Tracking expression and subcellular localization of RNA and protein species using high-throughput single cell imaging flow cytometry

We report a high-throughput application of multispectral imaging flow cytometry (MIFC) for analyzing the expression and localization of both RNA and protein molecules in a heterogeneous population of cells. The approach was developed using polyadenylated nuclear (PAN) RNA, an abundant, noncoding RNA expressed by Kaposi's sarcoma-associated herpesvirus (KSHV) during the lytic phase of infection. High levels of PAN RNA are, in part, dependent on its interaction with poly(A)-binding protein C1 (PABPC1), which relocalizes from the cytoplasm to the nucleus of lytically infected cells. We quantitatively tracked the cytoplasmic to nuclear translocation of PABPC1 and examined how this translocation relates to the expression and localization of viral RNA and protein molecules in KSHV-infected cells. This high-throughput approach will be useful for other systems in which changes in subcellular localization of RNA and protein molecules need to be monitored simultaneously.     

Interplay between Polyadenylate-Binding Protein 1 and Kaposi's Sarcoma-Associated Herpesvirus ORF57 in Accumulation of Polyadenylated Nuclear RNA,a Viral Long Noncoding RNA

Polyadenylate-binding protein cytoplasmic 1 (PABPC1) is a cytoplasmic-nuclear shuttling protein important for protein translation initiation and both RNA processing and stability. We report that PABPC1 forms a complex with the Kaposi''s sarcoma-associated herpesvirus (KSHV) ORF57 protein, which allows ORF57 to interact with a 9-nucleotide (nt) core element of KSHV polyadenylated nuclear (PAN) RNA, a viral long noncoding RNA (lncRNA), and increase PAN stability. The N-terminal RNA recognition motifs (RRMs) of PABPC1 are necessary for the direct interaction with ORF57. During KSHV lytic infection, the expression of viral ORF57 leads to a substantial decrease in overall PABPC1 expression, along with a shift in the cellular distribution of the remaining PABPC1 to the nucleus. Interestingly, PABPC1 and ORF57 have opposing functions in modulating PAN steady-state accumulation. The suppressive effect of PABPC1 specific to PAN expression is alleviated by small interfering RNA knockdown of PABPC1 or by overexpression of ORF57. Conversely, ectopic PABPC1 reduces ORF57 steady-state protein levels and induces aberrant polyadenylation of PAN and thereby indirectly inhibits ORF57-mediated PAN accumulation. However, E1B-AP5 (heterogeneous nuclear ribonucleoprotein U-like 1), which interacts with a region outside the 9-nt core to stimulate PAN expression, does not interact or even colocalize with ORF57. Unlike PABPC1, the nuclear distribution of E1B-AP5 remains unchanged by viral lytic infection or overexpression of ORF57. Together, these data indicate that PABPC1 is an important cellular target of viral ORF57 to directly upregulate PAN accumulation during viral lytic infection, and the ability of host PABPC1 to disrupt ORF57 expression is a strategic host counterbalancing mechanism.     

Crystal structure of a KSHV-SOX-DNA complex: insights into the molecular mechanisms underlying DNase activity and host shutoff

The early lytic phase of Kaposi's sarcoma herpesvirus infection is characterized by viral replication and the global degradation (shutoff) of host mRNA. Key to both activities is the virally encoded alkaline exonuclease KSHV SOX. While the DNase activity of KSHV SOX is required for the resolution of viral genomic DNA as a precursor to encapsidation, its exact involvement in host shutoff remains to be determined. We present the first crystal structure of a KSHV SOX-DNA complex that has illuminated the catalytic mechanism underpinning both its endo and exonuclease activities. We further illustrate that KSHV SOX, similar to its Epstein-Barr virus homologue, has an intrinsic RNase activity in vitro that although an element of host shutoff, cannot solely account for the phenomenon.     

A Lytic Viral Long Noncoding RNA Modulates the Function of a Latent Protein

Latent Kaposi''s sarcoma-associated herpesvirus (KSHV) episomes are coated with viral latency-associated nuclear antigen (LANA). In contrast, LANA rapidly disassociates from episomes during reactivation. Lytic KSHV expresses polyadenylated nuclear RNA (PAN RNA), a long noncoding RNA (lncRNA). We report that PAN RNA promotes LANA-episome disassociation through an interaction with LANA which facilitates LANA sequestration away from KSHV episomes during reactivation. These findings suggest that KSHV may have evolved an RNA aptamer to regulate latent protein function.     

The exonuclease and host shutoff functions of the SOX protein of Kaposi's sarcoma-associated herpesvirus are genetically separable

The Kaposi's sarcoma-associated herpesvirus (KSHV) SOX protein, encoded by ORF37, promotes shutoff of host cell gene expression during lytic viral replication by dramatically impairing mRNA accumulation. SOX is the KSHV homolog of the alkaline exonuclease of other herpesviruses, which has been shown to function as a DNase involved in processing and packaging the viral genome. Although the exonuclease activity of these proteins is widely conserved across all herpesviruses, the host shutoff activity observed for KSHV SOX is not. We show here that SOX expression sharply reduces the half-life of target mRNAs. Extensive mutational analysis reveals that the DNase and host shutoff activities of SOX are genetically separable. Lesions affecting the DNase activity cluster in conserved regions of the protein, but residues critical for mRNA degradation are not conserved across the viral family. Additionally, we present evidence suggesting that the two different functions of SOX occur within distinct cellular compartments-DNase activity in the nucleus and host shutoff activity in the cytoplasm.     

Kaposi's sarcoma-associated herpesvirus ORF57 protein enhances mRNA accumulation independently of effects on nuclear RNA export

The ORF57 protein expressed by Kaposi's sarcoma-associated herpesvirus (KSHV) during lytic replication is essential for KSHV virion production. ORF57 enhances gene expression by increasing accumulation of target gene mRNAs. ORF57 interacts with the cellular export factor REF and with RNA, suggesting that it may provide target mRNAs with access to REF, which mediates nuclear RNA export by binding to TAP/NXF1. A mutational analysis of ORF57 was performed to study the role of REF binding, RNA interaction, and multimerization in ORF57 function. ORF57 was shown to directly bind RNA. The ability to bind REF did not correlate with ORF57 function in enhancing mRNA accumulation. ORF57 enhanced the nuclear levels of mRNA and PAN, a nuclear KSHV RNA, and the activity of various ORF57 mutants on the levels of mRNA paralleled their ability to enhance nuclear PAN accumulation, suggesting that ORF57 may also act on messenger RNAs by export-independent effects on RNA stability. Finally, an ORF57 mutant lacking a region homologous to a nucleolar localization signal in herpesvirus saimiri was constructed. This mutant retained function, demonstrating that, unlike the ORF57 homolog in herpesvirus saimiri, nucleolar trafficking is not required for ORF57 function in enhancing mRNA accumulation.