Elsevier

Virus Research

Volume 95, Issues 1–2, September 2003, Pages 3-12
Virus Research

Trafficking of viral genomic RNA into and out of the nucleus: influenza, Thogoto and Borna disease viruses

https://doi.org/10.1016/S0168-1702(03)00159-X Get rights and content

Abstract

Most RNA viruses that lack a DNA phase replicate in the cytoplasm. However, several negative-stranded RNA viruses such as influenza, Thogoto, and Borna disease viruses replicate their RNAs in the nucleus, taking advantage of the host cell's nuclear machinery. A challenge faced by these viruses is the trafficking of viral components into and out of the nucleus through the nuclear membrane. The genomic RNAs of these viruses associate with proteins to form large complexes called viral ribonucleoproteins (vRNPs), which exceed the size limit for passive diffusion through the nuclear pore complex (NPC). To insure efficient transport across the nuclear membrane, these viruses use nuclear import and export signals exposed on the vRNPs. These signals recruit the cellular import and export complexes, which are responsible for the translocation of the vRNPs through the NPC. The ability to control the direction of vRNP trafficking throughout the viral life cycle is critical. Various mechanisms, ranging from simple post-translational modification to complex, sequential masking-and-exposure of localization signals, are used to insure the proper movement of the vRNPs.

Introduction

RNA viruses can be separated into three major classes based on the organization of their genomes: (i) single-stranded RNA of negative polarity, (ii) single-stranded RNA of positive polarity, and (iii) double-stranded RNA. Most negative-stranded RNA viruses replicate their genome in the cytoplasm. However, several of them have a nuclear phase, relying on some of the unique features of this compartment to complete their replication cycle. For example, influenza, Thogoto and Borna disease viruses (BDVs), which are the primary focus of this article, depend on the nuclear RNA splicing machinery to produce several of their transcripts. In the positive-stranded RNA virus class, only the retroviruses replicate in the nucleus. Among them, only the lentiviruses such as human immunodeficiency virus (HIV) rely on the active import of their genomes to reach the nucleus. However, the genome of lentiviruses enters the nucleus as a double-stranded DNA molecule after the genomic RNA has been reverse-transcribed. Thus, the genome trafficking of these viruses will not be described here in detail. Finally, no double-stranded RNA virus has been found to replicate its RNAs within the nucleus.

The cell nucleus is separated from the cytoplasm by a double membrane. Viruses that replicate their genome in the host cell nucleus have evolved strategies for moving viral components across this membrane barrier twice: first to reach the nucleus after uncoating, and then back to the cytoplasm after RNA replication has occurred. Trafficking through this membrane occurs only at specific locations, the nuclear pore complexes (NPCs) and it is tightly controlled. The NPC is a large complex of 125 MDa spanning both membranes with extensions into the cytoplasm and into the nucleus. Over 100 different polypeptides (nucleoporins) comprise this aqueous channel (for review, see Allen et al., 2000, Bodoor et al., 1999, Stoffler et al., 1999) and the size limit for passive diffusion through the NPC is believed to be 9 nm (Bonner et al., 1975, Paine et al., 1975).

Viral RNAs are always tightly associated with viral proteins, forming ribonucleoprotein (RNP) complexes. In the case of influenza virus, the vRNP is believed to be 10–20 nm wide (Compans et al., 1972). Recently, the structure of an influenza virus mini-RNP was partially revealed by electron microscopy (Martin-Benito et al., 2001). The vRNA, coated by nucleoproteins (NPs) (1 NP for each 24 nucleotides (Compans et al., 1972, Ortega et al., 2000)), forms a loop. The trimeric polymerase complex (PB1–PB2–PA) binds to the partially complementary ends of the vRNA, giving rise to a complex panhandle structure (Martin-Benito et al., 2001).

