Chapter 12: Intracellular Trafficking

I T Introduction Because of the structural and functional compartmentalization of eukaryotic cells, components of these particles are generally produced at multiple intracellular locations but must be brought together for assembly However, in the submicroscopic world inhabited by viruses, an animal cell is large: the distances over which virion components must be transported within a cell are roughly equivalent to 5 kilometers on a human scale The cellular highways most commonly used for movement of viral components for assembly are those formed by microtubules and actin filaments (as is also true during entry) Assembly of viral particles can occur at any one of several intracellular addresses, depending on whether the particles are enveloped or naked and on the site and mechanism of genome replication Assembly of enveloped viruses therefore requires delivery of some viral proteins to the appropriate membrane, as well as transport of other proteins and the nucleic acid genome Consequently, eukaryotic cells are constantly engaged in transport of macromolecules among their compartments via intracellular trafficking systems Assembly within the nucleus All structural proteins of these nonenveloped viruses are imported into the nucleus following synthesis In the infected cell cytoplasm, allowing complete assembly within this organelle These same pathways are responsible for import of both viral genomes (or nucleoproteins) and viral non-structural proteins that function in the nucleus early in the infectious cycle Import of viral proteins for assembly The majority of the sequences in viral structural proteins that have been shown to mediate nuclear import conform to the simple or bipartite nuclear localization signals A typical mammalian cell contains on the order of 3,000 to 4,000 nuclear pore complexes, each with a very high capacity, some 10^3 translocation events As large quantities of viral structural proteins must enter the nucleus prior to assembly, there is potential for competition among viral and cellular proteins for access to receptors or the nuclear pore complex proteins that mediate transport The increased density of these signals may allow more effective competition for essential components of the import pathway, or the nuclear localization signals may be more accessible in the complex Assembly at the plasma membrane The particles of many viruses that reproduce in animal cells include a lipid envelope derived from a membrane of the host cell, although this structural feature is rare among plant viruses Viruses with envelopes derived from the plasma membrane also contain internal proteins, which may be membrane associated, and, of course, nucleic acid genomes Transport of viral membrane proteins to the plasma membrane Entry into the first staging post of the secretory pathway, the endoplasmic reticulum (ER), is accompanied by membrane insertion of integral membrane proteins The extracellular domains include the binding sites for cellular receptors crucial for initiation of the infectious cycle, whereas cytoplasmic segments are important in virus assembly Oligomer assembly takes place during transit from the ER to the cell surface, as does the proteolytic processing necessary to produce some mature (functional) envelope glycoproteins from the precursors that enter the secretory pathway These characteristic covalent modifications take place as proteins travel through a series of specialized compartments that provide the chemical environments and enzymatic machinery necessary for their maturation Translocation of viral membrane proteins into the endoplasmic reticulum All proteins destined for insertion into the plasma membrane, or the membranes of intracellular organelles, enter the ER as they are translated The ER membrane demarcates a geometrically convoluted but continuous internal space, the ER lumen, from the remainder of the cytoplasm Polyribosomes engaged in synthesis of proteins that will enter the secretory pathway become associated with the cytoplasmic face of the ER membrane soon after translation begins. Areas of the ER to which polyribosomes are bound form the rough ER Proteins destined to enter the ER typically include a short sequence in the nascent protein, the signal peptide, that directs association of polyribosomes with the ER membrane Signal peptides are commonly found at the N termini of proteins) are usually about 20 amino acids in length, and contain a core of 15 hydrophobic residues The signal peptide is recognized by the signal recognition particle (SRP), which, in turn, binds to the cytoplasmic domain of an integral ER membrane protein termed the SRP receptor Binding of the signal recognition particle to the ribosome temporarily halts translation, the stalled translation complex associates with the ER membrane, and the ribosome becomes engaged with a protein translocation channel, which forms a gated, aqueous pore through the ER membrane Such coupling of translation and translocation ensures that the protein crosses the membrane as an unfolded chain that can be accommodated within the