Transactivation of programmed ribosomal frameshifting by a viral protein

Programmed −1 ribosomal frameshifting (−1 PRF) is a widely used translational mechanism facilitating the expression of two polypeptides from a single mRNA. Commonly, the ribosome interacts with an mRNA secondary structure that promotes −1 frameshifting on a homopolymeric slippery sequence. Recently, we described an unusual −2 frameshifting (−2 PRF) signal directing efficient expression of a transframe protein [nonstructural protein 2TF (nsp2TF)] of porcine reproductive and respiratory syndrome virus (PRRSV) from an alternative reading frame overlapping the viral replicase gene. Unusually, this arterivirus PRF signal lacks an obvious stimulatory RNA secondary structure, but as confirmed here, can also direct the occurrence of −1 PRF, yielding a third, truncated nsp2 variant named “nsp2N.” Remarkably, we now show that both −2 and −1 PRF are transactivated by a protein factor, specifically a PRRSV replicase subunit (nsp1β). Embedded in nsp1β’s papain-like autoproteinase domain, we identified a highly conserved, putative RNA-binding motif that is critical for PRF transactivation. The minimal RNA sequence required for PRF was mapped within a 34-nt region that includes the slippery sequence and a downstream conserved CCCANCUCC motif. Interaction of nsp1β with the PRF signal was demonstrated in pull-down assays. These studies demonstrate for the first time, to our knowledge, that a protein can function as a transactivator of ribosomal frameshifting. The newly identified frameshifting determinants provide potential antiviral targets for arterivirus disease control and prevention. Moreover, protein-induced transactivation of frameshifting may be a widely used mechanism, potentially including previously undiscovered viral strategies to regulate viral gene expression and/or modulate host cell translation upon infection.Among the repertoire of mechanisms that viruses use to control or regulate their gene expression, noncanonical translation plays an important role, in particular for positive-strand RNA viruses whose genomic RNA serves a dual function as mRNA and genome (reviewed in ref. 1). A commonly used strategy is −1 programmed ribosomal frameshifting (−1 PRF), in which mRNA signals induce a significant proportion of translating ribosomes to change reading frame, with ribosomes slipping back (in the 5′ direction) by 1 nt into an overlapping ORF before continuing translation, generating a fusion protein composed of the products of both upstream and downstream ORFs (reviewed in refs. 1–4). PRF was first described as the mechanism by which the Gag-Pol polyprotein of the retrovirus Rous sarcoma virus is expressed from overlapping gag and pol ORFs (5, 6) and related signals have since been documented in many other viruses of medical, veterinary, and agricultural importance (7–11). PRF has also been increasingly recognized in cellular genes of both prokaryotes and eukaryotes as well as in other replicating elements, such as insertion sequences and transposons (12).Recently, we identified an unusual −2 programmed ribosomal frameshifting (−2 PRF) event that operates during the translation of the genome of porcine reproductive and respiratory syndrome virus (PRRSV), a member of the arterivirus family in the order Nidovirales (13). PRRSV can be divided into distinct European (EU, type 1) and North American (NA, type 2) genotypes. The viral genome comprises a positive-sense RNA molecule, ∼15 kb in length (14). As in other nidoviruses, its 5′ proximal region contains two large replicase ORFs (ORF1a and ORF1b) (15), with the ORF1b product being expressed as a fusion with the ORF1a product following −1 PRF in the short ORF1a/ORF1b overlap region (Fig. 1). Four ORF1a-encoded proteinases (residing in nsp1α, nsp1β, nsp2, and nsp4) subsequently cleave the pp1a and pp1ab polyproteins into (at least) 14 different nonstructural proteins (nsps; Fig. 1A). The recently identified −2 PRF signal is located several kilobases upstream of the ORF1a/ORF1b −1 PRF signal, and maps to the part of ORF1a that encodes nsp2. This large, multifunctional replicase subunit is involved in diverse steps of the arterivirus replicative cycle, including replicase polyprotein processing (16), the formation of replication structures (17, 18), and innate immune evasion (19–22). At the PRRSV −2 PRF signal, a proportion of ribosomes back up 2 nt, to generate a transframe fusion protein (nsp2TF) comprising the N-terminal two-thirds of nsp2 and the product encoded by a conserved alternative ORF [transframe (TF)] in the −2 reading frame. Compared with full-length nsp2, the nsp2TF product is truncated, equipped with an alternative C-terminal transmembrane domain (Fig. 1A), and targeted to a different subcellular compartment (13). Mutations preventing nsp2TF expression reduce PRRSV replication efficiency in cell culture 50- to 100-fold, highlighting the biological importance of the frameshifting event and nsp2TF expression. The −2 PRF takes place at a highly conserved RG_GUU_UUU slippery sequence (R = G or A), and frameshifting is remarkably efficient (around 20% in virus-infected cells and up to 50% in expression systems) (13).Open in a separate windowFig. 