Double-stranded RNA binding by human cytomegalovirus pTRS1

Area of Science:

• Virology
• Molecular Biology
• Immunology

Background:

• Human cytomegalovirus (HCMV) TRS1 and IRS1 proteins can rescue vaccinia virus (VV) replication when the E3L gene is deleted.
• These HCMV proteins inhibit interferon-induced antiviral pathways activated by double-stranded RNA (dsRNA).

Purpose of the Study:

• To investigate if HCMV TRS1 protein binds to dsRNA.
• To identify the dsRNA-binding domain of TRS1.
• To determine the role of dsRNA binding and other TRS1 domains in rescuing VV replication and counteracting host antiviral responses.

Main Methods:

• In vitro binding assays using cell-free translated or infected cell-expressed pTRS1.
• Competition assays with dsRNA, double-stranded DNA, and single-stranded RNA.
• Deletion analyses of TRS1 to map dsRNA-binding and functional domains.
• Replication rescue assays using VVDeltaE3L and TRS1 mutants in HeLa cells.
• Analysis of protein synthesis shutoff and eIF2alpha phosphorylation in stable cell lines.

Main Results:

• Recombinant pTRS1 binds to dsRNA, with preferential binding over dsDNA or ssRNA.
• The dsRNA-binding domain was mapped to amino acids 74-248 of TRS1.
• Deletion of this domain abrogated dsRNA binding.
• Full-length TRS1 rescued VVDeltaE3L replication, but mutants affecting the C-terminal region failed to rescue.
• The C-terminal domain is essential for preventing protein synthesis shutoff and eIF2alpha phosphorylation post-infection.

Conclusions:

• pTRS1 possesses an unconventional dsRNA-binding domain at its N-terminus.
• A distinct C-terminal domain of pTRS1 is also required for inhibiting host antiviral responses, including protein synthesis shutoff.
• Both dsRNA binding and the C-terminal function are necessary for TRS1 to effectively counter host antiviral defenses and rescue viral replication.