E. coli Q-beta Replicase Bacteriophage Replication

Lizzy Apunda '22 and Isabel Jaffer '22


Contents:


I.Introduction




Bacteriophage QB is a member of the leviviridae family (Brown, Fiedler, & Finn, 2009). It is a small virus that is about 25nm thick and is a coliphage with an RNA that is 4217 nucleotides long. QB has 20 faces each composed of six subunits and 12 vertices each composed of 5 subunits. Members of the leviviridae family form icosahedral capsids from 180 coat protein subunits around a 4.2 kb sense-strand RNA genome (Singleton et at., 2018). Each of these coat proteins (capsomers) has about 132 residues of amino acids.

Bacteriophage QB is a positive strand RNA virus. Positive strand RNA viruses have genomes that are functional mRNAs (Payne, 2017). For instance, QB’s genome codes for 4 proteins: A1, A2, CP and qb replicase. QB has other proteins like the B-subunit of a replicase, the maturation protein A2 and a minor protein A1 (Singleton et al., 2018). The penetration of the virus into a host cell is quickly followed by translation to produce RdRps and other viral proteins that are required for the production of more viral RNAs. QB ssRNA adsorb to bacterial sex pili proteins and infect.

Like other RNA viruses, QB replicates its genome by utilizing virally encoded RNA polymerase (RdRp) (Payne, 2017). The genome is used as the template for the synthesis of other RNA strands. Upon infection, the B-subunit interacts with host proteins to form a complex. The complex contains RNA-helicases to unwind DNA and NTPases that are useful for polymerization. Once the complex forms, the transcription of the genome, a copy of the genome, and mRNAs begin (Payne, 2017). Phage MS2 has the same genome as QB.

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Figure 1: Q-beta phage particles under a microscope


II. Mechanism/Infection




Upon infection, the bacteriophage binds in large numbers to E.coli cells that have the F-pili (Grumet et al., 1987). The tubular sex pili are composed of oligomeric protein known as Pilin, which allow the empty genome to escape leaving behind an empty capsid (Grumet et al., 1987). At this point, the B-subunit recruits host translation factors EF-Tu and EF-Ts and ribosomal protein S1 to form a QB replicase holoenzyme that drives transcription. The viral genome acts as an mRNA that hijacks the host’s translation machinery to produce coat and replicase proteins. Even though the viral genome is linear, it contains hair loops even at the 5’ and 3’ends. Hence, the helicases in the complex unwind DNA, which makes it easier to transcribe.

One of the first replicase proteins to be transcribed include the RNA dependent Polymerases (RdRPs). The RdRPs contain the catalytic machinery necessary for polymerization, initiation and termination (Gytz et al., 2015). Initiation and termination require the recruitment of host proteins. RdRps of a number of positive sense RNA viruses oligomerize and have a stimulatory effect on RNA synthesis and viral viability (Gytz et al., 2015).

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Figure 2A: An image illustrating QB phage attachment to host entry receptor

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Figure 2B:Composition of QB replicase and replication cycle of QB RNA. ( a ) Composition of QB replicase. QB replicase consists of the virus-encoded RNA-dependent RNA polymerase (B -subunit), and three host translation factors: elongation factor (EF)-Tu, EF-Ts, and ribosomal protein S1; ( B ) Replication cycle of QB RNA. QB virus has a single positive strand RNA. The positive and negative strand QB RNAs both have 5'-GG and CCA-3' sequences. A-3' does not serve as a template nucleoside, and is added at the terminal stage of RNA synthesis without a nucleic acid template (Tomita, 2014).


III. General Structure




When E. coli is infected by Bacteriophage QB, a core complex consisting of the and host elongation factors (EF-Tu and EF-T) is formed. The EF-Tu and EF-T elongation factors, as well as ribosomal proteins, work in conjunction with the B subunit. The B subunit is the catalytic domain for RNA-dependent RNA polymerization. It consists of three domains: the palm (disordered), the
, and the fingers.

The thumb domain contains three segments. is alpha-helical and precedes the fingers domain, while comes after the palm domain. is known as the C-terminal segment. It comprises the three-stranded B-sheet at the tip of the thumb domain.


The fingers domain is comprised of four-stranded, antiparallel B-sheets. It contains three segments: , which precedes the palm domain, , which is inserted in the palm domain between motifs A and B, and , which is a single alpha-helix and is also known as the T-helix.

