Elsevier

Virus Research

Volume 95, Issues 1–2, September 2003, Pages 75-85
Virus Research

The interaction of cytoplasmic RNA viruses with the nucleus

https://doi.org/10.1016/S0168-1702(03)00164-3Get rights and content

Abstract

Mammalian cells infected with poliovirus, the prototype member of the picornaviridae family, undergo rapid macromolecular and metabolic changes resulting in efficient replication and release of virus from infected cells. Although this virus is predominantly cytoplasmic, it does shut-off transcription of all three cellular transcription systems. Both biochemical and genetic studies have shown that a virally encoded protease, 3Cpro, is responsible for host cell transcription shut-off. The 3C protease cleaves a number of RNA polymerase II transcription factors including the TATA-binding protein (TBP), the cyclic AMP-responsive element binding protein (CREB), the Octamer binding protein (Oct-1), p53, and RNA polymerase III transcription factor IIICα, and Polymerase I factor SL-1. Most of these cleavages occur at glutamine-glycine bonds. Additionally, a second viral protease, 2Apro, also cleaves TBP at a tyrosine-glycine bond. The latter cleavage could be responsible for shut-off of small nuclear RNA transcription. Recent studies indicate that the viral protease-polymerase precursor 3CD can enter nucleus in poliovirus-infected cells. The nuclear localization signal (NLS) present within the 3D sequence appears to play a role in the nuclear entry of 3CD. Thus, 3C may be delivered to the infected cell nucleus in the form the precursor 3CD or other 3C-containing precursors. Auto-proteolytic cleavage of these precursors could then generate 3C. Thus, for a small RNA virus that strictly replicates in the cytoplasm, a portion of its life cycle does include interaction with the host cell nucleus.

Introduction

Viruses are dependent upon the host cell they infect to provide a sustainable environment in which they can replicate. Different types of viruses have developed contrasting strategies in order to accomplish this simple goal. It has been theorized that the RNA viruses with their cytoplasmic site of replication and smaller genome size must resort to host evasion strategies that are more detrimental to the host organism. The majority of large DNA viruses replicate in the nucleus of their host and acquire host genes or become integrated into the host cell genome in order to regulate key cellular processes. In this way, these viruses can evade immune response and maintain a long-term persistent infection within the host organism without a large disease impact upon the host (Chaston and Lidbury, 2001). While the RNA viruses are genetically simpler and replication for many of them is confined to the cytoplasm, this does not mean that they have not evolved very interesting and diverse strategies for interacting with the nucleus and directing key cellular processes utilizing only a handful of proteins.

It should be noted that for certain groups of RNA viruses, most notably the family Retroviridae and the family Orthomyxoviridae, at least part of the replication cycle does take place within the nucleus of the host cell. As this differs significantly from the life cycle of the other cytoplasmically replicating RNA viruses, these families of viruses will not be dealt with in this review.

The majority of work involving RNA virus–nuclear interactions has been done with the family Picornaviridae. The picornaviruses include more than 200 clinically significant animal and human pathogens including the genus Enterovirus (species poliovirus (PV), echovirus, and coxsackievirus), Rhinovirus (the main cause of the common cold), Cardiovirus (species encephalomyocarditis and Theiler's murine encephalomyelitis viruses), Aphthovirus (species foot-and-mouth disease virus (FMDV)), and Hepatovirus (species hepatitis A virus) (reviewed in Rueckert, 1996). PV is the prototype virus of the Picornaviridae family. This lytic virus contains a small, positive-strand RNA genome which is replicated by a virus-encoded RNA-dependent RNA polymerase with no proofreading capability. Consequently, this virus is replicated in the cytoplasm of the host cell with a high rate of mutation, which provides the virus with an evolutionary advantage. Once inside the cytoplasm, the viral RNA is translated by host cell translation machinery. During translation each precursor protein (P1–P3) is released from the growing polyprotein by the virally encoded proteases (Fig. 1). The small size of this virus results in the condensation of genetic information. To increase the number of functional proteins that can be obtained from the concise genetic information, these viruses have adopted a unique processing mechanism. The viral RNA encodes a single open reading frame, which encodes a single 247 kDa polyprotein. This large polyprotein is subsequently processed into the individual viral peptides by the virally encoded proteases 2Apro and 3Cpro. Synthesis of the new viral RNA begins by copying the genomic RNA to form a complementary minus-strand RNA. This in turn serves as a template for synthesis of new plus strands. During the initial phase of replication these plus strands are used as additional templates, but as the viral life cycle progresses, more and more of the plus-strand RNAs are packaged into new virions (reviewed in Rueckert, 1996). A growing body of biochemical and genetic evidence indicates that most nonstructural proteins are involved in viral replication as well as other functions during infection. This demonstrates how the virus compensates for its small size by the efficient utilizations of the proteins it contains.

