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MEDICAL BIOLOGY: ON VIRAL ENTRY INTO HOST CELLS

The following points are made by Robert W. Doms (New Engl. J. Med. 2004 351:743):

1) Until recently, antiviral drugs were both uncommon and not terribly potent. This has changed: during the past decade, more than 30 antiviral drugs have been licensed, and many of them are very effective. Most of the drugs inhibit the activity of viral enzymes, but a new class of agents that block entry of the virus into the cell is being developed. The development of entry inhibitors is driven by the identification of the cell-surface receptors to which viruses bind and by new findings about viral protein structures that bind receptors and mediate viral entry. These advances offer opportunities for the development of agents that block viral transmission and treat viral infections, as well as for vaccine development.

2) Most entry inhibitors under clinical development are directed against viruses that are surrounded by a lipid membrane -- the so-called "enveloped viruses". Regardless of the type of enveloped virus, the fundamental steps of entry are the same. First, the virus attaches to the cell surface, often engaging a specific viral receptor. Viral receptors play a critical role in mediating the entry of the virus into the cell, and the distribution of receptors across specific cell types helps to determine viral tropism. Thus, most strains of the human immunodeficiency virus (HIV) need to engage CD4 and the chemokine receptor CCR5 sequentially to enter the cell, which largely restricts viral infection to certain T cells and macrophages.

3) Second, binding to the receptor induces the viral-envelope protein to undergo conformational changes that mediate fusion between the viral and cellular membranes in one of two ways. For some viruses, receptor binding leads to endocytosis of the viral particle and delivery to an acidic compartment. There, the low-pH environment triggers conformational changes that lead to membrane fusion. Influenzavirus, West Nile virus, and rabies virus are examples of viruses that use this pathway. For other viruses, the mere process of binding to one or more receptors leads to the needed conformational changes. These pH-independent viruses can fuse at the cell surface; HIV is the best-characterized example.

4) Since each step of the viral-entry pathway is a potential target for antiviral agents, entry inhibitors fall into several categories, depending on which step they target. The first category includes compounds that bind to viral receptors. Small-molecule inhibitors that target the HIV receptor CCR5, which are under clinical development, have been shown to reduce dramatically the levels of circulating virus in HIV-infected patients. Because they target an invariant cellular protein, use of these compounds obviates the difficulty of attempting to target a virus that has considerable genetic variability. The success of these compounds in early clinical trials, coupled with resistance to HIV infection in people who lack CCR5, fueled efforts to identify receptors that are engaged by other viruses. The recent identification of angiotensin-converting enzyme-2 as the receptor for the coronavirus that causes the severe acute respiratory syndrome(1) has already led to the identification of antibodies that prevent receptor binding and may lead to the discovery of small-molecule inhibitors as well.

5) The second category of entry inhibitors includes compounds that bind to the virus and prevent it from interacting with its receptors. Although some neutralizing antibodies have long been known to operate by this mechanism, small-molecule inhibitors that accomplish the same feat have been developed more recently. Crystallographic studies have helped enormously. For example, an understanding of the structure of picornaviruses (such as human rhinoviruses) has led to the development of a whole series of compounds that fit into a pocket on the viral surface. Some of these compounds block viral attachment, and several have been tested in clinical trials. A small-molecule inhibitor that prevents binding of the HIV envelope protein to the CD4 receptor is also being tested in clinical trials.

6) The final category of entry inhibitors prevents the conformational changes needed for membrane fusion and includes enfuvirtide, an inhibitor of HIV membrane fusion that has been licensed by the Food and Drug Administration. Enveloped viruses appear to use two classes of membrane-fusion proteins. Class I fusion proteins -- such as those found on HIV, influenzavirus, Ebola virus, and respiratory syncytial virus -- are trimers of identical subunits that project from the viral surface. On activation by either acid pH or receptor binding, a series of conformational changes occurs. Part of the fusion protein is inserted into the membrane of the cell, linking the viral and cellular membranes. Fusion is then caused by a conformational change in which the two helical regions of the fusion protein fold back on each other, winching the fusion peptide (inserted in the cell membrane) and the membrane-anchoring region of the viral envelope protein (anchored in the viral membrane) toward each other, a process that brings about lipid mixing.(2) Enfuvirtide is a peptide that, by binding to one of these helical regions, prevents the conformational change needed for fusion.(3)

