Phosphoprotein P of rinderpest virus : Its role in the virus life cycle

Abstract

Phosphoprotein P of Rinderpest Virus: A Key Player in the Virus Life Cycle Introduction

Negative-strand RNA viruses (NNSVs) are enveloped viruses infecting hosts across the animal and plant kingdoms. They exhibit diverse morphologies, host interactions, and genome structures. Despite this diversity, all NNSVs share a common replication and transcription strategy. Their genetic information exists exclusively in the form of a tight, helical ribonucleoprotein complex (RNP). Only the RNP, not naked RNA, serves as a template for viral propagation. The RNP proteins are essential for gene expression and function either as part of the template or as components of the polymerase. In essence, the genome of a negative-strand RNA virus is an RNP rather than a naked RNA molecule.

The phosphoprotein (P) gene of all paramyxoviruses exhibits high sequence homology, with several conserved motifs. As observed in some NNSVs, such as Vesicular Stomatitis Virus (VSV) and Sendai Virus (SeV), the P protein, although catalytically inactive, plays a major role in viral replication and transcription.

Rinderpest virus (RPV), a member of the Morbillivirus genus in the Paramyxoviridae family, is a significant pathogen of wild and domestic bovids. Its genome is a single-stranded, negative-sense RNA encapsidated by the viral nucleocapsid protein (N). The viral phosphoprotein (P) and the large polymerase subunit (L) together constitute the RNA-dependent RNA polymerase. Both proteins associate with the nucleocapsid to form the RNP core, which functions as the transcription complex of the virus.

Aims of the Present Investigation

The replication and transcription mechanisms of NNSVs are similar, occurring from the same genomic RNA template. However, the switch between mRNA transcription and genome replication remains poorly understood. Some hypotheses suggest that the N protein plays a role in this switch by encapsidating nascent leader RNA and shifting the process from transcription to replication.

The phosphoprotein P, also called the polymerase subunit, is an essential component of the transcriptional complex. Recent studies indicate that phosphorylation regulates P protein function in several NNSVs, but its exact role in RPV remains unclear.

To address this, we characterized RPV P protein in terms of phosphorylation and assessed its functional consequences.

Identification of Cellular Kinase and Phosphorylation Sites of RPV P Protein

RPV P protein was expressed in E. coli and found to be unphosphorylated. Its phosphorylation status was examined under in vitro and in vivo conditions. Using a transient expression system in mammalian cells with a T7 promoter and recombinant vaccinia virus expressing T7 polymerase, P protein was phosphorylated when cells were labeled with 32

32 P-inorganic phosphate and immunoprecipitated with anti-P antibody.

Similar results were obtained when bacterially expressed P protein was incubated with cellular extract in the presence of

32

32 P-ATP. Using two kinase inhibitors, Casein Kinase II (CKII) was identified as the cellular kinase responsible for P protein phosphorylation. Phosphoamino acid analysis confirmed serine as the phosphate acceptor.

Using deletion mutants, the phosphorylation domain was mapped to the N-terminal region. Site-directed mutagenesis identified serine residues at positions 49, 88, and 151 as phosphorylation sites. Transient in vivo expression of wild-type and mutant P proteins in mammalian cells showed phosphorylation patterns similar to those observed in vitro.

Role of Phosphorylation on P Protein Oligomerization and Conformational Changes

The oligomerization status of recombinant P protein before and after phosphorylation was examined in vitro. Bacterially expressed, unphosphorylated P protein exists as an oligomer. Gel filtration analysis revealed that a large fraction of P protein forms tetramers, while the monomeric fraction undergoes oligomerization after phosphorylation. Tetramer formation was further confirmed by tag-dilution assays.

The oligomerization domain maps to the C-terminal coiled-coil region, conserved across paramyxoviruses. Fluorescence spectroscopy was used to monitor conformational changes induced by phosphorylation. Acrylamide quenching indicated that phosphorylation induces conformational changes in the N-terminal region. ANS (hydrophobic probe) analysis showed decreased hydrophobicity in the C-terminal coiled-coil region upon oligomerization. Monomeric and oligomeric P protein fractions displayed distinct conformational states measured via tryptophan fluorescence and acrylamide quenching, correlating phosphorylation with oligomerization.

Effect of Phosphorylation on Virus Transcription and Replication

The role of P protein phosphorylation in transcription was evaluated using the RPV minigenome system with a reporter gene. Phosphorylation was essential for in vivo transcription. Site-directed mutants revealed:

Replacement of wild-type P with phosphorylation-null mutants abolished transcription.

Serine 151 (S151) mutation had no significant effect, as S151A mutants retained transcription similar to wild-type.

Double mutants (e.g., S49/S88A) showed reduced transcription, potentially due to residual low-level phosphorylation of serine 49.

Analysis of reporter gene expression with co-transfection of wild-type and triple mutants showed higher CAT expression than wild-type alone. Interestingly, phosphorylation-null P protein exhibited higher replication activity than wild-type. In vitro studies indicated that P protein binds leader RNA, with phosphorylation modulating its binding affinity.

These observations suggest a critical role for phosphoprotein P in regulating the switch between viral replication and transcription in host cells.