Conformations of the Single-Stranded DNA of Bacteriophage M13

At least two conformations of M13 single-stranded DNA have been demonstrated by measuring differences in sedimentation coefficient and by direct visualization in the electron microscope. Which form is obtained from infected cells and/or intact phage depends on the pH, ionic strength, and temperature. The slower-sedimenting form can be converted to the faster-sedimenting, single-stranded form by low ionic strength, alkali treatment, formamide, or formaldehyde, but not by exposure to 100 degrees C in 1.0 M NaCl. The ability to assume either conformation appears to be a function of the nucleic acid alone. Whether or not these different conformations are of biological significance is still unknown.     

The Role of the Coat Protein A-Domain in P22 Bacteriophage Maturation

Bacteriophage P22 has long been considered a hallmark model for virus assembly and maturation. Repurposing of P22 and other similar virus structures for nanotechnology and nanomedicine has reinvigorated the need to further understand the protein-protein interactions that allow for the assembly, as well as the conformational shifts required for maturation. In this work, gp5, the major coat structural protein of P22, has been manipulated in order to examine the mutational effects on procapsid stability and maturation. Insertions to the P22 coat protein A-domain, while widely permissive of procapsid assembly, destabilize the interactions necessary for virus maturation and potentially allow for the tunable adjustment of procapsid stability. Future manipulation of this region of the coat protein subunit can potentially be used to alter the stability of the capsid for controllable disassembly.     

Replication of Bacteriophage M13: Specificity of the Escherichia coli dnaB Function for Replication of Double-Stranded M13 DNA

Infection of the temperature-sensitive E. coli mutant HfrH 165/70 (dnaB) with the filamentous single-stranded DNA phage M13 is abortive at the restrictive temperature. Upon infection at 41 degrees , single-stranded phage DNA penetrates the cell and is converted in a rifampicin-sensitive step to the double-stranded replicative form (RF). The parental RF attaches to the cell membrane, but subsequent replication of the RF is blocked. It is concluded that in M13 infection semiconservative RF replication of a double strand to a double strand, in contrast to single-stranded DNA synthesis, depends specifically on the dnaB function.     

A Possible Role for RNA Polymerase in the Initiation of M13 DNA Synthesis

The conversion of single-stranded DNA of bacteriophage M13 to the double-stranded replicative form in Escherichia coli is blocked by rifampicin, an antibiotic that specifically inhibits the host-cell RNA polymerase. Chloramphenicol, an inhibitor of protein synthesis, does not block this conversion. The next stage in phage DNA replication, multiplication of the doublestranded forms, is also inhibited by rifampicin; chloramphenicol, although inhibitory, has a much smaller effect. An E. coli mutant whose RNA polymerase is resistant to rifampicin action does not show inhibition of M13 DNA replication by rifampicin. These findings indicate that a specific rifampicin-RNA polymerase interaction is responsible for blocking new DNA synthesis. It now seems plausible that RNA polymerase has some direct role in the initiation of DNA replication, perhaps by forming a primer RNA that serves for covalent attachment of the deoxyribonucleotide that starts the new DNA chain.     

Nucleotide Sequence at the Binding Site for Coat Protein on RNA of Bacteriophage R17

The binding of a few molecules [1-6] of RNA bacteriophage coat protein to 1 molecule of RNA represses in vitro translation of the RNA synthetase cistron. Digestion of the complex, R17 coat protein-R17 RNA, by T1 RNase yields an RNA fragment bound to the coat protein. The nucleotide sequence of this fragment (59 residues) reveals that it contains the punctuation signal between the coat protein and RNA synthetase cistrons, suggesting that this is the site on the RNA where the coat protein acts as a translational repressor.     

Membrane Insertion of the M13 Minor Coat Protein G3p Is Dependent on YidC and the SecAYEG Translocase

The minor coat protein G3p of bacteriophage M13 is the key component for the host interaction of this virus and binds to Escherichia coli at the tip of the F pili. As we show here, during the biosynthesis of G3p as a preprotein, the signal sequence interacts primarily with SecY, whereas the hydrophobic anchor sequence at the C-terminus interacts with YidC. Using arrested nascent chains and thiol crosslinking, we show here that the ribosome-exposed signal sequence is first contacted by SecY but not by YidC, suggesting that only SecYEG is involved at this early stage. The protein has a large periplasmic domain, a hydrophobic anchor sequence of 21 residues and a short C-terminal tail that remains in the cytoplasm. During the later synthesis of the entire G3p, the residues 387, 389 and 392 in anchor domain contact YidC in its hydrophobic slide to hold translocation of the C-terminal tail. Finally, the protein is processed by leader peptidase and assembled into new progeny phage particles that are extruded out of the cell.     

