The Heidelberg virologist Hans-Georg Kräusslich and his team are exploring the molecular architecture and morphogenesis of the HI-Virus and the processes occurring at the plasma membrane of the host cell that lead to the release of new viruses and new infections. The budding and maturation processes of HIV particles and the lipid composition of their envelope could be used as targets for the development of new drugs to combat AIDS.
Since the 1980s, when evidence accumulated that the AIDS epidemic was spreading in the USA, no other human pathogenic virus has been as intensively studied as the human immunodeficiency virus (HIV). However, many aspects of the biology of HIV are not yet known in detail. The search for new therapeutic strategies against AIDS continues, as the deadly disease has not been curbed in many parts of the world and antiretroviral drugs are only available for a fraction of the 34 million people infected with HIV.
HIV belongs to a family of viruses called retroviruses whose genome consists of single-stranded RNA which is transcribed by the viral reverse transcriptase enzyme into double-stranded DNA in the host cell. The DNA is then incorporated into the host’s genome which treats the viral DNA as part of its own and makes the proteins that are required to assemble new virus particles. Professor Dr. Hans-Georg Kräusslich and his team in the Virology Division in the Department of Infectiology at the University Hospital of Heidelberg are specifically focused on the virus’ molecular architecture and the morphogenesis of infectious HIV particles at the plasma membrane of the host cell. The researchers are also looking for drugs which inhibit the assembly of the individual constituents into a mature virus and aid in the development of novel antiviral therapies.
The morphogenesis of HIV can be divided into three stages: (1) assembly, wherein the essential viral components are assembled; (2) budding, i.e. the virion crosses the plasma membrane, taking a piece of the host’s cell membrane with it to form its own lipid membrane; and (3) maturation, wherein the virion changes its structure and becomes infectious. All of these stages take place at or close to the plasma membrane of the infected cell. All components that are required for a virus to become infective are assembled inside the host cell, just below the cell’s surface. The components include two copies of the viral genomic RNA, cellular tRNA (Lys,3) molecules, which serve as the primers for the synthesis of cDNA, viral envelope proteins (env), the structural Gag polyproteins (group-specific antigen) as well as three viral enzymes (a protease, a reverse transcriptase and an integrase).
The decisive steps of HIV morphogenesis are more or less simultaneously coordinated by the Gag polyprotein and its proteolytic maturation products, which are the major structural proteins of the virus: the binding to the plasma membrane, the protein-protein interactions that lead to the formation of a spherical protein capsid consisting of around 2,500 Gag proteins, the recruitment of viral envelope protein and the packaging of the genomic RNA. The virus then pushes itself out of the host cell, a process known as budding, and takes with it part of the host cell membrane. The viruses mature during budding or shortly after: newly synthesised Gag polyprotein is cleaved by the viral protease at 10 different sites, which leads to a dramatic rearrangement of the capsid. This second assembly step, which takes place at the plasma membrane or outside of the host cell close to it, produces infectious virus particles with their typical cone-shaped capsid core which contains the condensed viral genome and the enzymes that the virus needs to take with it (protease, reverse transcriptase, integrase) in order to replicate and infect a new cell.
Using purified Gag protein that was expressed in bacteria, the researchers were able to show that the viral Gag polyprotein is solely responsible for the assembly of immature HIV particles and the packaging of the co-factors. However, the budding and release of viral particles involves the ESCRT machinery of the host cell, which is a highly complex and evolutionarily conserved secretory pathway. The human ESCRT (endosomal sorting complex required for transport) pathway comprises thirty different proteins and is involved in the sorting of ubiquitin-modified proteins. Other regulatory mechanisms are also involved.
