Phosphorylation regulates viral biomolecular condensates to promote infectious progeny production.

Biomolecular condensates (BMCs) play important roles in diverse biological processes. Many viruses form BMCs which have been implicated in various functions critical for the productive infection of host cells. The adenovirus L1-52/55 kilodalton protein (52K) was recently shown to form viral BMCs that coordinate viral genome packaging and capsid assembly. Although critical for packaging, we do not know how viral condensates are regulated during adenovirus infection. Here we show that phosphorylation of serine residues 28 and 75 within the N-terminal intrinsically disordered region of 52K modulates viral condensates in vitro and in cells, promoting liquid-like properties. Furthermore, we demonstrate that phosphorylation of 52K promotes viral genome packaging and the production of infectious progeny particles. Collectively, our findings provide insights into how viral condensate properties are regulated and maintained in a state conducive to their function in viral progeny production. In addition, our findings have implications for antiviral strategies aimed at targeting the regulation of viral BMCs to limit viral multiplication.

Identification of Host Proteins Involved in Hepatitis B Virus Genome Packaging.

A critical part of the hepatitis B virus (HBV) life cycle is the packaging of the pregenomic RNA (pgRNA) into nucleocapsids. While this process is known to involve several viral elements, much less is known about the identities and roles of host proteins in this process. To better understand the role of host proteins, we isolated pgRNA and characterized its protein interactome in cells expressing either packaging-competent or packaging-incompetent HBV genomes. We identified over 250 host proteins preferentially associated with pgRNA from the packaging-competent version of the virus. These included proteins already known to support capsid formation, enhance viral gene expression, catalyze nucleocapsid dephosphorylation, and bind to the viral genome, demonstrating the ability of the approach to effectively reveal functionally significant host-virus interactors. Three of these host proteins, AURKA, YTHDF2, and ATR, were selected for follow-up analysis. RNA immunoprecipitation qPCR (RIP-qPCR) confirmed pgRNA-protein association in cells, and siRNA knockdown of the proteins showed decreased encapsidation efficiency. This study provides a template for the use of comparative RNA-protein interactome analysis in conjunction with virus engineering to reveal functionally significant host-virus interactions.

Therapeutic Application and Structural Features of Adeno-Associated Virus Vector.

Adeno-associated virus (AAV) is characterized by non-pathogenicity, long-term infection, and broad tropism and is actively developed as a vector virus for gene therapy products. AAV is classified into more than 100 serotypes based on differences in the amino acid sequence of the capsid protein. Endocytosis involves the uptake of viral particles by AAV and accessory receptors during AAV infection. After entry into the cell, they are transported to the nucleus through the nuclear pore complex. AAVs mainly use proteoglycans as receptors to enter cells, but the types of sugar chains in proteoglycans that have binding ability are different. Therefore, it is necessary to properly evaluate the primary structure of receptor proteins, such as amino acid sequences and post-translational modifications, including glycosylation, and the higher-order structure of proteins, such as the folding of the entire capsid structure and the three-dimensional (3D) structure of functional domains, to ensure the efficacy and safety of biopharmaceuticals. To further enhance safety, it is necessary to further improve the efficiency of gene transfer into target cells, reduce the amount of vector administered, and prevent infection of non-target cells.

Development and Evaluation of a Shrimp Virus (IHHNV)-Mediated Gene Transfer and Expression System for Shrimps.

An efficient gene transfer and expression tool is lacking for shrimps and shrimp cells. To solve this, this study has developed a shrimp DNA virus-mediated gene transfer and expression system, consisting of insect Sf9 cells for viral packaging, the shrimp viral vector of pUC19-IHHNV-PH-GUS and the baculoviral vector of Bacmid or Bacmid-VP28 encoding the shrimp WSSV envelope protein VP28. The pUC19-IHHNV-PH-GUS vector was constructed by assembling the genomic DNA of shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), which has shortened inverted terminal repeats, into a pUC19 backbone, and then an expression cassette of baculoviral polyhedron (PH) promoter-driven GUS (ß-glucuronidase) reporter gene was inserted immediately downstream of IHHNV for proof-of-concept. It was found that the viral vector of pUC19-IHHNV-PH-GUS could be successfully packaged into IHHNV-like infective virions in the Sf9 cells, and the gene transfer efficiency of this system was evaluated and verified in three systems of Sf9 cells, shrimp hemolymph cells and tissues of infected shrimps, but the GUS expression could only be detected in cases where the viral vector was co-transfected or co-infected with a baculovirus of Bacmid or Bacmid-VP28 due to the Bacmid-dependence of the PH promoter. Moreover, the packaging and infection efficiencies could be significantly improved when Bacmid-VP28 was used instead of Bacmid.