RNA Synthesis Initiates In Vitro Conversion of M13 DNA to Its Replicative Form

Soluble enzyme fractions from uninfected Escherichia coli convert M13 and varphiX174 viral single strands to their double-stranded replicative forms. Rifampicin, an inhibitor of RNA polymerase, blocks conversion of M13 single strands to the replicative forms in vivo and in vitro. However, rifampicin does not block synthesis of the replicative forms of varphiX174 either in vivo or in soluble extracts. The replicative form of M13 synthesized in vitro consists of a full-length, linear, complementary strand annealed to a viral strand. The conversion of single strands of M13 to the replicative form proceeds in two separate stages. The first stage requires enzymes, ribonucleoside triphosphates, and single-stranded DNA; the reaction is inhibited by rifampicin. The macromolecular product separated at this stage supports DNA synthesis with deoxyribonucleoside triphosphates and a fresh addition of enzymes; ribonucleoside triphosphates are not required in this second stage nor does rifampicin inhibit the reaction. We presume that in the first stage there is synthesis of a short RNA chain, which then primes the synthesis of a replicative form by a DNA polymerase.     

Direct Participation of dCMP Hydroxymethylase in Synthesis of Bacteriophage T4 DNA

In order to retain in an in situ system the control mechanisms involved in synthesis of bacteriophage T4 DNA, infected cells were made permeable to nucleotides by plasmolysis with concentrated sucrose. Such preparations use exogenous deoxyribonucleotides to synthesize T4 phage DNA. As has been observed with in vivo studies, DNA synthesis was drastically reduced in plasmolyzed preparations from cells infected by amber mutants of genes 1, 32, 41, 42, 43, 44, or 45. Added 5-hydroxymethyl dCTP did not bypass either a mutant of gene 42 (dCMP hydroxymethylase) or of gene 1 (phage-induced deoxyribonucleotide kinase). In a phage system lacking deoxycytidine triphosphatase (gene 56) and the gene-46 product, and therefore incorporating dCTP into DNA, dCTP incorporation did not require dCMP hydroxymethylase, in keeping with in vivo results. With a triple amber mutant of genes 1, 46, and 56 only slight incorporation of dCTP occurred. By contrast, in experiments performed in vivo the synthesis of cytosine-containing DNA was unaffected by an amber mutation in gene 1.These studies provide evidence that dCMP hydroxymethylase, in addition to its known catalytic function, has a second, more direct, role in phage T4 DNA synthesis, apparently in recognition of hydroxymethyl dCTP. The role of the phage-induced deoxyribonucleotide kinase in T4 DNA synthesis in the plasmolyzed system remains unresolved.     

An Enhancing Role for DNA Synthesis in Formation of Bacteriophage Lambda Recombinants

Recombination in some intervals of the map of phage lambda is associated with more DNA synthesis than in other intervals. Blockage of DNA synthesis by high temperature in a host temperature-sensitive for DNA synthesis results in the relative reduction of recombinant frequencies in those regions having the larger amounts of recombination-associated synthesis. Reduction of DNA synthesis at normal temperatures by a combination of the bacterial mutation and a mutation in one of the phage genes required for DNA synthesis has the same consequence. Therefore, DNA synthesis enhances recombinant particle formation more in some map intervals than in others.     

Requirements for the Packaging of Geminivirus Circular Single-Stranded DNA: Effect of DNA Length and Coat Protein Sequence

Geminivirus particles, consisting of a pair of twinned isometric structures, have one of the most distinctive capsids in the virological world. Until recently, there was little information as to how these structures are generated. To address this, we developed a system to produce capsid structures following the delivery of geminivirus coat protein and replicating circular single-stranded DNA (cssDNA) by the infiltration of gene constructs into plant leaves. The transencapsidation of cssDNA of the Begomovirus genus by coat protein of different geminivirus genera was shown to occur with full-length but not half-length molecules. Double capsid structures, distinct from geminate capsid structures, were also generated in this expression system. By increasing the length of the encapsidated cssDNA, triple geminate capsid structures, consisting of straight, bent and condensed forms were generated. The straight geminate triple structures generated were similar in morphology to those recorded for a potato-infecting virus from Peru. These finding demonstrate that the length of encapsidated DNA controls both the size and stability of geminivirus particles.     

Initiation and Reinitiation of DNA Synthesis during Replication of Bacteriophage T7

In its first round of replication, the T7 chromosome follows a simple pattern, as viewed in the electron microscope. The iniation of DNA synthesis occurs about 17% from the genetic left end of the viral DNA rod. Bidirectional DNA synthesis from this origin then generates a replicating intermediate that we call an "eye form." In the eye form, when synthesis in the leftward direction reaches the left end of the viral chromosome, the molecule is converted into a Y-shaped replicating rod. The remaining growing point continues synthesis rightward, until presumably it runs off the right end of the DNA rod, thus terminating replication.Numerous T7 chromosomes were found in which a second round of replication had begun before the first round had finished. Analysis of these reinitiated DNA molecules showed that the second round of replication, like the first, began 17% from the end of the chromosome and involved bidirectional DNA synthesis.     

