Schematic illustration of a classical nuclear import pathway. Data from Yang Q et al. 1998. Mol Cell 1:223–234.
Different groups of proteins are imported by specific nuclear transport receptor complexes. In what is known as the “classical system” of import, cargo proteins containing basic nuclear localization signals (NLS) bind to the cytoplasmic nuclear localization signal receptor protein importin-α (step 1 in the figure). This complex then binds importin-β1, which mediates docking with the nuclear pore complex by binding to members of a family of nucleoporins (step 2). It is likely that initial association involves nucleoporins present in the cytoplasmic filaments of the nuclear pore. Importin-β1 also interacts with RAN, a small RAS-related nucleotide-binding protein. RAN GTPase (step 3) is required for translocation of the complex into the nucleus through the central channel of the nuclear pore (step 4).
A single translocation through the nuclear pore complex does not require energy consumption. However, maintenance of a gradient of the guanosine nucleotide-bound forms of RAN is absolutely essential for continued transport. A RAN-specific guanine nucleotide exchange protein named RCC1 (regulator of chromosome condensation 1) resides in the nucleus and promotes the exchange of GDP to GTP. In contrast, a RAN-GTPase-activating protein (RANGAP-1) localized in the cytoplasm promotes hydrolysis of GTP. The nuclear pool of RAN-GDP is replenished by the action of nuclear transport factor 2 (NTF2), which transports RAN-GDP from the cytoplasm to the nucleus efficiently (step 5), where it can be converted to RAN-GTP. The asymmetric distribution of RCC1 and RANGAP-1 allows for the formation of a gradient of RAN-GTP/RAN-GDP. This gradient provides the driving force and directionality for nuclear transport.
Importin-β1 has a higher affinity for RAN-GTP, which is more abundant in the nucleus, than for RAN-GDP. Therefore, following import into the nucleus, importin-β1 binds to RAN-GTP and the complex disassembles, eventually releasing the cargo protein. The importin-β1 recycles to the cytoplasm bound to RAN-GTP (step 6). There, it is displaced by the action of two high-affinity RAN-GTP-binding proteins, RANBP1 and RANBP2 (or NUP358). This enables conversion of RAN-GTP to RAN-GDP and binding of importin-β1 to new substrates.
Figure 5.26 Different strategies for entering the nucleus. (A) Each segment of the influenza virus genome is small enough to be transported through the pore complex. (B) The herpes simplex virus 1 capsid docks onto the nuclear pore complex and is minimally disassembled to allow transit of the viral DNA into the nucleus. (C) The adenovirus subviral particle is dismantled at the nuclear pore, allowing transport of the viral DNA with core protein VII into the nucleus. (D) The capsids of some viruses (parvovirus and hepadnavirus) are small enough to enter the nuclear pore complex without disassembly but do not enter by this route. These virus particles bind the nuclear pore complex, which induces local disruption of the nuclear envelope, allowing nuclear entry.
Nuclear Import of DNA Genomes
The capsids of many DNA-containing viruses are larger than 39 nm in diameter and cannot be imported into the nucleus from the cytoplasm. One mechanism for crossing the nuclear membrane comprises docking of a capsid onto the nuclear pore complex, followed by delivery of the viral DNA into the nucleus. Adenoviral and herpesviral DNAs are transported into the nucleus via this mechanism, albeit with different strategies. Herpes simplex virus capsids dock onto the nuclear pore, where they remain largely intact, and the nucleic acid is injected into the nucleus through a portal in the nucleocapsid (Fig. 5.26B). The DNA of some bacteriophages is packaged in virus particles at high pressure, which provides sufficient force to insert the viral DNA genome into the bacterial cell (Box 5.6). A similar mechanism may allow injection of herpesviral DNA. Herpesvirus capsids also dock onto the nuclear pore complex, and interaction with nucleoporins destabilizes a viral protein, pUL25, which locks the genome inside the capsid. This event drives the naked viral DNA, which is packaged in the nucleocapsid under very high pressure, to exit through the portal. Ordinarily, the charged, hydrophilic viral nucleic acid would have difficulty passing through the pore, but this mechanism overcomes the requirement for hydrophobic interactions with nucleoporins.
In contrast to herpesvirus particles, partially disassembled adenovirus capsids dock onto the nuclear pore complex by interaction with NUP214 (Fig. 5.26C and 5.27). Release of the viral genome requires capsid protein binding to kinesin-1, the motor protein that mediates transport on microtubules from the nucleus to the cell periphery. As the capsid is held on the nuclear pore, movement of kinesin-1 toward the plasma membrane is thought to pull the capsid apart (Fig. 5.27). The released protein VII-associated viral DNA is then imported into the nucleus, where viral transcription begins.
The 26-nm capsid of parvoviruses is small enough to fit through the nuclear pore (39 nm), and it has been assumed that these virus particles enter by this route. However, there is no experimental evidence that parvovirus capsids pass intact through the nuclear pore. Instead, virus particles bind to the nuclear pore complex, followed by disruption of the nuclear envelope and the nuclear lamina, leading to entry of virus particles (Fig. 5.26D). After release from the endoplasmic reticulum, the 45-nm capsid of simian virus 40 also docks onto the nuclear pore, initiating disruption of the nuclear envelope and lamina. Such nuclear disruption appears to require cell proteins that also participate in the increased nuclear permeability that takes place during mitosis, raising the possibility that nuclear entry of these viral genomes is a consequence of remodeling a cellular process.
Import of Retroviral Genomes
Fusion of the viral membranes of most retroviruses with the plasma membrane releases the viral core into the cytoplasm. The retroviral core consists of the viral RNA genome, coated with NC protein, and the enzymes reverse transcriptase (RT) and integrase (IN), enclosed in a shell comprising the capsid (CA) protein. The RNA is reverse transcribed into DNA, which has to reach the nucleus in order to integrate and replicate as part of the host genome (see Chapter 7). The capsid core surrounding the viral RNA allows nucleotides necessary for reverse transcription to enter, but not larger molecules. It is thought that this core has to at least partially disassemble for DNA synthesis to continue but does not completely dissociate from the preintegration complex, comprising the viral DNA, IN, and other proteins. The mechanism of nuclear import of the preintegration complex is poorly understood, but it is quite clear that this structure is too large to pass through the nuclear pore complex. The betaretrovirus Moloney murine leukemia virus can efficiently infect only dividing cells when the nuclear membrane breaks down during mitosis. The viral preintegration complex has to then be tethered to chromatin so that it remains associated with cellular DNA when the nuclear membrane re-forms in daughter cells, circum-venting the need for active transport.
DISCUSSION
The bacteriophage DNA injection machine
The mechanisms by which the bacteriophage genome enters the bacterial host are unlike those for viruses of eukaryotic cells. One major difference is that the bacteriophage particle