1.PRRSV genome organization and location of ribosomal frameshifting signals. (A) Overview of the ∼15-kb PRRSV genome. The long 5′ ORFs 1a and 1b encode nonstructural polyproteins, and at least eight shorter 3′ ORFs (2a-7) encode structural proteins. The 3′ ORFs are translated from a nested set of subgenomic mRNAs, two of which are bicistronic. ORF1a and ORF1b are translated from the genomic RNA, with translation of ORF1b depending on −1 PRF at the end of ORF1a. The TF ORF overlaps the central ORF1a region in the −2 reading frame and is accessed via −2 PRF (13). A −1 frameshift at the same site generates the nsp2N product (see details under the section “Alternative −2 and −1 PRF at the Same PRRSV Slippery Sequence”). The vertical red line indicates the location of the RG_GUU_UUU shift site (R = A or G, in different arteriviruses). Domains in nsp2/nsp2TF: C, Cys-rich domain HVR, hypervariable region; PLP2, papain-like proteinase;TM/TM′, (putative) transmembrane domains. (B) Sequence of the SD01-08 RNA in the region of the −2/−1 PRF signal, with the slippery sequence (red) and C-rich motif (blue) highlighted. The −1 reading frame stop codon is underlined and codons for each of the reading frames are indicated. (C) Features of the canonical −1 PRF signal present in the PRRSV ORF1a/ORF1b overlap region. The stimulatory RNA pseudoknot is composed of two stems connected by single-stranded loops.As depicted in Fig. 1 B and C, the elements that promote PRF in PRRSV are quite distinct. The −1 PRF signal at the ORF1a/1b junction comprises a slippery sequence (generally U_UUA_AAC) where the ribosome changes frame, and a stimulatory RNA pseudoknot structure immediately downstream, an organization that is conserved throughout the Nidovirales order (23, 24) and widely used in other viral −1 PRF mechanisms. It is thought that interaction of the translating ribosome with the pseudoknot confounds its RNA-unwinding activity (25, 26) and may induce tension in the mRNA that assists in the uncoupling of codon–anticodon interactions at the shift site (27–29). In contrast, only a few cases of −2 PRF in mammalian cells have been documented thus far (13, 29) and the elements involved are poorly understood. Our previous computer-based RNA-folding analysis suggested that the RNA downstream of the slippery sequence (RG_GUU_UUU) used for −2 PRF in PRRSV is rather unstructured and does not fold into a structure compatible with canonical RNA-structure-stimulated PRF. However, mutations within a conserved CCCANCUCC motif located 11 nt downstream of the shift site can reduce or inhibit frameshifting, consistent with the presence of a 3′ stimulatory element of some form (13). Remarkably, our previous study also provided indications for the occurrence of efficient −1 frameshifting at (or near) the same slippery sequence. Due to the presence of a translation termination codon in the −1 reading frame immediately following the slippery sequence, this would yield a truncated form of nsp2, termed “nsp2N” (Fig. 1A).In this report, we identify PRRSV replicase subunit nsp1β as a transactivator of efficient −2 and −1 PRF at the same slippery sequence and provide evidence that its frameshift-stimulatory activity requires interaction with the viral mRNA. In support of this, a highly conserved putative RNA-binding motif (GKYLQRRLQ), integrated into the structure of nsp1β’s papain-like autoproteinase domain, was found to be critical for the stimulation of frameshifting and for interacting with the RNA sequence of the PRRSV PRF signal. The minimal RNA sequence required to direct efficient PRF was mapped within a 34-nt region of the PRRSV nsp2-coding sequence that includes the shift site and the conserved CCCANCUCC motif. Our findings reveal an unusual noncanonical translation mechanism in which a viral protein functions as a transactivator of efficient −2 and −1 PRF. This study advances our understanding of noncanonical translation, suggests that viruses may use additional strategies to modulate viral and potentially host cell translation during infection, and has practical implications in biotechnology and the design of antiviral strategies.     