Since vRNPs appear to exceed the size limit for passive import into the nucleus, they must be actively translocated. In general, intercompartmental transport of macromolecules within cells requires that such cargo molecules contain transport signals. Cargo molecules possessing import or export signals are recognized by the members of a large and diverse family of proteins called karyopherins. Importins such as karyopherin alpha and beta recruit cargoes for transport into the nucleus, and exportins such as Crm1 (chromosome maintenance region 1) bind cargoes directed to the cytoplasm. A small GTP-binding protein with GTPase activity, Ran, has been proposed to control the directionality of the transport (Görlich et al., 1996, Izaurralde et al., 1997, Nachury and Weis, 1999). The proposed mechanism involves a nucleocytoplasmic gradient of the GTP-bound form of Ran (high RanGTP/RanGDP ratio in the nucleus, low ratio in the cytoplasm). This gradient is maintained by the presence of the Ran nucleotide exchange factor RCC1 in the nucleus (triggering the exchange of GDP for GTP) and the RanGAP (Ran GTPase activating protein), which is preferentially located in the cytoplasm. Ran, in its GTP-associated form, binds to the exportin–cargo complex and mediates its translocation to the cytoplasm. The GTP hydrolysis in the cytoplasm results in the release of the cargo (for review, see Görlich and Kutay, 1999; see also Fig. 1).

Active translocation through the nuclear membrane is a process specific to cargoes containing transport signals: nuclear import signal (NLS) and/or nuclear export signal (NES). Classical NLSs contain either a short stretch of basic lysine residues, like that of the SV40 large T-antigen NLS (PKKKRKV), or two clusters of basic residues separated by a spacer of variable length, as found in nucleoplasmin (KRxxxxxxxxxxKKKK). Proteins carrying these signals recruit molecules of the karyopherin alpha family, which, after binding to karyopherin beta, form an import-competent trimeric complex that moves into the nucleus in a RanGDP-dependent manner. Other types of NLSs, possibly paired with different cellular partners, have been described for several cellular and viral proteins (for review, see Chook and Blobel, 2001). NES-containing cargoes that use the Crm1 export pathway, such as HIV Rev, bear leucine-rich export signals (LxxLxxLxL). However, the Crm1-dependent cytoplasmic translocation is only one of the export pathways, and different types of NESs have been described (see Görlich and Kutay, 1999, Weis, 2002).

This review will focus on the transport of the vRNPs of influenza, Thogoto and BDVs into and out of the nucleus, on the proteins and signals involved in this movement, and the transport process, including its regulation.

Section snippets

Import

Influenza virus, the prototype of the Orthomyxoviruses family, is an enveloped virus whose genome is composed of eight single-stranded RNA segments of negative polarity, encoding 11 proteins. During entry into cells, the virus first binds to sialic acid residues on cellular membrane glycoproteins through its receptor, hemagglutinin (HA), and then it is endocytosed. Acidification of the endocytic vesicle results in a conformational change of HA, exposing a short peptide responsible for the

Import

Another member of the Orthomyxoviridae family is Thogoto virus (TV), the prototype of the Thogotovirus genus. Like influenza virus, its genome is composed of several single-stranded RNA molecules (six) of negative sense. Viral NP, when expressed alone, can translocate to, and accumulate in, the nucleus (Weber et al., 1998). TV-NP seems to lack an influenza A virus NP-like unconventional NLS (Weber et al., 1998). Furthermore, TV-NP does not appear to bind karyopherin alpha (Weber et al., 1998).

Import

BDV is the prototype of the Bornaviridae family of the order Mononegavirales. This enveloped virus contains one genomic single-stranded RNA molecule of negative polarity. An interesting feature of BDV is that, although its genomic organization is very similar to that of other Mononegavirales, it differs from other members of the family in being replicated in the nucleus rather than the cytoplasm of the cells it infects (for review, see Tomonaga et al., 2002). After entry into the cell, the vRNP

Human immunodeficiency virus

The HIV genome is imported into the nucleus, not as a vRNP but as a cDNA molecule, after reverse transcription of the viral RNA in the cell cytoplasm. Since this review focuses on viruses whose genomes enter and leave the nucleus as RNP, the trafficking of the HIV genome falls beyond the scope of this review. Nonetheless, a brief discussion about intracellular trafficking of this virus is warranted.

The imported HIV cDNA molecule is associated with several viral and cellular proteins to form the

Conclusion

The crossing of nuclear membranes by RNA genomes is a key event for RNA viruses with a life cycle that includes a nuclear phase. As we come to understand the nucleocytoplasmic trafficking mechanisms of cellular proteins and the structure of the NPC, we see that the translocation of the vRNP cannot happen by simple diffusion. These active transport processes harness signals on viral proteins as well as cellular adapter molecules. A major effort has been undertaken to identify the viral proteins

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