Viral Packaging Research Articles

Screen for Modulation of Nucleocapsid Protein Condensation Identifies Small Molecules with Anti-Coronavirus Activity.

Biomolecular condensates formed by liquid-liquid phase separation have been implicated in multiple diseases. Modulation of condensate dynamics by small molecules has therapeutic potential, but so far, few condensate modulators have been disclosed. The SARS-CoV-2 nucleocapsid (N) protein forms phase-separated condensates that are hypothesized to play critical roles in viral replication, transcription, and packaging, suggesting that N condensation modulators might have anti-coronavirus activity across multiple strains and species. Here, we show that N proteins from all seven human coronaviruses (HCoVs) vary in their tendency to undergo phase separation when expressed in human lung epithelial cells. We developed a cell-based high-content screening platform and identified small molecules that both promote and inhibit condensation of SARS-CoV-2 N. Interestingly, these host-targeted small molecules exhibited condensate-modulatory effects across all HCoV Ns. Some have also been reported to exhibit antiviral activity against SARS-CoV-2, HCoV-OC43, and HCoV-229E viral infections in cell culture. Our work reveals that the assembly dynamics of N condensates can be regulated by small molecules with therapeutic potential. Our approach allows for screening based on viral genome sequences alone and might enable rapid paths to drug discovery with value for confronting future pandemics.

Mechanistic insight into the efficient packaging of antigenomic S RNA into Rift Valley fever virus particles.

Rift Valley fever virus (RVFV), a bunyavirus, has a single-stranded, negative-sense tri-segmented RNA genome, consisting of L, M and S RNAs. An infectious virion carries two envelope glycoproteins, Gn and Gc, along with ribonucleoprotein complexes composed of encapsidated viral RNA segments. The antigenomic S RNA, which serves as the template of the mRNA encoding a nonstructural protein, NSs, an interferon antagonist, is also efficiently packaged into RVFV particles. An interaction between Gn and viral ribonucleoprotein complexes, including the direct binding of Gn to viral RNAs, drives viral RNA packaging into RVFV particles. To understand the mechanism of efficient antigenomic S RNA packaging in RVFV, we identified the regions in viral RNAs that directly interact with Gn by performing UV-crosslinking and immunoprecipitation of RVFV-infected cell lysates with anti-Gn antibody followed by high-throughput sequencing analysis (CLIP-seq analysis). Our data suggested the presence of multiple Gn-binding sites in RVFV RNAs, including a prominent Gn-binding site within the 3' noncoding region of the antigenomic S RNA. We found that the efficient packaging of antigenomic S RNA was abrogated in a RVFV mutant lacking a part of this prominent Gn-binding site within the 3' noncoding region. Also, the mutant RVFV, but not the parental RVFV, triggered the early induction of interferon-β mRNA expression after infection. These data suggest that the direct binding of Gn to the RNA element within the 3' noncoding region of the antigenomic S RNA promoted the efficient packaging of antigenomic S RNA into virions. Furthermore, the efficient packaging of antigenomic S RNA into RVFV particles, driven by the RNA element, facilitated the synthesis of viral mRNA encoding NSs immediately after infection, resulting in the suppression of interferon-β mRNA expression.

Evaluation of sequencing and PCR-based methods for the quantification of the viral genome formula