translocation channel When a protein entering the ER is destined for secretion from the cell, the entire protein chain is translated in the ER lumen and the signal peptide is proteolytically removed by signal peptidase, releasing the soluble protein into the ER In proteins that span the membrane multiple times, the number, location, and orientation of stop and start transfer signals within a protein determine the topology with which it is organized in the ER membrane Reactions within the ER The lumen contains many enzymes that catalyze chemical modifications, such as disulfide bond formation and glycosylation, or that promote folding and oligomerization Glycosylation Viral envelope proteins that travel the secretory pathway, like their cellular counterparts, are generally modified by the addition of oligosaccharides to either asparagine (N-linked glycosylation) or serine or threonine (O-linked glycosylation) Consequently, the great majority of viral proteins are glycosylated by host cell components, but the structural proteins of some large DNA viruses are modified in this way by viral enzymes The presence of oligosaccharides on a protein can be detected as changes in the protein's electrophoretic mobility, following exposure of cells to inhibitors of glycosylation or of cell extracts to enzymes that clears the oligosaccharide Subsequently, several sugar residues are trimmed from N-linked core oligosaccharides in preparation for additional modifications that take place as the protein travels from the ER to the plasma membrane Each glycoprotein population therefore comprises a heterogeneous mixture of glycoforms, varying in whether a particular site is glycosylated, as well as in the composition and structure of the oligosaccharide present at each site Glycosylation has also been suggested to fulfill more general functions, such as protecting proteins and virus particles that circulate in body fluids from degradation and host immune defenses The hydrophilic oligosaccharides are present on the surfaces of such proteins, where they can form a sugar "shell", masking much of the proteins' surfaces, including epitopes recognized by antiviral antibodies Disulfide bond formation A second chemical modification generally restricted to proteins entering the secretary pathway, and essential for the correct folding of many, is the formation of intermolecular disulfide bonds between pairs of cysteine residues Disulfide bonds rarely form in the reducing environment of the cytoplasm: the more oxidizing ER lumen provides an appropriate chemical environment for this reaction As formation of the full and correct complement of disulfide bonds in a protein is often the rate-limiting step in its folding, these enzymes are important catalysts of this process Protein folding and quality control A number of other cellular proteins assist the folding of the extracellular domains of viral membrane glycoproteins as they enter the lumen of the ER Rather, their primary function is to facilitate folding, largely by preventing improper (dead-end) associations among unfolded or incompletely folded polypeptide chains, such as the non-specific, hydrophobic interactions The ER chaperones, which include GRP78 and calnexin, are also crucial for the quality control systems that determine the fate of newly synthesized proteins translated into the ER Binding of this chaperone [GRP78], generally at multiple sites in a nascent protein molecule, is thought to protect against misfolding and aggregation by sequestering sequences prone to nonspecific interaction, such as hydrophobic patches However, studies of specific viral glycoproteins in living cells indicate that where oligosaccharides occur within the protein chain is one important determinant of chaperone selectivity In contrast to GRP78, which recognizes protein sequences directly, calnexin distinguishes newly synthesized glycoproteins by binding to immature oligosaccharide chains Proteins with oligosaccharides that include a single glucose residue are recognized by calnexin but are released upon removal of the glucose by the enzyme glucosidase II Proteins that are misfolded or not modified correctly cannot escape covalent or non covalent associations with ER enzymes or molecular chaperones The quality control functions of resident ER chaperones and other proteins therefore ensure that nonfunctional proteins are cleared from the secretory pathway at an early step Oligomerization Most viral membrane proteins are oligomers that must assemble as their constituent protein chains are folded and covalently modified Several viral and some cellular heteromeric membrane proteins must oligomerize to travel beyond the ER, because folding of one subunit depends on association with the other(s) Nevertheless, oligomerization begins, and in some cases must be completed, within the ER, where it can be facilitated by the folding catalysts and chaperones characteristic of this compartment Vesicular transport to the cell surface The mechanism of vesicular transport Viral membrane proteins, like their cellular counterparts, travel to the cell surface through a