Section snippets

Inhibition of host cell transcription

Normal cells infected with PV undergo rapid and dramatic macromolecular and metabolic changes. Although this virus is predominately cytoplasmic, with all parts of the life cycle including viral replication and assembly occurring in the cytoplasm, it does inhibit transcription of all three host cell transcription systems, which are distinctly nuclear functions (Fig. 1). The original conclusion that viral proteins are involved in the shut off of host cell transcription came from the observation

Inhibition of polymerase I transcription by PV

The advent of in vitro transcription assays provided a powerful tool with which to study the mechanisms of transcriptional inhibition. The RNA polymerase I (Pol I) system is responsible for the synthesis of ribosomal RNA (rRNA). Transcription from this system comprises 70–90% of the total transcription carried out by the cell and directly influences the growth rate of the cell (Sollner-Webb and Tower, 1986, Sommerville, 1986). This is the first transcription system to be shut down during PV

Inhibition of polymerase III transcription by PV

The RNA polymerase III (Pol III) transcription system is responsible for the transcription of all the tRNAs and the 5S rRNAs. In addition to the transcription of these RNAs, Pol III is utilized by at least two double-stranded DNA viruses (adenovirus and Epstein-Barr virus) to transcribe genes encoded in their genomes. In addition to the RNA polymerase, two transcription factors, TFIIIB and TFIIIC, are also required for the transcription of tRNA genes, while the transcription of 5S rRNA requires

Inhibition of Pol II transcription by PV

Pol II transcription is also inhibited during PV infection. This is of broad interest because this is the system responsible for the transcription of mRNA and is the most complicated of the three transcription systems. In order for basal level transcription to occur, the polymerase must be recruited to the promoter by general transcription factors. In the Pol II transcription system, the factor TFIID is responsible for initial binding to the Pol II promoter TATA-box through one of its component

Inhibition of Pol II-activated transcription by PV

In addition to basal transcription from core promoters, activator-dependent Pol II transcription from complex promoters involving the core promoter in addition to an upstream cis binding site is possible. In activated Pol II transcription, transcriptional activator proteins such as SP-1, CREB and Oct-1 bind to the activator site while TFIID binds the TATA box. The binding of these activators to these upstream sites stimulates the activity of basal level Pol II transcription by promoting

Inhibition of host cell transcription by other RNA viruses

The picornavirus, FMDV, also inhibits host cell transcription. As described in Section 8, a 3ABC precursor was seen to localize to the nuclear periphery in transiently transfected cells. In addition, cleavage of the nuclear protein, histone H3, was observed. In HeLa cells transiently transfected with the homologous 3C from this virus, transcriptional inhibition was observed. In this case, the inhibition is believed to be caused by the cleavage of histone H3 (Falk et al., 1990, Tesar and

Nuclear-cytoplasmic transport

As outlined above, viral proteins such as PV 3Cpro do interact with nuclear proteins in order to shut down cellular processes. The question that must be addressed in looking at the interaction of RNA viruses with the nucleus is how the proteins of a virus, whose entire life cycle occurs in the cytoplasm, get to the nucleus? Or conversely how and why proteins normally found in the nucleus of a cell interact with a cytoplasmic RNA virus?

As outlined above, PV is a lytic RNA virus, which replicates

Nuclear localization of viral proteins

The complementary side of nucleocytoplasmic transport is the idea of viral proteins localizing to the nucleus. As is shown below, this generally requires NLS, but that itself is not always sufficient. One of the proteins of hepatitis C virus (HCV), NS5A, is known to have an NLS located in the COOH-terminal half of the protein. When the NLS of NS5A is fused to β-gal as a reporter construct, β-gal is found in the nucleus of transfected CV-1 cells. However, in cells transfected with a construct

Summary

The studies summarized here indicate that some lytic RNA viruses block nuclear import possibly by altering NPC. During infection nuclear proteins are relocalized to the cytoplasm. This suggests a possible mechanism by which RNA viruses may evade host cell defenses such as apoptosis, by blocking signal transduction to the nucleus.

In contrast to this, it appears that the localization of viral proteins is not blocked during infection. In the case of PV at least, infection is necessary for the

Acknowledgements

The authors gratefully acknowledge the NIH grant support (AI 27451 and AI 45733) for studies reported here. The authors wish to thank the past and present members of the Dasgupta Laboratory for their contributions and help.

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