Viruses show great diversity in their genome organization. Multipartite viruses package their genome segments into separate particles, most or all of which are required to initiate infection in the host cell. The benefits of such seemingly inefficient genome organization are not well understood. One hypothesised benefit of multipartition is that it allows for flexible changes in gene expression by altering the frequency of each genome segment in different environments, such as encountering different host species. The ratio of the frequency of segments is termed the genome formula (GF). Thus far, formal studies quantifying the GF have been performed for well-characterised virus-host systems in experimental settings using RT-qPCR. However, to understand GF variation in natural populations or novel virus-host systems, a comparison of several methods for GF estimation including high-throughput sequencing (HTS) based methods is needed. Currently, it is unclear how HTS-methods compare a golden standard, such as RT-qPCR. Here we show a comparison of multiple GF quantification methods (RT-qPCR, RT-digital PCR, Illumina RNAseq and Nanopore direct RNA sequencing) using three host plants (Nicotiana tabacum, Nicotiana benthamiana, and Chenopodium quinoa) infected with cucumber mosaic virus (CMV), a tripartite RNA virus. Our results show that all methods give roughly similar results, though there is a significant method effect on genome formula estimates. While the RT-qPCR and RT-dPCR GF estimates are congruent, the GF estimates from HTS methods deviate from those found with PCR. Our findings emphasize the need to tailor the GF quantification method to the experimental aim, and highlight that it may not be possible to compare HTS and PCR-based methods directly. The difference in results between PCR-based methods and HTS highlights that the choice of quantification technique is not trivial.

Role of DNA sliding friction and nonequilibrium dynamics in viral DNA ejection and packaging.

In many viruses DNA is packaged to high densities, resulting in high “internal force” inside the capsid shell. This helps drive DNA ejection through the portal channel when viruses infect cells. Imaging of the in vitro ejection of fluorescent-labeled DNA from phages T5 and lambda found that it was initially slow despite the internal force being highest when the capsid is full. This was attributed to hydrodynamic drag on the tightly-packed DNA, but this hypothesis has not been tested. Here we introduce a new method for probing the DNA mobility by using optical tweezers to pull it out from phage phi29 capsids. This allows us to measure the dynamics with higher precision, avoid using DNA-binding dyes that alter the dynamics, and apply variable forces. We measure even slower DNA exit than reported previously, a steeper, exponential velocity increase as the first 20% of the genome length exits, and find that exit velocity is not proportional to driving force as expected for hydrodynamic friction. Rather, our findings are consistent with theoretical predictions for DNA-DNA sliding friction. We also observe large heterogeneity in the exit dynamics and stochastic pausing, indicating that ejection is a nonequilibrium process, in qualitative agreement with simulations. Features of the pausing suggest it is related to the phenomenon of “clogging” in soft-matter physics, which occurs in the flow of disordered materials through constrictions. Our results indicate that intramolecular friction and nonequilibrium pausing control the kinetics of ejection but that this friction does not significantly affect DNA packaging.

Regulation of the phage lambda viral packaging motor's grip and DNA end-clamp mechanism

Many viruses utilize ATP-powered motors to package DNA into viral capsids. Here we use single DNA manipulation with optical tweezers and rapid solution exchange to interrogate the dynamics of DNA gripping by the phage lambda motor. Binding of poorly hydrolyzed ATP analogs results in persistent gripping and when slips occasionally occur, the release velocity is very slow, indicating significant friction between the motor and DNA. ADP induces more frequent transitions between gripping and faster slipping. Independent of nucleotides, an “end clamp” mechanism arrests slipping when the end of the DNA is about to exit the capsid. These features are qualitatively similar to those exhibited by the phage T4 motor, although there are some notable differences. Specifically, there are quantitative differences in the DNA grip/release kinetics and their dependence on nucleotides and applied force. But the most striking difference is that there are frequent long pauses during DNA exit from the capsid even in the absence of nucleotides. This pausing occurs at low capsid filling and does not increase with length of DNA packaged, indicating that it is not caused by jamming/knotting of the packaged DNA, but is attributable to fluctuation of apo-state motor subunits into a DNA gripping conformation. This finding supports our previous hypothesis that pauses which are frequently observed during packaging are caused by fluctuation of motor subunits into such a conformation. Overall, compared with T4, the lambda motor exhibits stronger interactions with DNA, which may be attributable to the small terminase subunit in the lambda packaging motor complex.

