The L-A dsRNA Virus and Its Satellites: Totiviruses and Killers in Saccharomyces cerevisiae

Dissecting the dsDNA viral packaging machinery: structural evolution and potential for therapeutics

ABSTRACT Some double-stranded DNA viruses, such as herpesviruses and bacteriophages, utilize a powerful molecular motor to package DNA into their capsids to liquid-crystalline density. The motor is composed of large and small terminase subunits that bind to a portal complex located at a unique vertex on the icosahedral capsid. The small terminase is critical for recognition of the viral genomic DNA. The enzymatic component of the motor is the large terminase that has two functional domains necessary for ATP hydrolysis and DNA cleavage. Bacteriophage large terminase amino acid sequences are highly divergent, but their overall structural architecture and function are conserved across both tailed bacteriophages and herpesviruses. There are limited antiviral drugs for herpesvirus infections. Therefore, the viral DNA packaging motor is being investigated as an alternative drug target. In this review, we discuss the past and recent genetic, biochemical, and structural research on viral terminase motors that has illuminated their role in DNA packaging and potential as candidates for antiviral drug targeting.

Influenza A virus initiates genome selection through promoter-dependent nuclear export

Identification of the SARS-CoV-2 genome packaging signal in the nsp12-coding region

A SARS-CoV-2 packaging signal is identified within the nsp12 coding region and shows that the nucleocapsid protein mediates selective genome packaging through its C-terminal domain, establishing the mechanistic basis for SARS-CoV-2 genome packaging and offer a potential antiviral target.

Characterizing Import of RNA Polymerase into a Phage Nucleus 2310316

Jumbo bacteriophages in the ϕKZ-like family have shown great potential as a therapeutic to treat infections from the human pathogen Pseudomonas aeruginosa, known for its high level of antibiotic resistance. ϕKZ-like phages form a proteinaceous “phage nucleus” upon infection that compartmentalizes and protects phage DNA replication and transcription mechanisms from host defenses. Transcription occurs within the phage nucleus, requiring RNA polymerase and other transcriptional components to be imported into the compartment. Selective import of phage-encoded proteins into this nucleus is critical for successful infection, yet the mechanisms governing this specificity remain incompletely understood. In this work I investigated how the ϕKZ-encoded non-virion RNA polymerase (nvRNAP) is selectively imported. Previously, a series of genetic selections identified the genes imp1 as the major import factor and imp7 as a nvRNAP-specific import factor. Therefore, we hypothesized that Imp1 or 7 may directly interact with nvRNAP. We then purified and mixed Imp7 with nvRNAP, in both its active and inactive forms (with and without sigma factor, respectively). We discovered that Imp7 does not directly bind to nvRNAP in vitro. Preliminary data also suggests Imp1 may bind to the catalytically inactive nvRNAP and not the active nvRNAP in the presence of Imp7. These findings suggest multiple potential models for nvRNAP import, including the independent and separate import of catalytically inactive nvRNAP and sigma factor, forming the catalytically active nvRNAP only when both are successfully imported. Our results highlight a complex import system for the phage nvRNAP and lay the groundwork for further mechanistic dissection of protein trafficking across the phage nuclear barrier. This knowledge could uncover new principles of protein trafficking across novel biological barriers and inform the development of phage-based therapeutics against antibiotic-resistant pathogens. NIH Microbial, Parasitic, and Fungal Immunology (MPF)

High-resolution structure of the vaccinia virus phospholipase D-fold endonuclease K4.

Vaccinia virus (VACV) is an orthopoxvirus closely related to mpox virus, which started global outbreaks in 2022. Poxvirus genomes are flanked by short, inverted complementary hairpin telomeres that feature mismatched bases and insertions essential for viral replication. In this context, a role of the late protein K4 has been proposed. K4 is present in the virion, is apparently non-essential and has a phospholipase D (PLD)-fold as has VACV F13 protein. It also shares fold and nuclease activity with its closest homologue, mammalian PLD3. We established an endonuclease activity against ssDNA and hairpin loops and bubbles in a dsDNA context while RNA is resistant to cleavage. The 2.4 Å cryo-EM structure of K4 shows an unusual octameric assembly, also present in solution. At low concentration, tetramers and dimers similar to the one of hPLD3 are also present. Despite its nuclease activity, in K4 a C-terminal extension blocks the DNA binding pockets. Using an inactive mutant, fortuitously, a DNA 19mer bound simultaneously to 2 sites of the octamer where it displaced the C-termini. DNA binding uses similar residues as the hPLD3 5'-exonuclease, despite different activities and orientations of the DNA. The role of K4 and the control of its activity by the observed auto-inhibition remain enigmatic.

Message in a Bottle: The Therapeutic Potential of Domesticated Capsid Proteins.

Domesticated gag genes derived from long terminal repeat (LTR) retrotransposons are widespread in mammals and occur in other metazoans, including Drosophila and zebrafish. Their protein products commonly contain a capsid (CA) domain, and many retain the ability to assemble capsid-like particles that package RNA, and potentially other biomolecules, with emerging roles in intercellular communication, neuronal signaling, placental gene regulation, and fertility. Biochemical, genetic, and structural studies have identified the core domains that are necessary and sufficient for capsid formation, even in the absence of accessory proteins. Several systems also exhibit selective self-RNA packaging directed by cis elements, suggesting rules that could be harnessed for programmable cargo selection. Building on this foundation, recent efforts have developed host-encoded Gag CA proteins for delivery applications, ranging from engineered systems to approaches that exploit naturally preloaded capsids. In addition to established platforms such as adeno-associated viruses (AAVs), lipid nanoparticles (LNPs), and nonintegrating lentiviral systems, domesticated Gag CA proteins may offer complementary features such as lower immunogenicity, flexible cargo capacity, and tissue-specific targeting. Open questions remain regarding release and cellular entry mechanisms, in vivo tropism, determinants of immunogenicity across family members, and safety.