The structure and physical properties of a packaged bacteriophage particle.

A string of nucleotides confined within a protein capsid contains all the instructions necessary to make a functional virus particle, a virion. Although the structure of the protein capsid is known for many virus species1,2, the three-dimensional organization of viral genomes has mostly eluded experimental probes3,4. Here we report all-atom structural models of an HK97 virion5, including its entire 39,732 base pair genome, obtained through multiresolution simulations. Mimicking the action of a packaging motor6, the genome was gradually loaded into the capsid. The structure of the packaged capsid was then refined through simulations of increasing resolution, which produced a 26 million atom model of the complete virion, including water and ions confined within the capsid. DNA packaging occurs through a loop extrusion mechanism7 that produces globally different configurations of the packaged genome and gives each viral particle individual traits. Multiple microsecond-long all-atom simulations characterized the effect of the packaged genome on capsid structure, internal pressure, electrostatics and diffusion of water, ions and DNA, and revealed the structural imprints of the capsid onto the genome. Our approach can be generalized to obtain complete all-atom structural models of other virus species, thereby potentially revealing new drug targets at the genome-capsid interface.

Structural and sequence analysis of the RPO30 gene of sheeppox and goatpox viruses from India.

Sheeppox and goatpox are transboundary viral diseases of sheep and goats that cause significant economic losses to small and marginal farmers worldwide, including India. Members of the genus Capripoxvirus (CaPV), namely Sheeppox virus (SPPV), Goatpox virus (GTPV), and Lumpy skin disease virus (LSDV), are antigenically similar, and species differentiation can only be accomplished using molecular approaches. The present study aimed to understand the molecular epidemiology and host specificity of SPPV and GTPV circulating in India through sequencing and structural analysis of the RNA polymerase subunit-30 kDa (RPO30) gene. A total of 29 field isolates from sheep (n = 19) and goats (n = 10) belonging to different geographical regions of India during the period: Year 2015 to 2023, were analyzed based on the sequence and structure of the full-length RPO30 gene/protein. Phylogenetically, all the CaPV isolates were separated into three major clusters: SPPV, GTPV, and LSDV. Multiple sequence alignment revealed a highly conserved RPO30 gene, with a stretch of 21 nucleotide deletion in all SPPV isolates. Additionally, the RPO30 gene of the Indian SPPV and GTPV isolates possessed several species-specific conserved signature residues/motifs that could act as genotyping markers. Secondary structure analysis of the RPO30 protein showed four α-helices, two loops, and three turns, similar to that of the E4L protein of vaccinia virus (VACV). All the isolates in the present study exhibited host preferences across different states of India. Therefore, in order to protect vulnerable small ruminants from poxviral infections, it is recommended to take into consideration a homologous vaccination strategy.

Characteristics of the a sequence of the duck Plague virus genome and specific cleavage of the viral genome based on the a sequence.

During the replication process, the herpesvirus genome forms the head-to-tail linked concatemeric genome, which is then cleaved and packaged into the capsid. The cleavage and packing process is carried out by the terminase complex, which specifically recognizes and cleaves the concatemeric genome. This process is governed by a cis-acting sequence in the genome, named the a sequence. The a sequence and genome cleavage have been described in some herpesviruses, but it remains unclear in duck plague virus. In this study, we analysed the location, composition, and conservation of a sequence in the duck plague virus genome. The structure of the DPV genome has an a sequence of (DR4)m-(DR2)n-pac1-S termini (32 bp)-L termini (32 bp)-pac2, and the length is 841 bp. Direct repeat (DR) sequences are conserved in different DPV strains, but the number of DR copies is inconsistent. Additionally, the typical DR1 sequence was not found in the DPV a sequence. The Pac1 and pac2 motifs are relatively conserved between DPV and other herpesviruses. Cleavage of the DPV concatemeric genome was detected, and the results showed that the DPV genome can form a concatemer and is cleaved into a monomer at a specific site. We also established a sensitive method, TaqMan dual qRT‒PCR, to analyse genome cleavage. The ratio of concatemer to total viral genome was decreased during the replication process. These results will be critical for understanding the process of DPV genome cleavage, and the application of TaqMan dual qRT‒PCR will greatly facilitate more in-depth research.

Structural basis of Gabija anti-phage defence and viral immune evasion.

Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation1-5. Gabija is one of the most prevalent anti-phage defence systems, occurring in more than 15% of all sequenced bacterial and archaeal genomes1,6,7, but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-electron microscopy (cryo-EM) to define how Gabija proteins assemble into a supramolecular complex of around 500 kDa that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defence complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajA-GajB assembly (hereafter, GajAB) that is essential for phage resistance in vivo. We show that a phage-encoded protein, Gabija anti-defence 1 (Gad1), directly binds to the Gabija GajAB complex and inactivates defence. A cryo-EM structure of the virally inhibited state shows that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defence complex and define a unique mechanism of viral immune evasion.

Joint Efforts of Replicative Helicase and SSB Ensure Inherent Replicative Tolerance of G-Quadruplex.

G-quadruplex (G4) is a four-stranded noncanonical DNA structure that has long been recognized as a potential hindrance to DNA replication. However, how replisomes effectively deal with G4s to avoid replication failure is still obscure. Here, using single-molecule and ensemble approaches, the consequence of the collision between bacteriophage T7 replisome and an intramolecular G4 located on either the leading or lagging strand is examined. It is found that the adjacent fork junctions induced by G4 formation incur the binding of T7 DNA polymerase (DNAP). In addition to G4, these inactive DNAPs present insuperable obstacles, impeding the progression of DNA synthesis. Nevertheless, T7 helicase can dismantle them and resolve lagging-strand G4s, paving the way for the advancement of the replication fork. Moreover, with the assistance of the single-stranded DNA binding protein (SSB) gp2.5, T7 helicase is also capable of maintaining a leading-strand G4 structure in an unfolded state, allowing for a fraction of T7 DNAPs to synthesize through without collapse. These findings broaden the functional repertoire of a replicative helicase and underscore the inherent G4 tolerance of a replisome.

Circular replication-associated protein-encoding single-stranded DNA virus with risk of spillover is widely prevalent in domestic animals in China.

Circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA viruses are highly diverse and have a broad range of hosts. In this study, we report the detection of Bo-Circo-like virus AH20-1 in the feces of diarrheal cattle. The virus has a circular genome of 3,912 nucleotides, three major putative open reading frames, and encodes a Rep gene of 310 amino acids. We found that the virus is closely related to the Bo-Circo-like virus CH strain, which belongs to the novel Kirkoviridae family. Furthermore, we conducted a nationwide surveillance program and found that the virus is prevalent in China (23.6%, 205/868), with the BCLa subtype being the predominant strain. Our findings suggest that the virus can infect sheep, highlighting the potential for cross-species transmission. Our pressure analysis indicates that the CRESS-DNA Kirkoviridae family has broad host adaptation, and that selection pressure played an important role in the evolution of its Rep genes. Our study underscores the need for continued epidemiological surveillance of this virus due to its widespread prevalence in our ruminant population and potential for cross-species transmission.

Genome sequence and characterization of a novel Pseudomonas putida phage, MiCath.

Pseudomonads are ubiquitous bacteria with importance in medicine, soil, agriculture, and biomanufacturing. We report a novel Pseudomonas putida phage, MiCath, which is the first known phage infecting P. putida S12, a strain increasingly used as a synthetic biology chassis. MiCath was isolated from garden soil under a tomato plant using P. putida S12 as a host and was also found to infect four other P. putida strains. MiCath has a ~ 61 kbp double-stranded DNA genome which encodes 97 predicted open reading frames (ORFs); functions could only be predicted for 48 ORFs using comparative genomics. Functions include structural phage proteins, other common phage proteins (e.g., terminase), a queuosine gene cassette, a cas4 exonuclease, and an endosialidase. Restriction digestion analysis suggests the queuosine gene cassette encodes a pathway capable of modification of guanine residues. When compared to other phage genomes, MiCath shares at most 74% nucleotide identity over 2% of the genome with any sequenced phage. Overall, MiCath is a novel phage with no close relatives, encoding many unique gene products.

'SAXS-osmometer' method provides measurement of DNA pressure in viral capsids and delivers an empirical equation of state.