Energetic and frustration analysis of SARS-CoV-2 nucleocapsid protein mutations

ABSTRACT The ongoing COVID-19 spreads worldwide with the ability to evolve in diverse human populations. The nucleocapsid (N) protein is one of the mutational hotspots in the SARS-CoV-2 genome. The N protein is an abundant RNA-binding protein critical for viral genome packaging. It comprises two large domains including the N-terminal domain (NTD) and the C-terminal domain (CTD) linked by the centrally located linker region. Mutations in N protein have been reported to increase the severity of disease by modulating viral transmissibility, replication efficiency as well as virulence properties of the virus in different parts of the world. To study the effect of N protein missense mutations on protein stability, function, and pathogenicity, we analyzed 228 mutations from each domain of N protein. Further, we have studied the effect of mutations on local residual frustration changes in N protein. Out of 228 mutations, 11 mutations were predicted to be deleterious and destabilized. Among these mutations, R32C, R191C, and R203 M mutations fall into disordered regions and show significant change in frustration state. Overall, this work reveals that by altering the energetics and residual frustration, N protein mutations might affect the stability, function, and pathogenicity of the SARS-CoV-2.

Characterization of a conserved cysteine residue in the papillomavirus E2 protein

Background:Human papillomaviruses (HPVs) are DNA tumor viruses that infect cutaneous and mucosal epithelium. While most infections are self-limiting, a small subset that infects the mucosal epithelium progresses to cancer. All papillomaviruses encode the protein E2 which regulates viral transcription and replication; a highly conserved cysteine residue in the DNA contact helix of E2 plays an unknown role. Previous research suggests the residue is not necessary for replication or binding to DNA. We hypothesize that post-translational modification of this conserved cysteine residue leads to release of viral DNA during packaging of progeny virions.
 Methods:Mutations of the murine papillomavirus conserved E2 C307 residue to serine and phenylalanine were used to investigate its role in E2 function. C33A, HPV negative cervical cancer cells, were transfected with an E2-responsive luciferase reporter and either wild type or mutant C307 E2 vectors; luciferase assays were performed 48 hours post-transfection to assess transcriptional activity. Whole cell lysates from overexpressed C307 mutants were separated by SDS-PAGE and immunoblotted to assess expression levels relative to wild type. To examine protein localization, C33A cells were transfected with equal amounts of wild type or mutant E2 and fixed 48 hours post-transfection for immunofluorescence.
 Results:C307S and C307F mutants are both capable of weakly activating transcription. Overexpression of the mutants resulted in a dose dependent increase in transcriptional activity. Both mutants are expressed at levels comparable to wild type E2 and are correctly localized to the nucleus.
 Conclusion/Impact:The deficient transcription function displayed by the C307 mutants cannot be explained by poor expression or mislocalization. Continued study of this conserved cysteine will help to further understanding of papillomavirus biology and may offer insight into novel avenues for treatment or prevention of HPV-associated cancers.

Initiation of HIV-1 Gag lattice assembly is required for recognition of the viral genome packaging signal.

The encapsidation of HIV-1 gRNA into virions is enabled by the binding of the nucleocapsid (NC) domain of the HIV-1 Gag polyprotein to the structured viral RNA packaging signal (Ψ) at the 5' end of the viral genome. However, the subcellular location and oligomeric status of Gag during the initial Gag-Ψ encounter remain uncertain. Domains other than NC, such as capsid (CA), may therefore indirectly affect RNA recognition. To investigate the contribution of Gag domains to Ψ recognition in a cellular environment, we performed protein-protein crosslinking and protein-RNA crosslinking immunoprecipitation coupled with sequencing (CLIP-seq) experiments. We demonstrate that NC alone does not bind specifically to Ψ in living cells, whereas full-length Gag and a CANC subdomain bind to Ψ with high specificity. Perturbation of the Ψ RNA structure or NC zinc fingers affected CANC:Ψ binding specificity. Notably, CANC variants with substitutions that disrupt CA:CA dimer, trimer, or hexamer interfaces in the immature Gag lattice also affected RNA binding, and mutants that were unable to assemble a nascent Gag lattice were unable to specifically bind to Ψ. Artificially multimerized NC domains did not specifically bind Ψ. CA variants with substitutions in inositol phosphate coordinating residues that prevent CA hexamerization were also deficient in Ψ binding and second-site revertant mutants that restored CA assembly also restored specific binding to Ψ. Overall, these data indicate that the correct assembly of a nascent immature CA lattice is required for the specific interaction between Gag and Ψ in cells.