series of membrane-bound counterparts and vesicles The first step in this pathway is transport of the folded protein from the ER to the Golgi apparatus, where proteins are sorted according to the addresses specified in their primary sequences or by their covalent modifications Fusion of the vesicle membrane with that of the target compartment releases the cargo into the lumen of that compartment A vesicle SNARE and an appropriate SNARE in the target membrane represent a minimal machinery for membrane fusion in vitro, but additional proteins allow efficient and regulated fusion Reactions within the Golgi apparatus The cis- and trans-Golgi networks are separated by a variable number of cisternae termed the cis, medial, and trans compartments The enzymes that catalyze sequential reactions in the synthesis of mature glycans are restricted to successively later Golgi compartments encountered by a protein traveling the secretory pathway Synthesis of O-linked Oligosaccharides by glycosyltransferases, which add one sugar residue at a time to specific serine or threonine residues, also takes place in the Golgi apparatus By providing an exit channel for protons, and hence increasing the pH of normally acidic compartments, such as those of the trans-Golgi network, this protein prevents premature switching of proteolytically processed HA to the fusion-active conformation Parameters determining the rate and efficiency of transport may include the complexity of the protein and the inherent asynchrony of protein folding and whether translation of cellular mRNAs is inhibited Consequently, competition among viral and cellular proteins, which may vary with the nature and physiological state of the host cell, is also likely to affect the transport of viral proteins to the cell surface The bulky fatty acid chains attached to the cytoplasmic tails may regulate envelope protein conformation or association with specific membrane domains Sorting of viral proteins in polarized cells However, this membrane is not uniform in all animal cells: differentiated cells often devote different parts of their surfaces to specialized functions, and the plasma membranes of such polarized cells are partitioned into correspondingly distinct regions During infection by many enveloped viruses, the asymmetric surfaces of such cells are distinguished during entry and when components of progeny virus particles are sorted to a specific plasma membrane region Epithelial cells Epithelial cells, which cover the external surfaces of vertebrates and line all their internal cavities (such as the respiratory and gastrointestinal tracts), and primary targets of virus infection The cells of an epithelium are organized into close-knit sheets, by both the tight contacts they make with one another and their interactions with the underlying basal lamina, a thin layer of extracellular matrix The former [apical domain] performs more specialized functions, whereas the latter [basolateral domain] is associated with cellular housekeeping These two domains differ in their protein and lipid contents, in part because they are separated by specialized cell-cell junctions (tight junctions), which prevent free diffusion and mixing of components in the outer leaflet of the lipid player Viruses have been important tools in elucidation of the molecular mechanisms responsible for the polarity of typical epithelial cells, because certain enveloped viruses bud asymmetrically Signals necessary for basolateral targeting comprise short amino acid sequences located in the cytoplasmic domains of membrane proteins Indeed, many basolateral targeting signals overlap with those that direct proteins for clathrindependent endocytosis, and certain proteins are transferred through endosomes from one membrane domain to the other, a process termed transcytosis Neurons Axons are specialized for the transmission of electrical and chemical impulses, controlled in part by the formation of synaptic vesicles and release of their contents Several mechanisms contribute to the establishment and maintenance of neuronal polarity, including transport of vesicles in specific directions along the highly organized microtubules of the axon, transport of mRNAs to specific regions of the neuron, and sorting and targeting of membrane proteins for delivery to axonal or dendritic surfaces The directional movement of vesicles and many cellular organelles in neurons is dependent on polarized microtubules and molecular motors that travel toward either their (-) or (+) ends Later in the infectious cycle, components of virus particles must be moved in the opposite direction (toward the synapse) upon association with proteins of the kinesin family Disruption of the secretory pathway in virus-infected cells Inhibition of transport of cellular proteins Some viral proteins interfere with transport to the plasma membrane of specific cellular proteins, notably major histocompatibility complex (MHC) class I molecules Prevention of transport of MHC class I proteins to the cell surface therefore helps prevent or delay the detection and destruction of infected cells Such degradation