Regulation of Hepatitis C Virion Production via Phosphorylation of the NS5A Protein

Abstract

Hepatitis C virus (HCV) is a significant pathogen, infecting some 170 million people worldwide. Persistent virus infection often leads to cirrhosis and liver cancer. In the infected cell many RNA directed processes must occur to maintain and spread infection. Viral genomic RNA is constantly replicating, serving as template for translation, and being packaged into new virus particles; processes that cannot occur simultaneously. Little is known about the regulation of these events. The viral NS5A phosphoprotein has been proposed as a regulator of events in the HCV life cycle for years, but the details have remained enigmatic. NS5A is a three-domain protein and the requirement of domains I and II for RNA replication is well documented. NS5A domain III is not required for RNA replication, and the function of this region in the HCV lifecycle is unknown. We have identified a small deletion in domain III that disrupts the production of infectious virus particles without altering the efficiency of HCV RNA replication. This deletion disrupts virus production at an early stage of assembly, as no intracellular virus is generated and no viral RNA and nucleocapsid protein are released from cells. Genetic mapping has indicated a single serine residue within the deletion is responsible for the observed phenotype. This serine residue lies within a casein kinase II consensus motif, and mutations that mimic phosphorylation suggest that phosphorylation at this position regulates the production of infectious virus. We have shown by genetic silencing and chemical inhibition experiments that NS5A requires casein kinase II phosphorylation at this position for virion production. A mutation that mimics phosphorylation at this position is insensitive to these manipulations of casein kinase II activity. These data provide the first evidence for a function of the domain III of NS5A and implicate NS5A as an important regulator of the RNA replication and virion assembly of HCV. The ability to uncouple virus production from RNA replication, as described herein, may be useful in understanding HCV assembly and may be therapeutically important.

Hepatitis c virus and host cell lipids: An intimate connection

“…[22][23][24] Core can be divided proximity of LDs. 59,60 It is assumed that newly synthesized viral RNA is transported to LDs by NS5A, 24 whose phosphorylation state possibly regulating the balance between viral RNA replication and particle assembly. 59,60,[64][65][66] NS5B, the RNA-dependent RNA polymerase, is composed of an N-terminal catalytic domain and a C-terminal TM segment anchoring it to intracellular membranes ( Fig.…”

“…54 It is composed of four domains: 55 an N-terminal AH essential for attachment to cellular membranes and contributing to LD targeting; 56,57 an RNA binding domain (D1); an intrinsically unfolded and poorly conserved D2 contributing to RNA replication; 58 and a C-terminally located D3, crucial for virus assembly (D3). 59,60 NS5A is the target of several cellular kinases, including casein kinase 1 (CK1), and CK2. Accordingly, it is detectable in infected cells as a basal and a hyperphosphorylated form with apparent molecular weights of 56 and 58 kDa, respectively.…”

“…61,66,151 Point mutations preventing NS5A hyperphosphorylation enhance RNA replication, but strongly impair virus production, arguing that NS5A phosphorylation status regulates the switch between RNA replication and assembly. 60 How could this regulation be achieved? HCV assembly is a process thought to occur on the surface of core-decorated LDs, 82 where NS5A is believed to 'deliver' newly synthesized viral genomes to trigger nucleocapsid formation.…”

“…However, this hypothesis is challenged by the observation that mutations in NS5A selectively impairing its hyperphosphorylation and virus assembly do not prevent NS5A localization to LDs. 59,60 Alternatively, NS5A hyperphosphorylation might release VAPs that in turn could contribute, together with PI(4) P to recruitment of OSBP. Obviously, further studies will be required to define the exact role of VAPs and NS5A phosphorylation status for HCV replication and assembly.…”

思いもよらなかった薬物標的

“…Our differentiating strategy centred on the identification of compounds functionally distinct from those acting on the traditional targets of antiviral research in this field, the NS3 protease and the NS5B RNA-dependent RNA polymerase [5][6][7] . Using a Con-1 genotype 1b replicon replicating in Huh-7 liver cells 8 , we screened over one million compounds from the Bristol-Myers Squibb proprietary collection in high throughput mode. We used a dual assay format that simultaneously evaluated replication of the related flavivirus bovine viral diarrhoea virus (BVDV) and host cell cytotoxicity as an efficient means of preliminarily eliminating compounds without specificity for HCV 9 .…”