Monosynaptic restriction of the anterograde herpes simplex virus strain H129 for neural circuit tracing.

Identification of synaptic partners is a fundamental task for systems neuroscience. To date, few reliable techniques exist for whole brain labeling of downstream synaptic partners in a cell-type-dependent and monosynaptic manner. Herein, we describe a novel monosynaptic anterograde tracing system based on the deletion of the gene UL6 from the genome of a cre-dependent version of the anterograde Herpes Simplex Virus 1 strain H129. Given that this knockout blocks viral genome packaging and thus viral spread, we reasoned that co-infection of a HSV H129 ΔUL6 virus with a recombinant adeno-associated virus expressing UL6 in a cre-dependent manner would result in monosynaptic spread from target cre-expressing neuronal populations. Application of this system to five nonreciprocal neural circuits resulted in labeling of neurons in expected projection areas. While some caveats may preclude certain applications, this system provides a reliable method to label postsynaptic partners in a brain-wide fashion.

An Epidemiological Analysis of Severe Acute Repiratory Syndrome Coronavirus 2 Genome Sequencing: A Hospital-based Retrospective Study

Introduction: Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is a positive-sense enveloped single-stranded Ribonucleic Acid (RNA) virus. Structural proteins help the virus package its RNA, while other proteins enable viral replication by facilitating host entry. Through constant mutation, the virus changes its emergence profile, and combinations of mutations can lead to increased transmissibility and receptor binding capacity, altering its surface structure. Whole genome sequencing is an important tool for studying these changes. In this study, the authors report on the genome sequencing of patients who tested positive for SARS-CoV-2 using real-time Reverse TranscriptasePolymerase Chain Reaction (RT-PCR) tests. Aim: To identify the different lineages circulating in specific districts of West Bengal, India, and perform an epidemiological analysis of the patients to control disease severity during the second wave of Coronavirus Disease-2019 (COVID-19). Materials and Methods: This retrospective hospital-based study was conducted at the Virus Research and Diagnostic Laboratory (VRDL) at RG Kar Medical College and Hospital, West Bengal, India from January 2021 to October 2021. Data collection started in January 2021 and was analysed in October 2021. Nasopharyngeal and oropharyngeal swabs were taken from patients with SARS-CoV-2, and the samples were tested using RT-PCR. Positive samples were sent to the Regional Virus Research and Diagnostic Laboratory-National Institute of Cholera and Enteric Diseases (NICED) for sequencing. Samples were collected from patients in different districts of West Bengal who were reported to the VRDL of RG Kar Medical College and Hospital. A total of 172,550 samples were tested for SARSCoV-2, and out of 13,764 positive samples, 230 were sent for genome sequencing. The primary inclusion criteria were SARSCoV-2 positive patients with CT values between 25 and 30 who were vaccinated. Patient information, address, gene variant, and gene mutation of the samples were analysed. Statistical analysis was performed using t-test with Statistical Package for Social Sciences (SPSS) software. Results: Whole-genome sequencing helped identify new trends and their prevalence in specific areas, aiding in prevention efforts. The most common type of mutation observed after double vaccination was the delta variant (B.1.617.2), followed by the kappa variant (B.1.617.1) and the alpha variant (B.1.1.7). Conclusion: Epidemiological genome sequencing studies help to identify emerging and changing viral trends, contributing to the mitigation of the spread of new variants. The delta, kappa, and alpha variants were the three primary sequences discovered in this study. The identification of these lineages facilitates the design of novel vaccines and diagnostic medications. Continuous monitoring and analysis of sequences from new cases in India and other affected countries are crucial for understanding the genetic evolution and substitution rates of SARS-CoV-2.