of CD4, the major receptor for this virus, contributes to assembly and release of virus particles: tight binding of this cellular protein to the viral Env glycoprotein in the ER prevents transit of both proteins to the cell surface Drastic effects on compartments of the secretory pathway The replication of most (+) strand RNA viruses takes place in association with membranous structures derived from various cytoplasmic membranes of the host cell The temporary acceleration in vesicular traffic from the ER may increase the supply of membranes and other components to the ER-Golgi intermediate compartment (ERGIC), the origin of the distinctive vesicles that serve as platforms for the replication of the viral RNA genome Induction or inhibition of the unfolded protein response When the capacity of the ER protein folding and removal machinery is exceeded, GRP78 is sequestered by association with the large quantities of misfolded or unfolded proteins These signal transduction cascades, collectively known as the unfolded protein response, lead to inhibition of translation and enhanced production of ER membranes and resident folding chaperones and catalysts, as well as of ER proteins that clear improperly folded proteins Activation of the unfolded protein response in virus-infected cells would seem to be a mixed blessing: increased ER capacity would facilitate the production of large quantities of viral proteins, but attenuation of translation, induction of apoptosis, and increased ER-associated degradation could limit virus reproduction Interactions with internal cellular membranes The envelopes of a variety of viruses are acquired from internal membranes of the infected cell, rather than from the plasma membrane The diversity of the internal membranes with which these viruses associate during envelope acquisition and exocytosis is the result of variations on a single mechanistic theme: the site of assembly of infectious virus particles is determined by the intracellular location of viral envelope proteins Transport of viral genomes to assembly sites Like the structural proteins and enzymes of virus particles, progeny genomes must be available, or concentrated, at the intracellular site of assembly Similarly, both assembly of virus particles and the replication of the genomes of many (+) strand viral RNAs and of large DNA viruses such as poxviruses are restricted to specialized cytoplasmic structures derived from host cell membranes In contrast, the genomes of many (-) strand RNA viruses must be transported to the cytoplasmic faces of the appropriate membrane, most commonly the plasma membrane Transport of genomic and pregenomic RNA from the nucleus to the cytoplasm Retroviral genomes are unspliced RNA transcripts synthesized In infected cell nuclei by host cell RNA polymerase II, as is hepadnaviral pregenomic RNA Perhaps the most elaborate requirements for transport of viral RNA species between the nucleus and cytoplasm are found in influenza A virus infected cells: both the direction of transport of genomic RNA and the nature of the viral RNA exported from the nucleus change as the infectious cycle progresses Export of these large viral ribonucleoproteins is facilitated late in infection by caspase-dependent degradation of A nuclear pore component (NUP153) with concomitant increases in nuclear pore diameter and the capacity to translocate large macromolecular assemblies across the nuclear membrane Transport of genomes from the cytoplasm to the plasma membrane Nevertheless, it is clear that there are multiple routes for transport of viral RNA genomes even to the same destination (the plasma membrane) and that the mode of cytoplasmic transport of RNAs made in the nucleus can be prescribed by the mechanism of nuclear export Entry into the latter pathway is determined by the viral M protein, which associates with RNPs containing genomic RNA to induce formation of a tightly coiled RNA-protein "skeleton" Formation of this structure precludes replication and mRNA synthesis and allows transport of nucleocapsids from the perinuclear region, where they assemble, towards the plasma membrane via both microtubules and actin filaments The functions of Gag include transport of unspliced RNA molecules to membrane assembly sites and packaging of the RNA into assembling capsids Such selective encapsidation requires packaging signals located at the 5' end of retroviral genomes, their binding by the NC domain of cytoplasmic Gag, and more extensive Gag-genomic RNA interactions established as virus particles assemble Although these RNA- and membrane-binding domains are present in all retroviral Gag proteins, the mode of transport of RNA genomes in the cytoplasm appears to be governed by their pathway of nuclear export Such interactions commit genomic RNA to the assembly pathway, direct the RNA to the plasma membrane, and promote interactions among internal and envelope components of virus particles The retroviral mechanism is rather different: following virus assembly and budding, Gag (and Gag-Pol) polyproteins are processed by viral protease to the individual structural proteins