Translational frameshifting generates the gamma subunit of DNA polymerase III holoenzyme.

The dnaX gene (previously called dnaZX) of Escherichia coli has only one open reading frame for a 71-kDa polypeptide from which two distinct DNA polymerase III holoenzyme subunits, tau (71 kDa) and gamma (47 kDa), are produced. To determine how the gamma subunit is generated, we examined the influence of mutations in the dnaX gene on the pattern of tau and gamma production in overproducing cells. Important structural elements in dnaX mRNA include a stretch of six adenines (nucleotides 1425-1430), a stable hairpin structure (nucleotides 1437-1466), and a UGA stop codon in a -1 frame (nucleotides 1434-1436) between the stretch of adenines and the hairpin structure. Disruption of this stop codon generates a slightly larger gamma subunit, indicative of the use of a -1 stop codon farther downstream (nucleotides 1470-1472). These results suggest that a -1 frameshift during translation allows the use of this UGA codon to terminate translation of the gamma polypeptide. The amino acid composition, sequence, and mass spectra of a C-terminal peptide from mild digestion of the purified gamma protein with endoproteinase Lys-C confirms that this frameshift occurs at either of the two lysine codons in the region of the adenine stretch. Remarkable features of this frameshifting are its high frequency (i.e., about 80% in an overproducing cell) and the striking structural similarity to the frameshifting signal responsible for expression of the pol and pro genes in many retroviruses.     

RNA sequence of astrovirus: distinctive genomic organization and a putative retrovirus-like ribosomal frameshifting signal that directs the viral replicase synthesis.

The genomic RNA of human astrovirus was sequenced and found to contain 6797 nt organized into three open reading frames (1a, 1b, and 2). A potential ribosomal frameshift site identified in the overlap region of open reading frames 1a and 1b consists of a "shifty" heptanucleotide and an RNA stem-loop structure that closely resemble those at the gag-pro junction of some retroviruses. This translation frame-shift may result in the suppression of in-frame amber termination at the end of open reading frame 1a and the synthesis of a nonstructural, fusion polyprotein that contains the putative protease and RNA-dependent RNA polymerase. Comparative sequence analysis indicated that the protease and polymerase of astrovirus are only distantly related to the respective enzymes of other positive-strand RNA viruses. The astrovirus polyprotein lacks the RNA helicase domain typical of other positive-strand RNA viruses of similar genome size. The genomic organization and expression strategy of astrovirus, with the protease and the polymerase brought together by predicted frameshift, most closely resembled those of plant leuteoviruses. Specific features of the sequence and genomic organization support the classification of astroviruses as an additional family of positive-strand RNA viruses, designated Astroviridae.     

Specific mutations in a viral RNA pseudoknot drastically change ribosomal frameshifting efficiency

Many viruses regulate protein synthesis by -1 ribosomal frameshifting using an RNA pseudoknot. Frameshifting is vital for viral reproduction. Using the information gained from the recent high-resolution crystal structure of the beet western yellow virus pseudoknot, a systematic mutational analysis has been carried out in vitro and in vivo. We find that specific nucleotide tertiary interactions at the junction between the two stems of the pseudoknot are crucial. A triplex is found between stem 1 and loop 2, and triplex interactions are required for frameshifting function. For some mutations, loss of one hydrogen bond is sufficient to abolish frameshifting. Furthermore, mutations near the 5' end of the pseudoknot can increase frameshifting by nearly 300%, possibly by modifying ribosomal contacts. It is likely that the selection of suitable mutations can thus allow viruses to adjust frameshifting efficiencies and thereby regulate protein synthesis in response to environmental change.     