Loss of furin site enhances SARS-CoV-2 spike protein pseudovirus infection

BackgroundSARS-CoV-2 has a significant impact on healthcare systems all around the world. Due to its high pathogenicity, live SARS-CoV-2 must be handled under biosafety level 3 conditions. Pseudoviruses are useful virological tools because of their safety and versatility, but the low titer of these viruses remains a limitation for their more comprehensive applications. MethodHere, we constructed a Luc/eGFP based on a pseudotyped lentiviral HIV-1 system to transduce SARS-CoV-2 S glycoprotein to detect cell entry properties and cellular tropism. ResultsThe furin cleavage site deletion of the S protein removed (SFko) can help SARS-CoV-2 S to be cleaved during viral packaging to improve infection efficiency. The furin cleavage site in SARS-CoV-2-S mediates membrane fusion and SFko leads to an increased level of S protein and limits S1/S2 cleavage to enhance pseudovirus infection in cells. Full-length S (SFL) pseudotyped with N, M, and E helper packaging can effectively help SFL infect cells. Finally, pseudotyped SFko particles were successfully used to detect neutralizing antibodies in RBD protein-immunized mouse serum. ConclusionOverall, our study indicates a series of modifications that result in the production of relatively high-titer SARS-COV-2 pseudo-particles that may be suitable for the detection of neutralizing antibodies from COVID-19 patients.

Molecular Mechanism of Adenovirus Late Protein L4-100K Chaperones the Trimerization of Hexon.

Assembly of the adenovirus capsid protein hexon depends on the assistance of the molecular chaperone L4-100K. However, the chaperone mechanisms remain unclear. In this study, we found that L4-100K was involved in the hexon translation process and could prevent hexon degradation by the proteasome in cotransfected human cells. Two nonadjacent domains, 84-133 and 656-697, at the N-terminal and C-terminal regions of human adenovirus type 5 L4-100K, respectively, were found to be crucial and cooperatively responsible for hexon trimer expression and assembly. These two chaperone-related domains were conserved in the sequence of L4-100K and in the function of hexon assembly across different adenovirus serotypes. Different degrees of cross-activity of hexon trimerization with different serotypes were detected in subgroups B, C, and D, which were proven to be controlled by the interaction between the C-terminal chaperone-related domain of L4-100K and hypervariable regions (HVR) of hexon. Additionally, HVR-chimeric hexon mutants were successfully assembled with the assistance of the 1-697 mutant. Structural analysis of 656-697 by nuclear magnetic resonance and structural prediction of L4-100K using Robetta showed that the two conserved domains are mainly composed of α-helices and are located on the surface of the highly folded core region. Our research provides a more complete understanding of hexon assembly and guidance for the development of hexon-chimeric adenovirus vectors that will be safer, smarter, and more efficient. IMPORTANCE Adenovirus vectors have been widely used in clinical trials of vaccines and gene therapy, although some deficiencies remain. Chimeric modification of the hexon was expected to improve the potency of preexisting immune evasion and targeting, but in many cases, viral packaging is prevented by the inability of the chimeric hexon to assemble correctly. So far, few studies have examined the mechanisms of hexon trimer assembly. Here, we show how the chaperone protein L4-100K contributes to the assembly of the adenovirus capsid protein hexon, and these data will provide a guide for novel adenovirus vector design and development, as we desired.

An Infectious Virus‐like Particle Built on a Programmable Icosahedral DNA Framework**

AbstractViral genomes can be compressed into a near‐spherical nanochamber to form infectious particles. In order to mimic the virus morphology and packaging behavior, we invented a programmable icosahedral DNA nanoframe with enhanced rigidity and encapsulated the phiX174 bacteriophage genome. The packaging efficiency could be modulated through specific anchoring strands adjustment, and the trapped phage genome remained accessible for enzymatic operations. Moreover, the packed complex could infect Escherichia coli (E. coli) cells through bacterial uptake to produce plaques. This rigid icosahedral DNA architecture demonstrated a versatile platform to develop virus mimetic particles for convenient functional nucleic acid entrapment, manipulation and delivery.