We present a novel method that provides a measurement of DNA pressure in viral capsids using small angle X-ray scattering (SAXS). This method, unlike our previous assay, does not require triggering genome release with a viral receptor. Thus, it can be used to determine the existence of a pressurized genome state in a wide range of virus systems, even if the receptor is not known, leading to a better understanding of the processes of viral genome uncoating and encapsidation in the course of infection. Furthermore, by measuring DNA pressure for a collection of bacteriophages with varying DNA packing densities, we derived an empirical equation of state (EOS) that accurately predicts the relation between the capsid pressure and the packaged DNA density and includes the contribution of both DNA-DNA interaction energy and DNA bending stress to the total DNA pressure. We believe that our SAXS-osmometer method and the EOS, combined, provide the necessary tools to investigate physico-chemical properties of confined DNA condensates and mechanisms of infection, and may also provide essential data for the design of viral vectors in gene therapy applications and development of antivirals that target the pressurized genome state.

Temperature-induced DNA density transition in phage λ capsid revealed with contrast-matching SANS.

Structural details of a genome packaged in a viral capsid are essential for understanding how the structural arrangement of a viral genome in a capsid controls its release dynamics during infection, which critically affects viral replication. We previously found a temperature-induced, solid-like to fluid-like mechanical transition of packaged λ-genome that leads to rapid DNA ejection. However, an understanding of the structural origin of this transition was lacking. Here, we use small-angle neutron scattering (SANS) to reveal the scattering form factor of dsDNA packaged in phage λ capsid by contrast matching the scattering signal from the viral capsid with deuterated buffer. We used small-angle X-ray scattering and cryoelectron microscopy reconstructions to determine the initial structural input parameters for intracapsid DNA, which allows accurate modeling of our SANS data. As result, we show a temperature-dependent density transition of intracapsid DNA occurring between two coexisting phases-a hexagonally ordered high-density DNA phase in the capsid periphery and a low-density, less-ordered DNA phase in the core. As the temperature is increased from 20 °C to 40 °C, we found that the core-DNA phase undergoes a density and volume transition close to the physiological temperature of infection (~37 °C). The transition yields a lower energy state of DNA in the capsid core due to lower density and reduced packing defects. This increases DNA mobility, which is required to initiate rapid genome ejection from the virus capsid into a host cell, causing infection. These data reconcile our earlier findings of mechanical DNA transition in phage.

Electron microscopy mapping of the DNA-binding sites of monomeric, dimeric, and multimeric KSHV RTA protein.

IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus associated with several human cancers, typically in patients with compromised immune systems. Herpesviruses establish lifelong infections in hosts in part due to the two phases of infection: the dormant and active phases. Effective antiviral treatments to prevent the production of new viruses are needed to treat KSHV. A detailed microscopy-based investigation of the molecular interactions between viral protein and viral DNA revealed how protein-protein interactions play a role in DNA-binding specificity. This analysis will lead to a more in-depth understanding of KSHV DNA replication and serve as the basis for anti-viral therapies that disrupt and prevent the protein-DNA interactions, thereby decreasing spread to new hosts.

A novel Queuovirinae lineage of Pseudomonas aeruginosa phages encode dPreQ0 DNA modifications with a single GA motif that provide restriction and CRISPR Cas9 protection in vitro.

Deazaguanine DNA modifications are widespread in phages, particularly in those with pathogenic hosts. Pseudomonas phage iggy substitutes ∼16.5% of its genomic 2'-deoxyguanosine (G) with dPreQ0, and the iggy deazaguanine transglycosylase (DpdA) is unique in having a strict GA target motif, not observed previously. The iggy PreQ0 modification is shown to provide protection against both restriction endonucleases and Cas9 (when present in PAM), thus expanding our understanding of the deazaguanine modification system, its potential, and diversity. Phage iggy represents a new genus of Pseudomonas phages within the Queuovirinae subfamily; which have very little in common with other published phage genomes in terms of nucleotide similarity (<10%) and common proteins (<2%). Interestingly, shared similarity is concentrated in dpdA and preQ0 biosynthesis genes. TEM imaging confirmed a siphovirus morphology with a prolate icosahedral head and a non-contractile flexible tail with one long central tail spike. The observed protective effect of the deazaguanine modification on the iggy DNA may contribute to its broad within-species host range. Phage iggy was isolated on Pseudomonas aeruginosa PAO1, but also infects PDO300, PAK, PA14, as well as 10 of 27 tested environmental isolates and 13 of 20 tested clinical isolates of P. aeruginosa from patients with cystic fibrosis.