Pervasive tertiary structure in the dengue virus RNA genome.

RNA virus genomes are efficient and compact carriers of biological information, encoding information required for replication both in their primary sequences and in higher-order RNA structures. However, the ubiquity of RNA elements with higher-order folds-in which helices pack together to form complex 3D structures-and the extent to which these elements affect viral fitness are largely unknown. Here we used single-molecule correlated chemical probing to define secondary and tertiary structures across the RNA genome of dengue virus serotype 2 (DENV2). Higher-order RNA structures are pervasive and involve more than one-third of nucleotides in the DENV2 genomic RNA. These 3D structures promote a compact overall architecture and contribute to viral fitness. Disrupting RNA regions with higher-order structures leads to stable, nonreverting mutants and could guide the development of vaccines based on attenuated RNA viruses. The existence of extensive regions of functional RNA elements with tertiary folds in viral RNAs, and likely many other messenger and noncoding RNAs, means that there are significant regions with pocket-containing surfaces that may serve as novel RNA-directed drug targets.

The ORF8 Protein of SARS-CoV-2 Modulates the Spike Protein and Its Implications in Viral Transmission.

COVID-19 is currently global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Accompanying the rapid spread of the error-prone RNA-based genome, several dominant SARS-CoV-2 variants have been genetically identified. The mutations in the spike protein, which are essential for receptor binding and fusion, have been intensively investigated for their contributions to viral transmission. Nevertheless, the importance of other viral proteins and their mutations in SARS-CoV-2 lifecycle and transmission remains fairly understood. Here, we report the strong potency of an accessory protein ORF8 in modulating the level and processing of the spike protein. The expression of ORF8 protein does not affect propagation but expression of spike protein, which may lead to pseudovirions with less spike protein on the surface, therefore less infection potential. At the protein level, ORF8 expression led to downregulation and insufficient S1/S2 cleavage of the spike protein in a dose-dependent manner. ORF8 exhibits a strong interaction with the spike protein mainly at S1 domains and mediates its degradation through multiple pathways. The dominant clinical isolated ORF8 variants with the reduced protein stability exhibited the increased capacity of viral transmission without compromising their inhibitory effects on HLA-A2. Although the increase in spike protein level and Spike pseudovirus production observed by using highly transmissible clinical spike variants, there was no significant compromise in ORF8-mediated downregulation. Because ORF8 is important for immune surveillance and might be required for viral fitness in vivo, the alteration of the spike protein might be an optional strategy used by SARS-CoV-2 to promote viral transmission by escaping the inhibitory effects of ORF8. Therefore, our report emphasized the importance of ORF8 in SARS-CoV-2 spike protein production, maturation, and possible evolution.

Improved third-generation lentiviral packaging with pLKO.1C vectors.

A widely used third-generation lentiviral packaging system produces virus with enhanced biosafety by eliminating HIV accessory genes and separating packaging elements into three different plasmids. However, for certain vectors such as pLKO.1, third-generation safety features reduce lentiviral titers due to the lack of the accessory gene tat. Here we present a way to improve virus production and target gene knockdown with a modified pLKO.1 CMV pLKO.1C) vector and optimized packaging construct ratios. Replacing the pLKO.1 RSV promoter with the Cytomegalovirus promoter yielded an average of threefold higher titer than standard pLKO.1 packaged using the third-generation system, while optimizing the packaging vector ratios further increased titer and yielded an average of tenfold higher titer than pLKO.1 packaged with the second-generation system.

Evidence for expression of promoterless GFP cassette: Is GFP an ideal reporter gene in biotechnology science?

Green fluorescent protein (GFP) has played an important role in biochemistry and cell biology as a reporter gene. It has been used to assess the potency of promoters for recombinant protein production. This investigation reveals evidences suggesting that the gfp GFP gene (EGFP) could be expressed without the promoter. In a study, a pLenti-F/GFP vector was constructed with the purpose to allow GFP expression in transduced cells but not in packaging cells; however, after transfecting the HEK293T cell line, GFP gene was expressed, compared to pLOX/CWgfp-transfected cells showed expression lag, lower levels and reduced percentage of GFP expression in the cells. This unexpected result could be due to auto transduction in packaging cell, possible retrotransposon activity in the cell line, possible contamination of pLenti-F/GFP with the pLOX/CWgfp and possible presence of a promoter within backbone of the vector. All the possibilities were ruled out. To exclude the possibility that a sequence within the region might act as a promoter, the fragment to be transfected was minimized to a region containing "from the start of the GFP gene to 5'LTR R". The GFP gene was again expressed. Therefore, our findings suggest the EGFP does not need promoter for expression. This should appeal to the researchers designing GFP based assays to evaluate the potency of promoters, since possible aberrant expression may have a potential to influence on the results of a planned experiment.