An Infectious Virus-like Particle Built on a Programmable Icosahedral DNA Framework.

Viral genomes can be compressed into a near-spherical nanochamber to form infectious particles. In order to mimic the virus morphology and packaging behavior, we invented a programmable icosahedral DNA nanoframe with enhanced rigidity and encapsulated the phiX174 bacteriophage genome. The packaging efficiency could be modulated through specific anchoring strands adjustment, and the trapped phage genome remained accessible for enzymatic operations. Moreover, the packed complex could infect Escherichia coli (E. coli) cells through bacterial uptake to produce plaques. This rigid icosahedral DNA architecture demonstrated a versatile platform to develop virus mimetic particles for convenient functional nucleic acid entrapment, manipulation and delivery.

Structural basis of sequence-specific RNA recognition by the antiviral factor APOBEC3G

An essential step in restricting HIV infectivity by the antiviral factor APOBEC3G is its incorporation into progeny virions via binding to HIV RNA. However, the mechanism of APOBEC3G capturing viral RNA is unknown. Here, we report crystal structures of a primate APOBEC3G bound to different types of RNAs, revealing that APOBEC3G specifically recognizes unpaired 5’-AA-3’ dinucleotides, and to a lesser extent, 5’-GA-3’ dinucleotides. APOBEC3G binds to the common 3’A in the AA/GA motifs using an aromatic/hydrophobic pocket in the non-catalytic domain. It binds to the 5’A or 5’G in the AA/GA motifs using an aromatic/hydrophobic groove conformed between the non-catalytic and catalytic domains. APOBEC3G RNA binding property is distinct from that of the HIV nucleocapsid protein recognizing unpaired guanosines. Our findings suggest that the sequence-specific RNA recognition is critical for APOBEC3G virion packaging and restricting HIV infectivity.

AAV2-hAADC (Eladocagene Exuparvovec) Biodistribution and Expression: Superiority of Intraputaminal versus Intracerebroventricular and Intrathecal (Lumbar) Routes of Administration.

Aromatic L-amino acid decarboxylase deficiency is a genetic disorder of enzyme loss with decreased neurotransmitter synthesis, and it is characterized by symptoms of impaired motor development and cognitive function, hypotonia, dystonia, and oculogyric crises. Though symptomatic severity varies, the majority of patients experience severe motor impairments, including an inability to sit, stand, or walk. One approved therapy for Aromatic L-amino acid decarboxylase deficiency involves intraputaminal delivery of an adeno-associated virus packaging the human Aromatic L-amino acid decarboxylase enzyme (hAADC) cDNA. The objective of this study in monkeys was to determine the acceptability of ICV/IT as minimally invasive dosing options by evaluating hAADC biodistribution and expression following intraputaminal, intracerebroventricular (ICV), or intrathecal (IT, lumbar) administration. Results show that all routes produced comparable CSF transgene levels and were well-tolerated. The intraputaminal route yielded the highest levels of transgene-derived mRNA expression in the putamen, caudate, and globus pallidus, while expression levels in the spinal cord and dorsal root ganglia (DRG, a target of special toxicological concern) were undetectable. In contrast, the highest transgene levels in ICV/IT groups were observed in the spinal cord and DRG, but levels were too low to result in expression in the putamen, caudate, and globus pallidus. Unlike ICV/IT, the intraputaminal route produced no transgene in blood, suggesting a lower likelihood of off-target toxicities. Additionally, intraputaminal dosing resulted in the lowest anti-AAV2 antibody (anti-drug antibody) levels. Together, these data demonstrate the superiority of intraputaminal administration over ICV/IT routes in achieving AAV2-hAADC transgene DNA distribution and mRNA expression in target therapeutic areas while minimizing risk of toxicity.