Other RdRPs have an F-motif in the N-terminal of their sequences where the thumb and fingers domain are connected (Kidmose et al., 2010). This motif may be responsible for template unwinding. In the Beta subunit, the function of the F-motif is fulfilled by highly in the fingers domain with assistance from the that connect the thumb and the fingers
.
The fingers domain and thumb domain are connected by the ‘bridge’ region which flank the in the fingers domain (Kidmose et al., 2010). The bridge region consists of two flexible segments . The bridge is responsible for preventing the unwound template and product strands from reannealing by limiting the cleft above the catalytic center. It is also located in the periphery of the predicted path of the duplex, acting as a strand separator.(Residues 520-532 are part of the bridge and are disordered) .

The B-subunit has the ability to interact with substrates and products. First, the template likely enters through the channel of the fingers domain . This template is bound by which are contained in a large loop. The template is also bound by one side of the four-stranded antiparallel Beta sheet, which is located in the fingers domain. The presumed substrate entrance channel for NTP is located between and from the palm domain along with and in the fingers domain .
The substrate channel through which NTP enters is located in motif D and A from the palm domain along with segments in the fingers domain . Five conserved lysine and arginine side chains, which are located on either side of the substrate channel, coordinate the incoming NTP .

At the catalytic site, Asp274 (Motif A) and Asp359-360 (Motif C) have the ability to synchronize two Mg2+ ions that can mediate catalysis . Lys214 and Arg220, which are tightly conserved, also have the ability to form electrostatic interactions with the NTP phosphates


IV. QB-RNA replication Enzyme




The replication enzyme is known as the QB replicase holoenzyme. This enzyme consists of four proteins: two B-subunits from the phage, ribosomal protein S1, EF-Tu and Ef-T’s. Intermolecular contacts in the monomeric and dimeric Qbeta replicase core complexes involve the interface between the two Beta subunits within the putative dimer of the core enzyme. The contribute to the dimer interface. The elongation factors and the protein are encoded by the host. The B-subunits interact via a symmetric network of salt bridges (Gytz et al., 2015). Residues Arg132 and Arg133 of each B-subunit
form with the opposing B-subunit’s Asp348. This dimer is also stabilized by .


The most significant interactions occur between EF-Tu domain 2 and the fingers domain, which includes the insertion of the T-helix into the binding pocket for CCA-aminoacyl group of aa-tRNA bound to EF-Tu: GTP ( Kidmose et al., 2010). Tyrosine forms hydrophobic interactions and a hydrogen bond with glutamic acid , while arginine residues of the Beta subunit form a salt bridge with EF-Tu glutamine residues.

The B-subunit and EF-Tu form an interface that is stabilized by a molecule of of the precipitant pentaerythritol propoxylate (PEP) (Kidmose et al., 2010). This results in increased hydrophobic interactions of the neighboring EF-Tu Phe 261 with a hydrophobic cluster of six phenylalanine and one leucine side chains from the fingers domain .



VI. References

Brown, S., Fiedler, J., & Finn, M. (2009). Assembly of Hybrid Bacteriophage Q? Virus-like Particles. Biochemistry, 48(47), 11155-11157. doi: 10.1021/bi901306p

Grumet, R., Sanford, J. C., & Johnston, S. A. (1987). Pathogen-derived resistance to viral infection using a negative regulatory molecule. Virology, 161(2), 561-569. 


Gytz, H., Mohr, D., Seweryn, P., Yoshimura, Y., Kutlubaeva, Z., Dolman, F., ... & Knudsen, C. R. (2015). Structural basis for RNA-genome recognition during bacteriophage QB replication. Nucleic acids research, 43(22), 10893-10906.

Kidmose, Rune T., Vasiliev, Nikita N., Chetverin, Alexander B., Andersen, Gregers Rom, and Knudsen, Charlotte R. (2010). Structure of the QB Replicase, an RNA-dependent RNA polymerase consisting of viral and host proteins. PNAS, 107(24), 10884-10889.

Payne, S. Viruses. 2017

Singleton, R. L., Sanders, C. A., Jones, K., Thorington, B., Egbo, T., Coats, M. T., & Waffo, A. B. (2018). Function of the RNA Coliphage QB Proteins in Medical In Vitro Evolution. Methods and protocols, 1(2), 18.

Tomita, K. (2014). Structures and Functions of Q? Replicase: Translation Factors beyond Protein Synthesis. International Journal Of Molecular Sciences, 15(9), 15552-15570. doi: 10.3390/ijms150915552

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