Date Published: August 24, 2018
Publisher: Public Library of Science
Author(s): Dylan Flather, Joseph H. C. Nguyen, Bert L. Semler, Paul D. Gershon, Ann Palmenberg.
Protein production, genomic RNA replication, and virion assembly during infection by picornaviruses like human rhinovirus and poliovirus take place in the cytoplasm of infected human cells, making them the quintessential cytoplasmic pathogens. However, a growing body of evidence suggests that picornavirus replication is promoted by a number of host proteins localized normally within the host cell nucleus. To systematically identify such nuclear proteins, we focused on those that appear to re-equilibrate from the nucleus to the cytoplasm during infection of HeLa cells with human rhinovirus via quantitative protein mass spectrometry. Our analysis revealed a highly selective re-equilibration of proteins with known mRNA splicing and transport-related functions over nuclear proteins of all other functional classes. The multifunctional splicing factor proline and glutamine rich (SFPQ) was identified as one such protein. We found that SFPQ is targeted for proteolysis within the nucleus by viral proteinase 3CD/3C, and a fragment of SFPQ was shown to migrate to the cytoplasm at mid-to-late times of infection. Cells knocked down for SFPQ expression showed significantly reduced rhinovirus titers, viral protein production, and viral RNA accumulation, consistent with SFPQ being a pro-viral factor. The SFPQ fragment that moved into the cytoplasm was able to bind rhinovirus RNA either directly or indirectly. We propose that the truncated form of SFPQ promotes viral RNA stability or replication, or virion morphogenesis. More broadly, our findings reveal dramatic changes in protein compartmentalization during human rhinovirus infection, allowing the virus to systematically hijack the functions of proteins not normally found at its cytoplasmic site of replication.
Viruses of the Picornaviridae are characterized by a positive polarity, single-stranded RNA genome of 7–10 kb within a non-enveloped icosahedral capsid. The genome contains a single open reading frame flanked by a long (>500 nucleotide) 5’-noncoding region (NCR), a shorter 3’-NCR, and a 3’-terminal poly(A) tract. Although not unique to picornaviruses, another feature of these viruses is the use of a viral protein, VPg, to prime viral RNA synthesis. As a result, VPg is covalently linked to the 5’-terminus of the viral RNA and is the only viral protein known to be encapsidated. Being of mRNA polarity, the viral genome is translated immediately following virus attachment, uncoating, and release into the cell cytoplasm. The 5’-NCR contains extensive RNA secondary structure elements, including an internal ribosome entry site (IRES) which drives the cap-independent translation of the viral genome. The multiple stem-loop structures of the IRES interact with cellular proteins to recruit ribosomes, initiating the synthesis of a polyprotein which is processed by viral proteinases 2A and 3CD/3C to produce both incompletely processed (yet functional) polyprotein intermediates and mature viral proteins [1–3]. Viral RNA replication, like translation, occurs in the cytoplasm of infected cells and employs the newly synthesized RNA-dependent RNA polymerase, 3D, in concert with non-structural viral proteins and host proteins. Genome replication is promoted by elements of secondary structure present at the termini of both positive- and negative-stranded viral RNA. These structures serve as the interaction sites for cellular RNA-binding proteins that are thought to promote intermolecular architectures conducive to this process [4–6]. RNA synthesis initially yields RNA intermediates of negative polarity, which then serve as templates for the production of genomic RNA. Nascent RNA genomes can then serve as templates for further rounds of translation and RNA replication and, upon production of sufficient viral protein, are encapsidated to yield mature, infectious virions. The resulting viral progeny then exit the cell via lysis and/or non-lytic release within extracellular vesicles .
In this study we have applied an unbiased quantitative protein mass spectrometry approach to investigate global changes in protein distribution during HRV16 infection. We detected a coordinated redistribution of proteins, many of which normally function in RNA-related processes such as mRNA splicing, from the nucleus to the cytoplasm by 8 hours post-HRV16 infection of HeLa cells. This suggested that infection with an RNA virus, HRV16, results in the enrichment of a specific functional class of proteins at the site of viral replication. Although a number of the proteins listed in Table 2 may be considered predominantly cytoplasmic, several (e.g., the filamins) also have an important nuclear role that has been recognized more recently [64, 65]. Whether others in our list have nuclear functions that remain to be identified, or whether IRES structures within their mRNAs stimulate their translation during HRV16 infection combined with a virus-induced blockage of nuclear import, remains to be seen.