References:

1. Sui J, Li W, Murakami A, et al. Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association. Proc Natl Acad Sci U S A 2004;101:2536-2541

2. Moore JP, Doms RW. The entry of entry inhibitors: a fusion of science and medicine. Proc Natl Acad Sci U S A 2003;100:10598-10602

3. Modis Y, Ogata S, Clements D, Harrison SC. Structure of the dengue virus envelope protein after membrane fusion. Nature 2004;427:313-319

New Engl. J. Med. http://www.nejm.org

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VIROLOGY: ON VIRUS ENTRY INTO ANIMAL CELLS

The following points are made by A.E. Smith and A. Helenius (Science 2004 304:237):

1) Although extremely simple in structure and composition, viruses are masters of camouflage and deception. Devoid of any means of independent locomotion, they disseminate by exploiting cells and organisms. Aided by rodents, insects, and migratory birds, and passed along by global trade and travel, they move around the world with amazing speed. Once they enter the body of a potential host, they can penetrate mucus layers, move through the blood stream, and disperse with the help of motile cells and neuronal pathways.

2) A critical moment occurs when a virus particle reaches a potential host cell and attaches itself to the surface. It must now deliver its capsid and accessory proteins into the cell in a replication-competent form, ideally with minimal damage to the cell and leaving little evidence of its entry for detection by the immune defenses. This is not a trivial problem because cell membranes are impermeable to macromolecules.

3) Viral particles mediate the transfer of the viral genome and accessory proteins from an infected host cell to a noninfected host cell. The task involves packaging the viral genome (RNA or DNA) and accessory proteins, releasing the package from the infected cell, protecting the essential components during extracellular transmission, and delivering them into a new host cell. Many viruses with a DNA genome must enter the nucleus, whereas RNA viruses, with a few exceptions, replicate in the cytosol. Overall, viruses use a "Trojan horse" strategy in which the victim assists the intruder. To extract assistance from the host cell, viruses use the detailed "insider information" that they have acquired during millions of years of coevolution with their hosts.

4) In a typical animal virus particle, the viral RNA or DNA is condensed in icosahedral or helical nucleoprotein complexes called capsids. In enveloped viruses, the capsids are surrounded by a lipid bilayer that contains viral spike glycoproteins. In addition, some viruses contain reverse transcriptases, RNA polymerases, kinases, and other proteins that are important during uncoating, replication, or other early intracellular steps.

5) To infect a target cell, a virus particle proceeds through a multistep entry process, during which each step is preprogrammed and tightly regulated in time and space. The entry steps are virus binding to the cell, endocytosis, and nuclear import. Another critical step in the infection process is uncoating, during which the lipid envelope must be shed and the capsids must be at least partially disassembled to expose a replication-competent genome. Once uncoating has occurred, the mobility of the genome within the cell is restricted.

6) In summary: Viruses replicate within living cells and use the cellular machinery for the synthesis of their genome and other components. To gain access, they have evolved a variety of elegant mechanisms to deliver their genes and accessory proteins into the host cell. Many animal viruses take advantage of endocytic pathways and rely on the cell to guide them through a complex entry and uncoating program. In the dialogue between the cell and the intruder, the cell provides critical cues that allow the virus to undergo molecular transformations that lead to successful internalization, intra-cellular transport, and uncoating.(1-5)

References (abridged):

1. S. Pohlmann, F. Baribaud, R. W. Doms, Trends Immunol. 22, 643 (2001)

2. A. A. Bashirova et al., J. Exp. Med. 193, 671 (2001)

3. Y. Feng, C. C. Broder, P. E. Kennedy, E. A. Berger, Science 272, 872 (1996)

4. C. M. Carr, P. S. Kim, Cell 73, 823 (1993)

5. S. A. Gallo et al., Biochim. Biophys. Acta 1614, 36 (2003)

Science http://www.sciencemag.org

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VIROLOGY: ON THE BUDDING OF VIRUSES FROM HOST-CELL MEMBRANES

The following points are made by M.W Yap and J.P Stoye (J. Biol. 2003 3:3):