Venezuelan encephalitis emergence mediated by a phylogenetically predicted viral mutation

RNA viruses are notorious for their genetic plasticity and propensity to exploit new host-range opportunities, which can lead to the emergence of human disease epidemics such as severe acute respiratory syndrome, AIDS, dengue, and influenza. However, the mechanisms of host-range change involved in most of these viral emergences, particularly the genetic mechanisms of adaptation to new hosts, remain poorly understood. We studied the emergence of Venezuelan equine encephalitis virus (VEEV), an alphavirus pathogen of people and equines that has had severe health and economic effects in the Americas since the early 20th century. Between epidemics, VEE disappears for periods up to decades, and the viral source of outbreaks has remained enigmatic. Combined with phylogenetic analyses to predict mutations associated with a 1992-1993 epidemic, we used reverse genetic studies to identify an envelope glycoprotein gene mutation that mediated emergence. This mutation allowed an enzootic, equine-avirulent VEEV strain, which circulates among rodents in nearby forests to adapt for equine amplification. RNA viruses including alphaviruses exhibit high mutation frequencies. Therefore, ecological and epidemiological factors probably constrain the frequency of VEE epidemics more than the generation, via mutation, of amplification-competent (high equine viremia) virus strains. These results underscore the ability of RNA viruses to alter their host range, virulence, and epidemic potential via minor genetic changes. VEE also demonstrates the unpredictable risks to human health of anthropogenic changes such as the introduction of equines and humans into habitats that harbor zoonotic RNA viruses.     

Net -1 frameshifting on a noncanonical sequence in a herpes simplex virus drug-resistant mutant is stimulated by nonstop mRNA

Ribosomal frameshifting entails slippage of the translational machinery during elongation. Frameshifting permits expression of more than one polypeptide from an otherwise monocistronic mRNA, and can restore expression of polypeptides in the face of frameshift mutations. A common mutation conferring acyclovir resistance in patients with herpes simplex virus disease deletes one cytosine from a run of six cytosines (C-chord) in the viral thymidine kinase (tk) gene. However, this mutation does not abolish TK activity, which is important for pathogenicity. To investigate how this mutant retains TK activity, we engineered and analyzed viruses expressing epitope-tagged TK. We found that the mutant's TK activity can be accounted for by low levels of full-length TK polypeptide produced by net -1 frameshifting during translation. The efficiency of frameshifting was relatively high, 3-5%, as the polypeptide from the reading frame generated by the deletion, which lacks stop codons (nonstop), was poorly expressed mainly because of inefficient protein synthesis. Stop codons introduced into this reading frame greatly increased its expression, but greatly decreased the level of full-length TK, indicating that frameshifting is strongly stimulated by a new mechanism, nonstop mRNA, which we hypothesize involves stalling of ribosomes on the polyA tail. Mutational studies indicated that frameshifting occurs on or near the C-chord, a region lacking a canonical slippery sequence. Nonstop stimulation of frameshifting also occurred when the C-chord was replaced with a canonical slippery sequence from HIV. This mechanism thus permits biologically and clinically relevant TK synthesis, and may occur more generally.     

Cell-specific viral targeting mediated by a soluble retroviral receptor-ligand fusion protein

TVA, the cellular receptor for subgroup A avian leukosis viruses (ALV-A) can mediate viral entry when expressed as a transmembrane protein or as a glycosylphosphatidylinositol-linked protein on the surfaces of transfected mammalian cells. To determine whether mammalian cells can be rendered susceptible to ALV-A infection by attaching a soluble form of TVA to their plasma membranes, the TVA-epidermal growth factor (EGF) fusion protein was generated. TVA-EGF is comprised of the extracellular domain of TVA linked to the mature form of human EGF. Flow cytometric analysis confirmed that TVA-EGF is a bifunctional reagent capable of binding simultaneously to cell surface EGF receptors and to an ALV-A surface envelope-Ig fusion protein. TVA-EGF prebound to transfected mouse fibroblasts expressing either wild-type or kinase-deficient human EGF receptors, rendered these cells highly susceptible to infection by ALV-A vectors. Viral infection was blocked specifically in the presence of a recombinant human EGF protein, demonstrating that the binding of TVA-EGF to EGF receptors was essential for infectivity. These studies have demonstrated that a soluble TVA-ligand fusion protein can mediate viral infection when attached to specific cell surfaces, suggesting an approach for targeting retroviral infection to specific cell types.     

Translational enhancement of the poliovirus 5'' noncoding region mediated by virus-encoded polypeptide 2A.

