5.23 Entry of reovirus into cells. (A) The different stages in cell entry of reovirus. After the attachment of σ1 protein to the cell receptor, the virus particle enters the cell by clathrin-mediated endocytosis. Proteolysis in the late endosome produces the infectious subviral particle (ISVP). The viral μ1, a myristoylated protein, is located at the surface of these particles and interacts with membranes. Consequently, subviral particles penetrate the lysosomal membrane and escape into the cytosol. (Insets) Close-up views of the emerging turret-like structure as the virus progresses through the ISVP and core stages. This structure may facilitate the entry of nucleotides into the core and the exit of newly synthesized viral mRNAs. (B) Schematic of the μ1 protein, showing locations of myristate and the protease cleavage sites flanked by the amphipathic α-helices. Virus images based on studies performed with mammalian reovirus type I Lang, reprinted from Dryden KA et al. 1993. J Cell Biol 122:1023–1041, with permission. Courtesy of Norm Olson and Tim Baker, Purdue University.
Import of Viral Genomes into the Nucleus
The reproduction of most DNA viruses, and some RNA viruses including retroviruses and influenza viruses, begins in the cell nucleus. The genomes of these viruses must therefore be imported from the cytoplasm. One way to accomplish this movement is via the cellular pathway for protein import into the nucleus. An alternative, observed in cells infected by some retroviruses, is to enter the nucleus after the nuclear envelope breaks down during cell division.
The Nuclear Pore Complex
The nuclear envelope is composed of two typical lipid bilayers separated by a luminal space. Like all other cellular membranes, it is impermeable to macromolecules such as proteins. However, the nuclear pore complexes that stud the nuclear envelopes of all eukaryotic cells provide aqueous channels that span both the inner and outer nuclear membranes for exchange of small molecules, macromolecules, and macromolecular assemblies between nuclear and cytoplasmic compartments. Numerous experimental techniques, including direct visualization of gold particles attached to proteins or RNA molecules as they are transported, have established that nuclear proteins enter and RNA molecules exit the nucleus by transport through the nuclear pore complex. The functions of the nuclear pore complex in these processes are not completely understood, not least because this important cellular machine is large (molecular mass, approximately 125 × 106 kDa in vertebrates), built from many different proteins, architecturally elaborate, and dynamic (Fig. 5.24).
The nuclear pore complex allows passage of cargo in and out of the nucleus by either passive diffusion or facilitated translocation. Passive diffusion does not require interaction between the cargo and components of the nuclear pore complex and becomes inefficient as molecules approach 9 nm in diameter. Objects as large as 39 nm in diameter can pass through nuclear pore complexes by facilitated translocation via specific interactions between the cargo and components of the nuclear pore complex. Many subviral particles are too large to pass through the nuclear pore complex, but several strategies overcome this limitation.
Nuclear Localization Signals
Proteins that reside within the nucleus are characterized by the presence of specific nuclear targeting sequences. Such nuclear localization signals are both necessary for nuclear entry of the proteins in which they are present and sufficient to direct heterologous, nonnuclear proteins to enter this organelle. Nuclear localization signals identified by these criteria share a number of common properties: they are generally fewer than 20 amino acids in length, and are usually rich in basic amino acids. Although no consensus nuclear localization sequence can be defined, most nuclear localization signals belong to one of two classes, simple or bipartite sequences (Fig. 5.25). A particularly well-characterized example of a simple nuclear localization signal is that of simian virus 40 large T antigen, which comprises five contiguous basic residues flanked by hydrophobic amino acids.
Figure 5.24 Structure and organization of the nuclear pore complex. (Bottom left) Cartoon depiction of the nuclear membrane, showing the topology of the nuclear pore complexes. The entire structure of the nuclear pore complex of Saccharomyces cerevisiae, comprising 552 proteins, was determined using cryo-electron tomography and modeling (see Kim SJ et al. 2018. Nature 555:475–482). The yeast and human nuclear pore complexes are highly conserved. (Top) Side view of the nuclear pore showing three spokes. Spokes are high-ordered subcomplexes formed by NUPs (nucleoporins). Spokes in turn assemble into larger complexes to form two outer and two inner rings flanking a membrane ring. The complex is completed by a nuclear basket composed of NUPs. The outer ring comprises export complexes (olive green) and connectors (tan) with the inner ring (magenta, pink, and green). The inner rings sandwich the membrane ring (light yellow). The nucleoplasmic outer ring links to the nuclear basket (only partly depicted as light blue filaments). (Bottom right) Top view of the nuclear pore from the cytoplasm showing a model of the central transporter (green). Structure reconstructions courtesy of Michael Rout, Rockefeller University, New York.
Figure 5.25 Nuclear localization signals. The general form and specific examples of simple and bipartite nuclear localization signals are shown in the one-letter amino acid code, where X is any residue. Bipartite nuclear targeting signals are defined by the presence of two clusters of positively charged amino acids separated by a spacer region of variable length and sequence. Both clusters of basic residues, which often resemble the simple targeting sequences of proteins like simian virus 40 T antigen, are required for efficient import of the proteins in which they are found. The subscript indicates either length (3–7) or composition (e.g., 3/5 means at least 3 residues out of 5 are basic). Gold particles with diameters as large as 26 nm are readily imported following their microinjection into the cytoplasm, as long as they are coated with proteins or peptides containing a nuclear localization signal.
Import of a protein into the nucleus via nuclear localization signals occurs in two distinct, and experimentally separable, steps (Box 5.5). A protein containing such a signal first binds to a soluble cytoplasmic receptor protein. This complex then engages with the cytoplasmic surface of the nuclear pore complex, a reaction often called docking, and is then translocated through the nuclear pore. In the nucleus, the complex is disassembled, releasing the protein cargo.
Nuclear Import of RNA Genomes
Influenza virus is among the few RNA-containing viruses with genomes that are replicated in the cell nucleus. The influenza virus genome, which consists of eight segments, is uncoated in the cytoplasm. After vRNPs separate from M1 and are released into the cytosol, they are imported rapidly into the nucleus (Fig. 5.26A). Import depends on the presence of a nuclear localization signal in the NP protein, a component of vRNP: naked viral RNA does not dock onto the nuclear pore complex, nor does it enter the nucleus.
BACKGROUND
Transport through the nuclear pore