Protein Fusion: A Novel Reaction in Bacteriophage λ Head Assembly

Parts of two phage-coded head proteins, pE and pC, become fused during bacteriophage lambda head assembly. pE is the main structural component of lambda heads and pC is a minor head protein that is not found as such in mature heads. The bond joining the two proteins appears to be covalent and is not a disulfide bond. Only a specific subset of the sequences of each protein is found in the fusion products, and these sequences are found in the products in equimolar amounts. Two nearly identical fusion products; X1 and X2, are detected; X2 is slightly smaller than X1 and appears to be a proteolytic cleavage product of X1. The fusion reaction probably takes place on a nascent head structure.     

Genetic Mapping of the Inversion Loop in Bacteriophage Mu DNA

An inversion loop seen in heteroduplex mapping of the DNA of mature Mu phage induced from a lysogen is observed also in defective lambda phage carrying one end of Mu. 14% of the DNA of Mu, including this region, designated the G loop, is shown to be to the right of all known genes in the prophage map. The inhomogeneous ends of Mu are observed as a separate phenomenon and appear in all mutants investigated. The recA and recBC functions of the host are not needed for the inversion responsible for the G loop to take place. Deletions of Mu DNA in the G-loop region have been isolated and are under study.     

Effect of Inactivation Methods on SARS-CoV-2 Virion Protein and Structure

The risk posed by Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) dictates that live-virus research is conducted in a biosafety level 3 (BSL3) facility. Working with SARS-CoV-2 at lower biosafety levels can expedite research yet requires the virus to be fully inactivated. In this study, we validated and compared two protocols for inactivating SARS-CoV-2: heat treatment and ultraviolet irradiation. The two methods were optimized to render the virus completely incapable of infection while limiting the destructive effects of inactivation. We observed that 15 min of incubation at 65 °C completely inactivates high titer viral stocks. Complete inactivation was also achieved with minimal amounts of UV power (70,000 µJ/cm²), which is 100-fold less power than comparable studies. Once validated, the two methods were then compared for viral RNA quantification, virion purification, and antibody detection assays. We observed that UV irradiation resulted in a 2-log reduction of detectable genomes compared to heat inactivation. Protein yield following virion enrichment was equivalent for all inactivation conditions, but the quality of resulting viral proteins and virions were differentially impacted depending on inactivation method and time. Here, we outline the strengths and weaknesses of each method so that investigators might choose the one which best meets their research goals.     

Discriminatory Ribosome Rebinding of Isolated Regions of Protein Synthesis Initiation from the Ribonucleic Acid of Bacteriophage R17

To determine whether bacterial ribosomes recognize a distinguishing feature in the immediate vicinity of actual initiator codons or are directed to these sites through involvement of other portion(s) of the mRNA molecule, the interaction between ribosomes and defined (32)P-labeled initiator fragments from R17 RNA was studied. When incubated with mixtures of the three sites, ribosomes from Bacillus stearothermophilus (which initiate only the A protein on intact phage RNA) are able to select out the A fragment and discriminate against the coat and replicase initiator regions. By contrast, Escherichia coli ribosomes do not rebind that coat-protein region of R17 most efficiently, as they in the native RNA, but likewise prefer the A-protein initiator fragment. In both cases, ribosome binding of the isolated A site is comparable by several criteria to normal polypeptide-chain initiation on an intact R17 messenger RNA in vitro. E. coli ribosomal preference for the A site is confirmed in experiments with randomly fragmented R17 RNA, by both the initiation dipeptide and ribosome protection assay. Thus the A-protein ribosome-binding site of R17 RNA appears intrinsically to be a good initiator, while efficient recognition of the coat and replicase regions requires the participation of some portion of the remainder of the phage RNA molecule.     

Structure of nascent replicative form DNA of coliphage M13.

Nascent replicative form type II (RFII) DNA of coliphage M13 synthesized in an Escherichia coli mutant deficient in the 5' leads to 3' exonuclease associated uith DNA polymerase I contains ribonucleotides that are retained in the covalently closed RFI DNA sealed in vitro by the joint action of T5 phage DNA polymerase and T4 phage DNA ligase. These RFI molecules are labile to alkali and RNase H, unlike the RFI produced either in vivo or from RFII with E. coli DNA polymerase I and E. coli DNA ligase. The ribonucleotides are located at one site and predominantly in one strand of the nascent RF DNA. Furthermore, these molecules contain multiple small gaps, randomly located, and one large gap in the intracistronic region.     

Bacteriophage T7 DNA Replication: A Linear Replicating Intermediate

The T7 chromosome in the first round of replication is a Y-shaped DNA rod. Thus, it differs from previously observed bacterial and viral replicating chromosomes that are circular.     

Structure of the Replicating DNA from Bacteriophage T4

At an early stage of replication, parental T4 DNA shows a loop structure often displaying two 3'-ended, single-stranded "whiskers", located in trans configuration at the branching-points. Several such loops have been observed within a single T4 molecule. Occasionally, reinitiation occurred in the middle of a loop, which suggests that the loop was growing in both directions.