Vital for Viruses: Intrinsically Disordered Proteins.

Viruses infect all kingdoms of life; their genomes vary from DNA to RNA and in size from 2kB to 1 MB or more. Viruses frequently employ disordered proteins, that is, protein products of virus genes that do not themselves fold into independent three-dimensional structures, but rather, constitute a versatile molecular toolkit to accomplish a range of functions necessary for viral infection, assembly, and proliferation. Interestingly, disordered proteins have been discovered in almost all viruses so far studied, whether the viral genome consists of DNA or RNA, and whatever the configuration of the viral capsid or other outer covering. In this review, I present a wide-ranging set of stories illustrating the range of functions of IDPs in viruses. The field is rapidly expanding, and I have not tried to include everything. What is included is meant to be a survey of the variety of tasks that viruses accomplish using disordered proteins.

Modulation of the functional interfaces between retroviral intasomes and the human nucleosome.

Infection by retroviruses as HIV-1 requires the stable integration of their genome into the host cells. This process needs the formation of integrase (IN)-viral DNA complexes, called intasomes, and their interaction with the target DNA wrapped around nucleosomes within cell chromatin. To provide new tools to analyze this association and select drugs, we applied the AlphaLISA technology to the complex formed between the prototype foamy virus (PFV) intasome and nucleosome reconstituted on 601 Widom sequence. This system allowed us to monitor the association between both partners and select small molecules that could modulate the intasome/nucleosome association. Using this approach, drugs acting either on the DNA topology within the nucleosome or on the IN/histone tail interactions have been selected. Within these compounds, doxorubicin and histone binders calixarenes were characterized using biochemical, in silico molecular simulations and cellular approaches. These drugs were shown to inhibit both PFV and HIV-1 integration in vitro. Treatment of HIV-1-infected PBMCs with the selected molecules induces a decrease in viral infectivity and blocks the integration process. Thus, in addition to providing new information about intasome-nucleosome interaction determinants, our work also paves the way for further unedited antiviral strategies that target the final step of intasome/chromatin anchoring. IMPORTANCE In this work, we report the first monitoring of retroviral intasome/nucleosome interaction by AlphaLISA. This is the first description of the AlphaLISA application for large nucleoprotein complexes (>200 kDa) proving that this technology is suitable for molecular characterization and bimolecular inhibitor screening assays using such large complexes. Using this system, we have identified new drugs disrupting or preventing the intasome/nucleosome complex and inhibiting HIV-1 integration both in vitro and in infected cells. This first monitoring of the retroviral/intasome complex should allow the development of multiple applications including the analyses of the influence of cellular partners, the study of additional retroviral intasomes, and the determination of specific interfaces. Our work also provides the technical bases for the screening of larger libraries of drugs targeting specifically these functional nucleoprotein complexes, or additional nucleosome-partner complexes, as well as for their characterization.

Mutations in the S gene of hepatitis B virus in three generations of patients with chronic hepatitis B.

Failure to neutralize HBsAg and subsequent escape from the host immune system may be caused by HBsAg mutations, particularly in the "a" determinant, which alters the antigenicity of the protein. The purpose of this study was to examine the frequency of S gene mutations in three generations of HBV cases in northeastern Iran. In this study, 90 patients with chronic HBV were assigned to three groups according to the inclusion criteria. The plasma were utilized to extract viral DNA, and the PCR was applied. Direct sequencing and alignment were performed on the S gene, using reference sequence. The results indicated that all HBV genomes were categorized as the genotype D/ayw2. Among 79 point mutations detected, 36.8% were silent, and 56.2% were missense. In the S region, mutations were observed in 88.9% of CHB subjects studied. In the three-generation group, 21.5% of mutations were in the "a" determinant, and 2.6%, 19.5%, and 87.0% of these mutations were observed in antigenic epitopes of CTLs, CD4+, and B cells, respectively. In addition, 56.7% of mutations occurred at Major Hydrophilic Region. S143L and G145R mutations which the most prevalent in the three-generation (36.7%, 20%), and two-generation (42.5%, 20%) groups, related to the failure of HBsAg detection, vaccine, and immunotherapy escape. The findings showed that most of the mutations were concentrated in the B cell epitope. Most CHB cases from the three-generation, especially grandmothers, had HBV S gene mutations and subsequent amino acid mutations, suggesting that these mutations may be critical for pathogenesis and vaccine evasion.

