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Virus entry is initiated by recognition by receptors present on the surface of host cells. Receptors can be major mediators of virus tropism, and in many cases receptor interactions occur in an apparently programmed series of events utilizing multiple receptors. After receptor interaction, both enveloped and nonenveloped viruses must deliver their genome across either the endosomal or plasma membrane for infection to proceed. Genome delivery occurs either by membrane fusion (in the case of enveloped viruses) or by pore formation or other means of permeabilizing the lipid bilayer (in the case of nonenveloped viruses). For those viruses that enter cells via endosomes, specific receptor interactions (and the signaling events that ensue) may control the particular route of endocytosis and/or the ultimate destination of the incoming virus particles. Our conception of virus entry is increasingly becoming more complex; however, the specificity involved in entry processes, once ascertained, may ultimately lead to the production of effective antiviral agents.

Every enveloped virus fuses its membrane with a host cell membrane, thereby releasing its genome into the cytoplasm and initiating the viral replication cycle. In each case, one or a small set of viral surface transmembrane glycoproteins mediates fusion. Viral fusion proteins vary in their mode of activation and in structural class. These features combine to yield many different fusion mechanisms. Despite their differences, common principles for how fusion proteins function are emerging: In response to an activating trigger, the metastable fusion protein converts to an extended, in some cases rodlike structure, which inserts into the target membrane via its fusion peptide. A subsequent conformational change causes the fusion protein to fold back upon itself, thereby bringing its fusion peptide and its transmembrane domain—and their attached target and viral membranes—into intimate contact. Fusion ensues as the initial lipid stalk progresses through local hemifusion, and then opening and enlargement of a fusion pore. Here we review recent advances in our understanding of how fusion proteins are activated, how fusion proteins change conformation during fusion, and what is happening to the lipids during fusion. We also briefly discuss the therapeutic potential of fusion inhibitors in treating viral infections.

Upon infection, virions or subviral nucleoprotein complexes are transported from the cell surface to the site of viral transcription and replication. During viral egress, particles containing viral proteins and nucleic acids again move from the site of their synthesis to that of virus assembly and further to the plasma membrane. Because free diffusion of molecules larger than 500 kDa is restricted in the cytoplasm, viruses as well as cellular organelles employ active, energy-consuming enzymes for directed transport. This is particularly evident in the case of neurotropic viruses that travel long distances in the axon during retrograde or anterograde transport. Viruses use two strategies for intracellular transport: Viral components either hijack the cytoplasmic membrane traffic or they interact directly with the cytoskeletal transport machinery. In this review we describe how viruses—particularly members of the , and —make use of the microtubule and the actin cytoskeleton. Analysing the underlying principles of viral cytosolic transport will be helpful in the design of viral vectors to be used in research as well as human gene therapy, and in the identification of new antiviral target molecules.

The separation of transcription in the nucleus and translation in the cytoplasm requires nucleo-cytoplasmic exchange of proteins and RNAs. Viruses have evolved strategies to capitalize on the nucleo-cytoplasmic trafficking machinery of the cell. Here, we first discuss the principal mechanisms of receptor-mediated nuclear import of proteinaceous cargo through the nuclear pore complex, the gate keeper of the cell nucleus. We then focus on viral strategies leading to nuclear import of genomes and subgenomic particles. Nucleo-cytoplasmic transport is directly important for those viruses that are replicating in the nucleus, such as DNA tumor viruses and RNA viruses, including parvoviruses, the DNA retroviruses hepadnaviruses, RNA-retro-transposons and retroviruses, adenoviruses, herpesviruses, papovaviruses, and particular negative-sense RNA viruses, such as the orthomyxovirus influenza virus. The viral strategies of nuclear import turn out to be surprisingly diverse. Their investigation continues to give insight into how nucleic acids pass in and out of the nucleus.