In order to obtain a greater understanding of viral assembly, budding and maturation, Kräusslich and his team use several different methods, including transmission electron microscopy, cryoelectron microscopy, tomography and fluorescence microscopy (see BIOPRO article of 8th Oct. 2012: "Martin Beck and the nuclear pore atlas"). Moreover, they use different biochemical and cell-biological approaches to elucidate the functions of the cellular ESCRT proteins, as well as the processes of ubiquitinylation and phosphorylation. They also use a method known as ‘random siRNA screening’ to identify further proteins involved in viral assembly, budding and replication. Working with Professor Hell’s group at the neighbouring German Cancer Research Center (DKFZ), which has access to so-called STED fluorescence microscopes with a remarkably high resolution, the researchers uncovered how infectious HI viruses dock to cells.
Kräusslich believes that the viral assembly process is a highly promising target for new drugs for the treatment of HIV infection and AIDS. Using the phage display technique, the researchers from Heidelberg identified the peptide CAI (capsid assembly inhibitor) that efficiently prevents the assembly of the viral capsid in vitro. Structural analyses have led to the identification of a previously unknown site where CAI needs to bind to the capsid protein CA and prevents it from interacting with other capsid proteins, thereby preventing the virus particles from assembling. Other investigations are focused on the identification of low molecular CAI analogues which have the potential to inhibit viral assembly and maturation. These analogues can also be used as lead compounds for the development of antiviral drugs.
When the virus pushes itself out of the host cell (budding), it takes with it part of the membrane of the host cell. This lipid membrane then covers the capsid. When the virus infects a new cell, its lipid membrane fuses with the plasma membrane of the infected cell. It plays a crucial role for the stability of the virus as well as its infectious ability, but little is yet known about its precise function. Much is however known about the HIV proteins. In cooperation with Britta Brügger and Felix Wieland from the Heidelberg University Biochemistry Centre, Kräusslich analysed the lipid components of HIV using mass spectrometry and thin-layer chromatography and compared them to the lipids of the host cell membranes. This cooperative project was part of the CellNetworks cluster of excellence at the University of Heidelberg, which is coordinated by Professor Kräusslich.
The lipidome (all lipids of a cell or organism) of the viral membrane differs considerably from the lipid composition of MT-4 cell plasma membranes (ed. note: MT-4 cells are human T-cell leukaemia cells that are used as host cells for culturing HIV). The researchers found that the cholesterol-phosolipid ratio of the viral membrane was more than twice as high as that of the plasma membrane. Moreover, the viral membrane contained even larger quantities of sphingomyelin, dihydrosphingomyelin and plasmologen PE (plasmaenyl-ethanolamine). These findings confirmed the ‘lipid raft’ hypothesis which proposes that budding and fusion processes take place at specific microdomains of the plasma membrane, which are due to the ‘rafting’ of lipid complexes that have a relatively low fluidity and therefore move around in the more fluid lipid film of the plasma membrane like a raft. Cholesterol, sphingolipids and plasmalogen PE are typical raft lipids. Similar lipid distribution patterns have in the meantime also been identified for other viruses with membrane envelopes. The researchers assume that certain lipid components also play a role in directing proteins like Gag to their assembly site or binding them to the membrane.
The HIV lipidome is not only of great interest for basic researchers; it also has the potential to be used as a target for antiviral drugs. Researchers have already shown that the inhibition of sphingomyelin biosynthesis leads to the loss of the infectious capability of viruses: changes in the lipid composition and use of specific synthetic peptides have been shown to prevent the viral membrane from fusing with the plasma membrane, thereby preventing viruses from being released from or entering host cells.
References:Sundquist WI, Kräusslich HG : HIV-1 assembly, budding and maturation. Cold Spring Harb Perspect Med. 2012 July; 2(7): a006924.Lorizate M, Kräusslich HG: Role of lipids in virus replication. Cold Spring Harb Perspect Biol. 2011 Oct 1;3(10):a004820.Brügger B, Glass B, Haberkant P, Leibrecht I, Wieland FT, Kräusslich, HG: The HIV lipidome: A raft with an unusual composition. Proc. Natl. Acad. Sci. USA 103: 2641-2646 (2006).