Genetic and biochemical studies have revealed that the 5' noncoding region of poliovirus mediates translation of the viral mRNA by an unusual mechanism involving entry of ribosomes in internal sequences of mRNA molecules. We have found that mRNAs bearing the 5' noncoding region of poliovirus were translated at an enhanced rate in poliovirus-infected mammalian cells at a time when translation of cellular mRNAs was not yet inhibited. This translational enhancement of the polioviral 5' noncoding region was mediated by the expression of virus-encoded polypeptide 2A. This indicates that 2A is a multifunctional protein involved directly or indirectly in the activation of viral mRNA translation, in addition to its known roles in viral polyprotein processing and in inhibition of cellular protein synthesis. Thus, 2A represents an activator of translation of a viral mRNA that is translated by an internal ribosome binding mechanism. A likely consequence of this role of 2A is the efficient translation of viral mRNAs early in the infectious cycle, when host cell mRNAs can still compete with viral mRNAs for the host cell translation apparatus.     

Translational control in lysates of Drosophila melanogaster cells.

Cell-free protein-synthesizing systems made from Drosophila melanogaster cells were used to study the translational control induced in these cells by heat shock. Lysates of normally growing cells, termed 25 degrees C cells, translate both normal and heat shock mRNAs. Lysates of cells heat shocked at 36 degrees C for 1 hr, termed 36 degrees C cells, translate preferentially heat-shock mRNAs and a few 25 degrees C cell mRNAs. Thus, both lysates appear to reproduce the control displayed in vivo. Both lysates are optimally active at 28 degrees C, and all translations are done at that temperature, demonstrating that, once established, the discrimination system does not require heat-shock temperature for its activity. Addition of crude ribosome fractions from 25 degrees C cell lysates to lysates from heat-shocked cells "rescues" translation of 25 degrees C cell mRNA, which suggests that the discriminating elements are associated with ribosomes. Neither the heat-shock crude ribosome supplements nor the soluble fractions have any effect on either lysate. The experiments also show that RNA selection is determined by some feature of the RNA structure that is insensitive to protease digestion and phenol/chloroform extraction. The essential structural feature may not be unique to Drosophila mRNAs because the Drosophila lysate is capable of discriminating among mRNAs from other organisms.     

Migration of engrafted neural stem cells is mediated by CXCL12 signaling through CXCR4 in a viral model of multiple sclerosis

Multiple sclerosis (MS) is a human demyelinating disease characterized by multifocal regions of inflammation, progressive myelin loss within the central nervous system (CNS), and eventual failure to remyelinate damaged axons. These problems suggest deficiencies in recruiting and/or maturation of oligodendrocyte progentior cells (OPCs) and highlight cell replacement therapies to promote remyelination. We have used a model of viral-induced demyelination to characterize signaling cues associated with positional migration of transplanted remyelination-competent cells. Although successful transplantation of rodent-derived glial cell types into models of MS has been performed, the mechanisms by which these cells navigate within an inflammatory environment created by a persistent virus has not been defined. Infection of the mouse CNS with the neurotropic JHM strain of mouse hepatitis virus (JHMV) results in an immune-mediated demyelinating disease with clinical and histologic similarities to MS. Surgical engraftment of GFP+ neural stem cells (NSCs) into spinal cords of JHMV-infected mice with established demyelination results in migration, proliferation, and differentiation of the cells into OPCs and mature oligodendrocytes that is associated with increased axonal remyelination. Treatment with anti-CXCL12 [stromal derived factor–1α, (SDF-1α)] blocking serum resulted in a marked impairment in migration and proliferation of engrafted stem cells. Moreover, small molecule–mediated antagonism of CXCR4, but not CXCR7, impaired migration and proliferation, to an extent similar to that with anti-CXCL12 treatment. These data highlight the importance of the CXCL12:CXCR4 pathway in regulating homing of engrafted stem cells to sites of tissue damage within the CNS of mice persistently infected with a neurotropic virus undergoing immune-mediated demyelination.     

Signature pattern analysis: a method for assessing viral sequence relatedness.

Signature pattern analysis identifies particular sites in amino acid or nucleic acid alignments of variable sequences that are distinctly representative of a query set of sequences relative to a background set. We explore the merits of using signature patterns for analysis of HIV-1 (human immunodeficiency virus type 1) sequences in cases of epidemiological linkage and potential superinfection. For these purposes, query sets are viral sequences that are all derived from one HIV-1 infected individual, hence the signature pattern is the array of sites that are characteristic of the range of viral variants obtained from that person. Once a signature pattern has been objectively defined, it can be used to examine other viral sequences from other individuals for evidence of genetic relatedness. A computer program to facilitate this analysis, VESPA, is described and applied to sequence data gathered during the investigation of HIV-1 transmission in a dental practice. The implications of signature polymorphisms seen within an infected individual, and shared polymorphisms between linked individuals, are also considered. VESPA may also be applied to the molecular analysis of biological phenotypes.     

