Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 280

Host cell components required for viral RNA synthesis were initially called “host factors,” because nothing was known about their chemical composition. Evidence that cellular proteins are essential components of a viral RdRP first came from studies of the bacteriophage Qβ enzyme. This viral RdRP is a multisubunit enzyme, consisting of a 65-kDa virus-encoded protein and four host proteins: ribosomal protein S1, translation elongation proteins (EF-Tu and EF-Ts), and an RNA-binding protein. Proteins S1 and EF-Tu contain RNA-binding sites that enable the RNA polymerase to recognize the viral RNA template. The 65-kDa viral protein has sequence and structural similarity to known RdRPs, but exhibits no RNA polymerase activity in the absence of the host proteins.

Figure 6.15 Structure of a viral RNA helicase. The RNA helicase of the flavivirus yellow fever virus is shown in surface representation, colored red, white, or blue depending on the distance of the amino acid from the center of the molecule. A model for melting of double-stranded RNA is shown (PDB file 1YKS).

Polioviral RNA synthesis also requires host cell proteins. When purified polioviral RNA is incubated with a cytoplasmic extract prepared from uninfected permissive cells, the genomic RNA is translated and the viral RNA polymerase is made. If the RNA synthesis inhibitor guanidine hydrochloride is included in the reaction, the polymerase assembles on the viral genome, but initiation is blocked. The RdRP-template assembly can be isolated free of guanidine, but RNA synthesis does not occur unless a new cytoplasmic extract is added, indicating that soluble cellular proteins are required for initiation. A similar conclusion comes from studies in which polioviral RNA was injected into oocytes derived from the African clawed toad Xenopus laevis: the viral RNA cannot replicate in Xenopus oocytes unless it is coinjected with a cytoplasmic extract from human cells. These observations can be explained by the requirement of the viral RNA polymerase for one or more mammalian proteins that are absent in toad oocytes.

One of these host cell proteins required for poliovirus RNA synthesis is poly(rC)-binding protein, which binds to a cloverleaf structure that forms in the first 108 nucleotides of the viral (+) strand RNA (Fig. 6.10). Formation of a ribonucleoprotein composed of the 5′ cloverleaf, 3CD, and poly(rC)-binding protein is essential for initiation of viral RNA synthesis. Interaction of poly(rC)-binding protein with the cloverleaf facilitates the binding of polioviral protein 3CD to the opposite side of the same cloverleaf.

Another host protein that is essential for polioviral RNA synthesis is poly(A)-binding protein 1. This protein brings together the ends of the viral genome by interacting with poly(rC)-binding protein 2, 3CD^pro, and the 3′ poly(A) tail of poliovirus RNA (Fig. 6.10). Formation of this circular ribonucleoprotein is required for (−) strand RNA synthesis.

Interactions among cellular and viral proteins can now be identified readily by mass spectrometry, and their function in viral genome replication can be determined by silencing their production by RNA interference or disrupting the gene using CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9. These approaches have been used to identify diverse cell proteins that participate in viral RNA-directed RNA synthesis in cells infected with a variety of (+), (–), and double-stranded RNA viruses.