals and to prevent the further spread of the virus.

The HIV glycoproteins gp120 and gp41 appear as spikes on the surface of the virus envelope and play an indispensable role in one of the very first steps of the HIV replication cycle: the entry of the virus particle into its susceptible target cells. Due to the crucial role of these envelope glycoproteins during HIV replication, gp120 and gp41 are interesting targets for the development of anti-HIV drugs. These drugs might enable the inhibition of the virus at a very early event during its replication cycle, and thus, before the virus gets incorporated into the cellular genome. Therefore, the aim of this PhD thesis was to study the antiviral potential of two different classes of HIV entry inhibitors (chapter 3) and to perform an in-depth investigation on the HIV envelope glycoproteins, particularly focusing on the N-linked glycans and disulfide bridges of HIV gp120 and gp41 (chapter 4).

In chapter 3, the antiviral potential of two classes of HIV entry inhibitors was studied, namely carbohydrate-binding agents (CBAs) and tellurium(Te)-containing oxidoreductase inhibitors. The former class of HIV entry inhibitors constitutes a broad variety of compounds from diverging origin and with a variable structure, and are able to block HIV entry by interacting with N-linked glycans that are abundantly present on HIV gp120. On the other hand, the Te-containing oxidoreductase inhibitors interfere with the reduction of particular disulfide bridges in HIV gp120 by cellular enzymes, which obligatorily occurs during the HIV entry step and induces conformational changes in gp120 and gp41, ultimately resulting in a blockade of the HIV entry.

It could be demonstrated that CBAs are highly potent in blocking four processes that are involved in HIV infection and transmission: cell-free virion infection of CD4+ T lymphocytes, syncytium formation between infected and uninfected CD4+ T cells, capture of virus particles by DC-SIGN-expressing cells and transmission of DC-SIGN-captured virus particles to CD4+ T cells (section 3.1). Furthermore, we showed that these compounds have no measurable harmful effects on the growth, survival and epithelial adhesion of a broad variety of vaginal and non-vaginal Lactobacillus strains (section 3.1). These bacteria are the predominant constituents of the healthy vaginal microbiome and significantly contribute to the vaginal homeostasis and its intrinsic antimicrobial potential. Taken together, these data demonstrate the potential of CBAs as broadly acting antiretroviral drugs, including the development of topical microbicides.

We also demonstrated that the development of viral resistance towards CBAs could be delayed by rationally combining well-defined CBAs with a different glycan-binding profile. Such resistance development led to the generation of mutant virus strains lacking several (predominantly high-mannose-type) N-linked glycans on gp120 (section 3.2). It was shown that CBA-resistant variants often had a significantly decreased viral infectivity as compared to the corresponding wild-type virus. These data indicate that the antiviral potential of CBAs (as shown in section 3.1) could potentially be enhanced by rationally combining well-defined CBAs with a distinct glycan-binding profile, and that CBAs may select for virus variants with a compromised infectivity potential.

The other class of anti-HIV compounds that was studied, namely tellurium(Te)-containing oxidoreductase inhibitors (section 3.3), was previously shown to target thioredoxin reductase-1 (TrxR1), which acts in concert with thioredoxin-1 (Trx1) for the reduction of well-defined disulfide bridges in HIV gp120 during the entry process. The reduction of these disulfide bridges in gp120 induces oxidoreduction-driven conformational changes in gp120, which are needed to allow efficient HIV entry. Our detailed investigation on the antiviral mechanism of action of these Te-compounds indeed revealed that the compounds interfere with HIV entry by blocking the Trx1/TrxR1-mediated reduction of disulfide bridges in gp120. Interestingly, we found TrxR1 to be closely associated with the virus particles. Although these compounds were found to markedly block HIV infection, most of them were also associated with significant cytotoxicity. It was concluded that the Trx1/TrxR1 system might hold promise for the development of novel highly efficient HIV entry inhibitors, but that the development of additional TrxR1 inhibitors with an improved activity/toxicity window is warranted.

In chapter 4, a thorough investigation of the N-glycans and disulfide bridges of HIV gp120 and gp41 was performed, aiming at improving our knowledge on the functional and spatial role of these structural entities in the functionality of the HIV envelope glycoproteins. These studies were performed using various mutant HIV strains in which N-linked glycans or disulfide bridges on the viral Env gene were deleted by site-directed mutagenesis.

In section 4.2, the N-glycan on Asn-262 of HIV gp120 was examined in more detail. This glycan was previously shown to be crucial for the viral infectivity and the correct expression of the viral glycoproteins and their incorporation in the viral envelope. We here demonstrate that this phenomenon is the result of the increased lysosomal degradation of the N262-glycan-deleted precursor envelope glycoprotein gp160, thereby precluding the further biosynthesis and envelope incorporation of gp120 and gp41.

In section 4.1, we discovered that one particular N-glycan on gp41 (i.e. Asn-616) is highly indispensable for the infectivity of several HIV-1 strains. Loss of the other N-glycans of gp41 (even combinations of 2-3 gp41 N-glycan deletions) had no or only a limited detrimental effect on the infectivity of the virus and only slightly affected the susceptibility of the mutant virus strains towards CBAs. These findings reveal that the antiviral activity of CBAs is based predominantly on their interaction with (high-mannose-type) glycans on gp120 rather than on gp41.

In addition, a detailed analysis on the spatial organization and interrelationship of N-glycans and disulfide bridges in HIV gp120 was performed (section 4.3). It was demonstrated that the N-glycan environment around disulfide bridges in gp120 is often tightly regulated and that several disulfide bridges in gp120 are obligatorily surrounded by at least one conserved N-glycan. The loss of a particular glycan in proximity to a disulfide bridge, or the insertion of a non-native N-glycan close to a disulfide bridge at a position otherwise disfavored for N-glycosylation, often has a detrimental effect on the viral functions. These data indicate that disulfide bridges and N-glycans are not randomly scattered across gp120 and that the co-localization of these structural features contributes to the efficiency of viral infection and transmission. The strictly regulated glycan environment at particular disulfide bridges of gp120 may thus represent hot-spots for a potential highly focused therapeutic intervention.

In conclusion, we demonstrated that the HIV envelope glycoproteins are interesting targets for the development of novel antiretroviral drugs, using compounds that either bind to the carbohydrate structures on gp120 or that interfere with oxidoreduction-driven conformational changes in gp120, enabling the inhibition of one of the very first steps of the HIV replication cycle. Furthermore, we demonstrated that some N-glycans on the HIV envelope glycoproteins are highly indispensable to preserve the viral functions, offering more insights in the fundamental role of the N-glycans on HIV gp120 and gp41 for the biosynthesis and function of the HIV envelope glycoproteins, and suggesting that the indispensable N-glycans on HIV gp120 and gp41 might be valuable hot-spots for the development of highly potent and selective antiretroviral drugs.

Date:29 Sep 2011 →  30 Sep 2015
Keywords:HIV
Disciplines:Microbiology, Systems biology, Laboratory medicine
Project type:PhD project