Validation of Promoters and Codon Optimization on CRISPR/Cas9-Engineered Jurkat Cells Stably Expressing αRep4E3 for Interfering with HIV-1 Replication

Persistent and efficient therapeutic protein expression in the specific target cell is a significant concern in gene therapy. The controllable integration site, suitable promoter, and proper codon usage influence the effectiveness of the therapeutic outcome. Previously, we developed a non-immunoglobulin scaffold, alpha repeat protein (αRep4E3), as an HIV-1 RNA packaging interference system in SupT1 cells using the lentiviral gene transfer. Although the success of anti-HIV-1 activity was evidenced, the integration site is uncontrollable and may not be practical for clinical translation. In this study, we use the CRISPR/Cas9 gene editing technology to precisely knock-in αRep4E3 genes into the adeno-associated virus integration site 1 (AAVS1) safe harbor locus of the target cells. We compare the αRep4E3 expression under the regulation of three different promoters, including cytomegalovirus (CMV), human elongation factor-1 alpha (EF1α), and ubiquitin C (UbC) promoters with and without codon optimization in HEK293T cells. The results demonstrated that the EF1α promoter with codon-optimized αRep4E3mCherry showed higher protein expression than other promoters with non-optimized codons. We then performed a proof-of-concept study by knocking in the αRep4E3mCherry gene at the AAVS1 locus of the Jurkat cells. The results showed that the αRep4E3mCherry-expressing Jurkat cells exhibited anti-HIV-1 activities against HIV-1NL4-3 strain as evidenced by decreased capsid (p24) protein levels and viral genome copies as compared to the untransfected Jurkat control cells. Altogether, our study demonstrates that the αRep4E3 could interfere with the viral RNA packaging and suggests that the αRep4E3 scaffold protein could be a promising anti-viral molecule that offers a functional cure for people living with HIV-1.

CryoEM structure and assembly mechanism of a bacterial virus genome gatekeeper

Numerous viruses package their dsDNA genome into preformed capsids through a portal gatekeeper that is subsequently closed. We report the structure of the DNA gatekeeper complex of bacteriophage SPP1 (gp612gp1512gp166) in the post-DNA packaging state at 2.7 Å resolution obtained by single particle cryo-electron microscopy. Comparison of the native SPP1 complex with assembly-naïve structures of individual components uncovered the complex program of conformational changes leading to its assembly. After DNA packaging, gp15 binds via its C-terminus to the gp6 oligomer positioning gp15 subunits for oligomerization. Gp15 refolds its inner loops creating an intersubunit β-barrel that establishes different types of contacts with six gp16 subunits. Gp16 binding and oligomerization is accompanied by folding of helices that close the portal channel to keep the viral genome inside the capsid. This mechanism of assembly has broad functional and evolutionary implications for viruses of the prokaryotic tailed viruses-herpesviruses lineage.

Intravenous Lentiviral Gene Therapy for Hemophilia a: Endothelial-Specific Expression and Increased Glycosylation for Improved FVIII Function