Molecular determinants for Rous sarcoma virus intasome assemblies involved in retroviral integration.

Integration of retroviral DNA into the host genome involves the formation of integrase (IN)-DNA complexes termed intasomes. Further characterization of these complexes is needed to understand their assembly process. Here, we report the single-particle cryo-EM structure of the Rous sarcoma virus (RSV) strand transfer complex (STC) intasome produced with IN and a preassembled viral/target DNA substrate at 3.36 Å resolution. The conserved intasome core region consisting of IN subunits contributing active sites interacting with viral/target DNA has a resolution of 3 Å. Our structure demonstrated the flexibility of the distal IN subunits relative to the IN subunits in the conserved intasome core, similar to results previously shown with the RSV octameric cleaved synaptic complex intasome produced with IN and viral DNA only. An extensive analysis of higher resolution STC structure helped in the identification of nucleoprotein interactions important for intasome assembly. Using structure-function studies, we determined the mechanisms of several IN-DNA interactions critical for assembly of both RSV intasomes. We determined the role of IN residues R244, Y246, and S124 in cleaved synaptic complex and STC intasome assemblies and their catalytic activities, demonstrating differential effects. Taken together, these studies advance our understanding of different RSV intasome structures and molecular determinants involved in their assembly.

Diverse infective and lytic machineries identified in genome analysis of tailed coliphages against broad spectrum multidrug-resistant Escherichia coli.

The emergence of multidrug-resistant (MDR) E. coli with deleterious consequences to the health of humans and animals has been attributed to the inappropriate use of antibiotics. Without effective antimicrobials, the success of modern medicine in treating infections would be at an increased risk. Bacteriophages could be used as an alternative to antibiotics for controlling the dissemination of MDR bacteria. However, before their use, the bacteriophages have to be assessed for the safety aspect. In this study, three broad host range highly virulent coliphage genomes were sequenced, characterized for infective and lytic potential, and checked for the presence of virulence and resistance genes. The genome sequencing indicated that coliphages ϕEC-S-21 and ϕEC-OE-11 belonged to Myoviridae, whereas coliphage ϕEC-S-24 belonged to the Autographiviridae family derived from the Podoviridae family. The genome size of the three coliphages ranged between 24 and 145 kb, with G + C content ranging between 37 and 51%. Coding sequences (CDS) ranged between 30 and 251 amino acids. The CDS were annotated and the proteins were categorized into different modules, viz., phage structural proteins, proteins associated with DNA replication, DNA modification, bacterial cell lysis, phage packaging, and uncharacterized proteins. The presence of tRNAs was detected only in coliphage ϕEC-OE-11. All three coliphages possessed diverse infective and lytic mechanisms, viz., lytic murein transglycosylase, peptidoglycan transglycosylase, n-acetylmuramoyl-l-alanine amidase, and putative lysozyme. Furthermore, the three coliphage genomes showed neither the presence of antibiotic resistance genes nor virulence genes, which makes them desirable candidates for use in phage therapy-based applications.

Characteristics of a novel temperate bacteriophage against Staphylococcus arlettae (vB_SarS_BM31).

BACKGROUND: Staphylococcus arlettae is a rarely reported coagulase-negative staphylococcus (CoNS) isolated from infected humans and livestock. Observing phage-bacteria interaction could improve the understanding of bacterial pathogenetic mechanisms, providing foundational evidence for phage therapy or phage detection. Herein, we aimed to characterise and annotate a novel bacteriophage, vB_SarS_BM31 (BM31), specific to S. arlettae. This bacteriophage was isolated from a milk sample associated with bovine mastitis and collected in the Sichuan Province, China. RESULTS: The BM31 genome comprised a linear double-stranded DNA of 42,271 base pair in length with a G + C content of 34.59%. A total of 65 open reading frames (ORFs) were assembled from phage DNA, of which 29 were functionally annotated. These functional genes were divided into four modules: the structural, DNA packing and replication, lysis, and lysogeny modules. Holin (ORF25), lysin (ORF26), and integrase (ORF28) were located closely in the entire BM31 genome and were important for lyse or lysogeny cycle of BM31. The phage was identified as a temperate phage according to whole genome analysis and life cycle assay, with basic biological characteristics such as small burst size, short latency period, and narrow host range, consistent with the characteristics of the family Siphoviridae, subcluster B14 of the Staphylococcus bacteriophage. CONCLUSIONS: The present isolation and characterisation of BM31 contributes to the Staphylococcus bacteriophage database and provides a theoretical foundation for its potential applications. To the best of our knowledge, BM31 is the only shared and completely reported phage against S. arlettae in the entire public database.