All plus-strand RNA viruses replicate in association with cytoplasmic membranes of infected cells. The RNA replication complex of many virus families is associated with the endoplasmic reticulum membranes, for example, picorna-, flavi-, arteri-, and bromoviruses. However, endosomes and lysosomes (togaviruses), peroxisomes and chloroplasts (tombusviruses), and mitochondria (nodaviruses) are also used as sites for RNA replication. Studies of individual nonstructural proteins, the virus-specific components of the RNA replicase, have revealed that the replication complexes are associated with the membranes and targeted to the respective organelle by the ns proteins rather than RNA. Many ns proteins have hydrophobic sequences and may transverse the membrane like polytopic integral membrane proteins, whereas others interact with membranes monotopically. Hepatitis C virus ns proteins offer examples of polytopic transmembrane proteins (NS2, NS4B), a “tip-anchored” protein attached to the membrane by an amphipathic -helix (NS5A) and a “tail-anchored” posttranslationally inserted protein (NS5B). Semliki Forest virus nsP1 is attached to the plasma membrane by a specific binding peptide in the middle of the protein, which forms an amphipathic -helix. Interaction of nsP1 with membrane lipids is essential for its capping enzyme activities. The other soluble replicase proteins are directed to the endo-lysosomal membranes only as part of the initial polyprotein. Poliovirus ns proteins utilize endoplasmic reticulum membranes from which vesicles are released in COPII coats. However, these vesicles are not directed to the normal secretory pathway, but accumulate in the cytoplasm. In many cases the replicase proteins induce membrane invaginations or vesicles, which function as protective environments for RNA replication.

Viruses use the host cellular machinery to translate viral proteins. Similar to cellular proteins directed to the secretory pathway, viral (glyco)proteins are synthesized on polyribosomes and targeted to the endoplasmic reticulum (ER). For viruses that encode polyproteins, folding of the individual proteins of the precursor often is coordinated. Translocation and the start of folding coincide and are assisted by cellular folding factors present in the lumen of the ER. The protein concentration a newborn protein finds in this compartment is enormous (hundreds of mg/ml) and the action of molecular chaperones is essential to prevent aggregation. Viral envelope proteins also undergo the cellular quality control mechanisms, which ensure, with variable stringency, that only proteins with the correct structure will proceed through the secretory pathway. Proteins that are misfolded, or not yet folded, are retained in the ER until they reach the native conformation or until their retrotranslocation into the cytosol for degradation. Peculiar characteristic of viruses is their ability to interfere with the cellular machinery to ensure virus production and, moreover, to pass through the body unobserved by the host immune system. This section describes some mechanisms of genetic variation and viral immune evasion that involve the secretory pathway.

Evasion of host immunity is a common objective of viruses that cause chronic infections. Viruses involved in sexually transmitted infections constitute no exception to this phenomenon. HIV, HPV, HSV, and HHV-8 subvert the class I major histocompatibility complex (MHC-I) antigen presentation pathway, thereby evading the cellular immune response. Although the goal of these viruses is the same and efficient MHC-I downregulation in infected cells is achieved, their techniques vary considerably. Whether viral inhibition occurs at the transcriptional level, during assembly of MHC-I complexes in the endoplasmic reticulum, during its journey to the cell surface, or after reaching the cell surface, each one of these viruses ingeniously achieves MHC-I downregulation and avoids the cellular immune response. Unraveling the mechanisms of interference with MHC-I antigen presentation employed by these viruses is not only crucial to understand their pathogenesis, but also reveals novel mechanisms of regulation of cellular receptors. When employed as modulators of cellular trafficking pathways, viruses become tools to dissect fundamental cell processes. In return, the precise dissection of these processes may offer new weapons against the ruses viruses employ to propagate and establish chronic infections.

Many viruses express membrane proteins. For enveloped viruses in particular, membrane proteins are frequently structural components of the virus that mediate the essential tasks of receptor recognition and membrane fusion. The functional activities of these proteins require that they are sorted correctly in infected cells. These sorting events often depend on the ability of the virus to mimic cellular protein trafficking signals and to interact with the cellular trafficking machinery. Importantly, loss or modification of these signals can influence virus infectivity and pathogenesis.