Background: Hemophilia A (HA) is an X-linked monogenic coagulation disorder in the intrinsic coagulation cascade resulting from deficiency of the factor VIII (FVIII, F8) gene. The current treatment of HA is based on protein replacement therapy (PRT) with limitations of short half-life, high cost, and life-time requirement of the treatment. Gene therapy has become a promising treatment option for HA. Methods: An advanced lentiviral vector (LV) system for intravenous (iv) F8 gene therapy has been established based on a novel B domain deleted (BDD) F8 gene, which restored all N-glycosylation sites (F8-299) in the B domain with increased coagulation function (DOI:10.1016/j.jbc.2021.101397). LV-F8-299 containing a universal (EF1a) or endothelial-specific (VEC) promoter was investigated in vitro using a human endothelial cell line, EA-hy926, and biologically and immunologically characterized in vivo in F8 knockout (KO) mice and in a non-human primate. Results: We found that LV-VEC-F8-299 (VEC-299) exhibited higher virus packaging (100 folds) and transduction efficiencies than EF1a-299. The F8 protein expression was verified in EA-hy926 by RT-PCR, Western blot and ELISA. We detected F8 activities close to 4 folds above the normal plasma level from the EF1a-299 transduced cells, whereas the VEC-299 expressed only 0.1 fold of the normal plasma F8 level. In vivo studies in F8 KO mice via iv injection of LVs after non-myeloablative radiation conditioning illustrated variable phenotypic corrections as well as anti-F8 immune responses from the two LVs. The iv gene therapy of EF1a-299 and VEC-299 LVs illustrated therapeutic F8 activities over time, in the range of 90% and 40%, respectively, in 60 days. Importantly, VEC-299 steadily reached a very high level (180%) of F8 activities, whereas EF-299 dropped to 3% after 120 days (Figure A). Kinetic analyses of anti-F8 IgG and inhibitor titer showed that the VEC-299 treated mice exhibited the lowest F8 inhibitory immune response over time. We next assessed the distribution of LV F8 gene in a macaque monkey (Macaca fascicularis). The monkey was chemo-conditioned with methylprednisolone (10 mg/kg) and prednisone (2 mg/kg) and iv injected with 1x109 TU/kg LV-VEC-299. The vector copy number (VCN) in body fluids was closely monitored by qPCR. From day 1 to week 14 after injection, the VCN in the blood remained stable (~1%), while in feces and saliva was high on day 1 (~3%), and in urine reached peak (~5%) on day 7, but all of which decreased with time. The VCN in body fluids (blood, urine, faces, saliva and tear) became nearly undetectable after 14 weeks. A second LV injection of 1x109TU/kg LV-VEC-299 was performed using a modified non-myeloablative conditioning with busulfan (2 mg/kg), cyclophosphamide (40 mg/kg) and dexamethasone (3 mg/kg). The VCN in the blood reached 20% from day 1 to day 7, then reduced to 5% and remained stable to week 6 (Figure B). The blood biochemical analyses including Alanine Transaminase (ALT), Aspartate Transaminase (AST), Total Protein (TP) and Albumin (ALB) remained in the normal range before and after treatment. Conclusion: These results demonstrated that the LV-VEC-299 exhibited delivery consistency, high coagulation function and low immunogenicity. The in vivo study supported that iv LV-VEC-299 gene therapy could be a safe, convenient and effective HA gene therapy strategy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Antimicrobial peptides: natural or synthetic defense peptides against HBV and HCV infections.

According to the literature, treatment of HCV and HBV infections faces challenges due to problems such as the emergence of drug-resistant mutants, the high cost of treatment, and the side effects of current antiviral therapy. Antimicrobial peptides (AMPs), a group of small peptides, are a part of the immune system and are considered as an alternative treatment for microbial infections. These peptides are water-soluble with amphiphilic (hydrophilic and hydrophobic surfaces) characteristics. AMPs are produced by a wide range of organisms including both prokaryotic and eukaryotic cells. The antiviral mechanisms of AMPs include inhibiting virus entry, inhibiting intracellular virus replication, inhibiting intracellular viral packaging, and inducing immune responses. In addition, AMPs are a new generation of antiviral biomolecules that have very low toxicity for human host cells, particularly liver cell lines. AMPs can be considered as one of the most important strategies for developing new adjuvant drugs in the treatment of HBV and HCV infections. In the present study, several groups of AMPs (with a net positive charge) such as Human cathelicidin, Claudin-1, Defensins, Hepcidin, Lactoferrin, Casein, Plectasin, Micrococcin P1, Scorpion venom, and Synthetic peptides were reviewed with antiviral properties against HBV and HCV.