1) How do enveloped viruses bud from their host cells? To understand how this process is achieved, several fundamental steps must be considered. First, viral structural components must be transported to the appropriate site, typically just under a cell membrane, and there assembled [1]. Second, the plasma membrane must be distorted to make a succession of curved budding structures; this requires overcoming the mechanical bending resistance of the plasma membrane [2]. Third, following the formation of the bud, the virus has to pinch off and escape from the cell [3]. This involves machinery that constricts the neck of the bud, resulting in fusion between the membranes on either side of the neck and the release of the virus from the plasma membrane. Studies with a number of virus types, most prominently retroviruses, have now revealed that cellular proteins that are intimately involved in intracellular membrane trafficking and receptor re-localization play key roles in facilitating these processes.

2) For a long time, it has been known that the only retroviral component required for assembly and budding is the Gag polyprotein, which ultimately forms the viral core [1]. Gag is cleaved into a variety of smaller components as the virus matures. These include, from amino terminus to carboxyl terminus, the matrix (MA), capsid (CA) and nucleocapsid (NC). Depending on the virus analyzed, a variety of other protein products are seen after cleavage of Gag. For example, in human immunodeficiency virus-1 (HIV-1) a short peptide called p6 is cleaved from the carboxy-terminal end of NC, whereas in murine leukemia virus (MuLV) a p12 peptide is cleaved from between MA and CA.

3) Three types of functional domain of Gag can be identified: M, sequences required for transport to and binding of membranes; I, involved in Gag-Gag interactions; and L, late sequences [1,3]. The L domains are short peptide motifs located in different regions of Gag in different viruses; mutation in these sequences results in failure to release budded viruses [4,5]. Many L domains are interchangeable between viruses, suggesting that their role in the late stages of budding is to act as docking sites for cellular proteins [5]. A key step in understanding the late budding process came with the demonstration that the L domain of HIV-1 Gag interacted with a component of the cellular machinery responsible for sorting cargo into multivesicular bodies (MVBs).

4) Although significant steps have been taken towards understanding virus budding during the past couple of years, there are still a number of important issues that remain to be addressed. How is the initial bud formed? It may be that energetic requirements for membrane distortion can be met simply by the I-domain-mediated assembly of Gag molecules, resulting in movement of associated membrane lipid molecules. But what happens in the case of viruses like MPMV that assemble in the cytoplasm? Is there a need for cellular enzymes such as endophilin to introduce negative curvature (bending towards the outside of the cell) by modifying the lipid composition of the membrane? How does membrane pinching-off take place? The ESCRT complex is intimately involved, but is the whole complex required and what is the role of other factors such as the ubiquitin ligase, Nedd4, that are clearly involved in the budding of certain viruses? How is the plasma membrane targeted for budding? In macrophages, HIV-1 can bud into vacuoles, but what targets Gag and associated ESCRT complexes to the cell surface in HIV-infected T cells?

References (abridged):

1. Swanstrom R, Wills JW: Synthesis, assembly, and processing of viral proteins. In Retroviruses (Edited by: Coffin JM, Hughes SH, Varmus HE). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press 1997, 263-334

2. Hurley JH, Wendland B: Endocytosis: driving membranes around the bend. Cell 2002, 111:143-146

3. Freed EO: Viral late domains. J Virol 2002, 76:4679-4687

4. Goettlinger HG, Dorfman T, Sodroski JG, Haseltine WA: Effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release. Proc Natl Acad Sci USA 1991, 88:3195-3199.

5. Yuan B, Campbell S, Bacharach E, Rein A, Goff SP: Infectivity of Moloney murine leukemia virus defective in late assembly events is restored by late assembly domains of other retroviruses. J Virol 2000, 74:7250-7260.

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