Establishment of a High-Yield Recombinant Adeno-Associated Virus/Human Bocavirus Vector Production System Independent of Bocavirus Nonstructural Proteins.

The genome of recombinant adeno-associated virus 2 (rAAV2) remains a promising candidate for gene therapy for cystic fibrosis (CF) lung disease, but due to limitations in the packaging capacity and the tropism of this virus with respect to the airways, strategies have evolved for packaging an rAAV2 genome (up to 5.8 kb) into the capsid of human bocavirus 1 (HBoV1) to produce a chimeric rAAV2/HBoV1 vector. Although a replication-incompetent HBoV1 genome has been established as a trans helper for capsid complementation, this system remains suboptimal with respect to virion yield. Here, a streamlined production system is described based on knowledge of the involvement of HBoV1 nonstructural (NS) proteins NS1, NS2, NS3, NS4, and NP1 in the process of virion production. The analyses reveal that NS1 and NS2 negatively impact virion production, NP1 is required to prevent premature termination of transcription of the cap mRNA from the native genome, and silent mutations within the polyadenylation sites of the cap coding sequence can eliminate this requirement for NP1. It is further shown that preventing the expression of all NS proteins significantly increases virion yield. Whereas the expression of capsid proteins VP1, VP2, and VP3 from a codon-optimized cap mRNA was highly efficient, optimal virion assembly, and thus potency, required enhanced VP1 expression, entailing a separate VP1 expression cassette. The final NS protein-free production system uses three-plasmid co-transfection of HEK293 cells, with one trans helper plasmid encoding VP1 and the AAV2 Rep proteins, and another encoding VP2-3 and components from adenovirus. This system yielded >16-fold more virions than the prototypic system, without reducing transduction potency. This increase in virion production is expected to facilitate greatly both research on the biology of rAAV2/HBoV1 and preclinical studies testing the effectiveness of this vector for gene therapy of CF lung disease in large animal models.

Genetics of critical contacts and clashes in the DNA packaging specificities of bacteriophages λ and 21.

The cos sites in λ and 21 chromosomes contain binding sites that recruit terminase to initiate DNA packaging. The small subunits of terminase, gpNu1 (λ) and gp1 (21), have winged helix-turn-helix DNA binding domains, where the recognition helixes differ in four of nine residues. To initiate packaging, the small subunit binds three R sequences in the cosB subsite. λ and 21 cannot package each other׳s DNA, due to recognition helix and R sequence differences. In λ and 21 cosBs, two bp, tri1 and tri2, are conserved in the R sequences yet differ between the phages; they are proposed to play a role in phage-specific packaging by λ and 21. Genetic experiments done with mixed and matched terminase and cosB alleles show packaging specificity depends on favorable contacts and clashes. These interactions indicate that the recognition helixes orient with residues 20 and 24 proximal to tri2 and tri1, respectively.

Fluorescently-labeled RNA packaging into HIV-1 particles: Direct examination of infectivity across central nervous system cell types.

HIV penetrates the central nervous system (CNS), and although it is clear that microglia and to a lesser extent astrocytes are infected, whether certain other cell types such as neurons are infected remains unclear. Here, we confirmed the finding that RNAs of both cellular and viral origins are present in native HIV-1 particles and exploited this phenomenon to directly examine HIV-1 infectivity of CNS cell types. Using in vitro transcribed mRNAs that were labeled with a fluorescent dye, we showed that these fluorescent mRNAs were packaged into HIV-1 particles by directly examining infected cells using fluorescence microscopy. Cells in culture infected with these labeled virions showed the fluorescent signals of mRNA labels by a distinct pattern of punctate, focal signals within the cells which was used to demonstrate that the CXCR4-tropic NL4-3 strain was able to enter microglia and to a lesser extent astrocytes, but not neurons. The strategy used in the present study may represent a novel approach of simplicity, robustness and reliability for versatile applications in HIV studies, such as the determination of infectivity across a broad range of cell types and within sub-populations of an individual cell type by direct visualization of viral entry into cells.