Viral Small Terminase: A Divergent Structural Framework for a Conserved Biological Function.

The genome packaging motor of bacteriophages and herpesviruses is built by two terminase subunits, known as large (TerL) and small (TerS), both essential for viral genome packaging. TerL structure, composition, and assembly to an empty capsid, as well as the mechanisms of ATP-dependent DNA packaging, have been studied in depth, shedding light on the chemo-mechanical coupling between ATP hydrolysis and DNA translocation. Instead, significantly less is known about the small terminase subunit, TerS, which is dispensable or even inhibitory in vitro, but essential in vivo. By taking advantage of the recent revolution in cryo-electron microscopy (cryo-EM) and building upon a wealth of crystallographic structures of phage TerSs, in this review, we take an inventory of known TerSs studied to date. Our analysis suggests that TerS evolved and diversified into a flexible molecular framework that can conserve biological function with minimal sequence and quaternary structure conservation to fit different packaging strategies and environmental conditions.

Phylogenetic analysis and searching bovine papillomaviruses in teat papillomatosis cases in cattle by performing histopathology, immunohistochemistry, and transmission electron microscopy.

Papillomaviruses are epitheliotropic in nature and cause proliferation in the skin, mucosa, and various internal organs of various animal species. The lesions they cause, specifically in cattle teats, lead to significant economic losses in the milk industry. In this study, we identified the bovine papillomaviruses (BPVs) responsible for teat papillomas in cattle. The tissue damage caused by the virus was examined histopathologically using immunohistochemical, transmission electron microscopy (TEM), and molecular methods. Additionally, sequence analyses were performed on the isolated field strains to better understand their genetic and phylogenetic relationships with previously reported isolates. Teat papillomatosis was confirmed in the collected samples by histopathological and immunohistochemical methods, which were followed by other diagnostic methods. Intranuclear virus particles were found in the epithelial cells during a TEM examination of teat lesions. BPV was detected in seven samples by performing PCR using degenerate primers and specific primers. The positive samples were used for typing through sequence analysis/PCR with type-specific primers. Three isolates from teat tissues with BPV infection were identified as BPV-6, two as BPV-10, one as BPV-2, and one as BPV-8. The five isolates identified through sequence analysis of positive samples belonged to the Xipapillomavirus 1 genus (one), the Epsilonpapillomavirus 1 genus (one), and the Deltapapillomavirus genus (one) (three). Furthermore, type-specific primers were found to be useful for molecular diagnosis of BPV, which occurs in the etiology of teat papillomas, followed by genotyping and primer generation during characterization. The detection of BPV types and their prevalence, biosafety measures in animal breeding, and the importance of vaccine research are all important.

B-to-A transition in target DNA during retroviral integration.

Integration into host target DNA (tDNA), a hallmark of retroviral replication, is mediated by the intasome, a multimer of integrase (IN) assembled on viral DNA (vDNA) ends. To ascertain aspects of tDNA recognition during integration, we have solved the 3.5 Å resolution cryo-EM structure of the mouse mammary tumor virus (MMTV) strand transfer complex (STC) intasome. The tDNA adopts an A-like conformation in the region encompassing the sites of vDNA joining, which exposes the sugar-phosphate backbone for IN-mediated strand transfer. Examination of existing retroviral STC structures revealed conservation of A-form tDNA in the analogous regions of these complexes. Furthermore, analyses of sequence preferences in genomic integration sites selectively targeted by six different retroviruses highlighted consistent propensity for A-philic sequences at the sites of vDNA joining. Our structure additionally revealed several novel MMTV IN-DNA interactions, as well as contacts seen in prior STC structures, including conserved Pro125 and Tyr149 residues interacting with tDNA. In infected cells, Pro125 substitutions impacted the global pattern of MMTV integration without significantly altering local base sequence preferences at vDNA insertion sites. Collectively, these data advance our understanding of retroviral intasome structure and function, as well as factors that influence patterns of vDNA integration in genomic DNA.