Efficient translational frameshifting occurs within a conserved sequence of the overlap between the two genes of a yeast Ty1 transposon.

The Ty (transposon yeast) family of retroviral-like transposons include two genes, TYA and TYB, analogous to the gag and pol genes of metazoan retroviruses. TYB lies downstream of TYA, the two genes overlapping by 38 base pairs. The primary translation product of TYB is a TYA/TYB fusion protein whose expression has been inferred to occur by translational frameshifting within the overlap region. We show that the event leading to expression of TYB is very efficient, resulting in 20% read-through into TYB from TYA. We demonstrate that the Ty mRNA is colinear with the DNA sequence of the element, eliminating any pretranslational model for TYB expression. Frameshifting requires no particular sequence of the upstream TYA gene, nor any global RNA structure. Surprisingly, it can be promoted by a 14-base-pair oligonucleotide of the overlap region. The ability of this oligonucleotide to function is inhibited when it is positioned immediately downstream of an initiator AUG. We conclude that the TYB gene is expressed by an efficient ribosomal frameshifting event requiring a small oligonucleotide sequence derived from the TYA/TYB overlap region.     

Polyoma large tumor antigen is not required for tumorigenesis mediated by viral DNA.

The arrangement of viral DNA sequences in a hamster cell line derived from a tumor induced by a recombinant plasmid DNA preparation containing the entire polyoma virus genome was examined. In the recombinant plasmid employed, viral DNA sequences specifying the large species of polyoma tumor antigen but not the small and middle tumor antigens were interrupted by the insertion of plasmid DNA at the EcoRI restriction endonuclease site. Blot-hybridization analyses of tumor cell DNA indicated that the "joints" linking viral and plasmid DNAs in the original recombinant plasmid used in animal inoculation had been preserved. Integration into the hamster cell genome had apparently occurred within plasmid DNA sequences. These results indicate that polyoma large tumor antigen is not required for tumorigenesis mediated by viral DNA.     

Ribosomal movement impeded at a pseudoknot required for frameshifting.

Translational frameshifting sometimes occurs when ribosomes encounter a "shift" site preceding a region of unusual secondary structure, which in at least three cases is known to be a pseudoknot. We provide evidence that ribosomes have a decreased rate of movement through a pseudoknot required for frameshifting. These paused ribosomes are directly situated over the shift sequence. Ribosomal pausing appears to be necessary but not sufficient for frameshifting.     

Systemic movement of an RNA plant virus determined by a point substitution in a 5'' leader sequence.

The ability of viruses to move through infected plants is an important determinant of host range and pathogenicity. We have investigated the genetic basis for the inability of the Type strain of barley stripe mosaic hordeivirus to undergo long-range systemic movement in the tobacco Nicotiana benthamiana. We show that, in this model system, a short open reading frame in the 5' leader of the smallest viral genomic RNA prevents long-range vascular movement. As predicted by the ribosome scanning model, the leader open reading frame decreases the efficiency with which the 5'-proximal gene is translated in vitro. Thus, systemic pathogenicity in this system may be determined by the efficiency of translation of a viral gene in vivo and is not determined by the primary sequence of the encoded protein.     

Disclosing the in vivo organization of a viral histone-like protein in Bacillus subtilis mediated by its capacity to recognize the viral genome

Organization of replicating prokaryotic genomes requires architectural elements that, similarly to eukaryotic systems, induce topological changes such as DNA supercoiling. Bacteriophage 29 protein p6 has been described as a histone-like protein that compacts the viral genome by forming a nucleoprotein complex and plays a key role in the initiation of protein-primed DNA replication. In this work, we analyze the subcellular localization of protein p6 by immunofluorescence microscopy and show that, at early infection stages, it localizes in a peripheral helix-like configuration. Later, at middle infection stages, protein p6 is recruited to the bacterial nucleoid. This migrating process is shown to depend on the synthesis of components of the 29 DNA replication machinery (i.e., terminal protein and DNA polymerase) needed for the replication of viral DNA, which is required to recruit the bulk of protein p6. Importantly, the double-stranded DNA-binding capacity of protein p6 is essential for its relocalization at the nucleoid. Altogether, the results disclose the in vivo organization of a viral histone-like protein in bacteria.