Genome Encapsidation Research Articles

Intracellular localization of adeno‐associated viral proteins expressed in insect cells

Production of vectors derived from adeno-associated virus (AAVv) in insect cells represents a feasible option for large-scale applications. However, transducing particles yields obtained in this system are low compared with total capsid yields, suggesting the presence of genome encapsidation bottlenecks. Three components are required for AAVv production: viral capsid proteins (VP), the recombinant AAV genome, and Rep proteins for AAV genome replication and encapsidation. Little is known about the interaction between the three components in insect cells, which have intracellular conditions different to those in mammalian cells. In this work, the localization of AAV proteins in insect cells was assessed for the first time with the purpose of finding potential limiting factors. Unassembled VP were located either in the cytoplasm or in the nucleus. Their transport into the nucleus was dependent on protein concentration. Empty capsids were located in defined subnuclear compartments. Rep proteins expressed individually were efficiently translocated into the nucleus. Their intranuclear distribution was not uniform and differed from VP distribution. While Rep52 distribution and expression levels were not affected by AAV genomes or VP, Rep78 distribution and stability changed during coexpression. Expression of all AAV components modified capsid intranuclear distribution, and assembled VP were found in vesicles located in the nuclear periphery. Such vesicles were related to baculovirus infection, highlighting its role in AAVv production in insect cells. The results obtained in this work suggest that the intracellular distribution of AAV proteins allows their interaction and does not limit vector production in insect cells.

Assembly of the Adenoviral IVa2 and L4-22K Proteins on a Viral DNA Packaging Sequence

The human adenoviral (Ad) life cycle is divided into early and late phases.In the early phase, the virus optimizes the host environment for its replication. Once the virus starts replicating its genome, the life cycle enters the late phase. Many viral proteins, including capsid proteins, are encoded in the viral late gene region and expressed during the late phase. At the end of the late phase, the viral genomes are encapsidated to form the infectious viral particles. This encapsidation process is a critical step which requires threshold levels of progeny viral genomes and capsid proteins. To optimize assembly of viral particles, the viral late gene transcription and genome encapsidation processes must proceed as ordered events, i.e. they must be precisely regulated. In this scenario, the activation of the encapsidation pathway behaves like a classic genetic switch. Previous studies have shown that two viral proteins, IVa2 and L4-22K, recognize a specific DNA binding site within both the promoter region of viral late genes and the packaging sequence (PS) DNA which are required for encapsidation. However, the DNA architecture is different in these two regions. Multiple copies of this specific binding site have been identified in the PS DNA while only a single copy has been identified in the late gene promoter. We hypothesize the interaction of IVa2, L4-22K, and these specific DNA binding sites regulates the viral decision to initiate encapsidation. To develop a predictive model to describe this process, we have initiated a physical-chemical study of the assembly of the IVa2 and L4-22K proteins on these specific DNA sequences.

VP4 Protein Appears as a Product of RNA Encapsidation in Triatoma Virus (TRV)

Triatoma Virus (TrV) is a non-enveloped +ssRNA virus belonging to the insect virus family Dicistroviridae. Its non-enveloped capsid is composed of four proteins named VP1-VP4, plus the minoritary, uncleaved protein precursor VP0, which comprises VP4 and VP3. While the smaller protein VP4 (5.5 kDa versus around 30 kDa for VP1-3) remained undetected in past studies by standard biochemical analyses, the icosahedral (T=1, pseudo-T=3) crystallographic structure of TrV (PDB ID: 3NAP) raised even more suspicions about its existence since no electron density could be attributed to this peptide. In the present work, mass spectrometry (MS) and Tricine-SDS gel electrophoresis were used to detect the previously elusive capsid protein VP4. Its cleavage sites were established by sequencing the N-terminus of the protein precursor and MS, and its stoichiometry with respect to the other major capsid proteins (VP1-3) was found to be 1:1. We also characterized the polypeptides comprising the naturally occurring non-infectious empty capsids, i.e., RNA-free TrV particles. The empty particles were composed of VP0-VP3 plus at least seven additional polypeptides, which were identified as products of the capsid precursor polyprotein (P1). We conclude that VP4 protein appears as a product of RNA encapsidation, and that defective processing of capsid proteins precludes genome encapsidation. Our results also suggest that the TrV capsid can be built without the scaffolding aid of the nucleic acid.

Comparative Production Analysis of Three Phlebovirus Nucleoproteins under Denaturing or Non-Denaturing Conditions for Crystallographic Studies

Nucleoproteins (NPs) encapsidate the Phlebovirus genomic (-)RNA. Upon recombinant expression, NPs tend to form heterogeneous oligomers impeding characterization of the encapsidation process through crystallographic studies. To overcome this problem, we set up a standard protocol in which production under both non-denaturing and denaturing/refolding conditions can be investigated and compared. The protocol was applied for three phlebovirus NPs, allowing an optimized production strategy for each of them. Remarkably, the Rift Valley fever virus NP was purified as a trimer under native conditions and yielded protein crystals whereas the refolded version could be purified as a dimer. Yields of trimeric Toscana virus NP were higher from denaturing than from native condition and lead to crystals. The production of Sandfly Fever Sicilian virus NP failed in both protocols. The comparative protocols described here should help in rationally choosing between denaturing or non-denaturing conditions, which would finally result in the most appropriate and relevant oligomerized protein species. The structure of the Rift Valley fever virus NP has been recently published using a refolded monomeric protein and we believe that the process we devised will contribute to shed light in the genome encapsidation process, a key stage in the viral life cycle.

Capsid protein identification and analysis of mature Triatoma virus (TrV) virions and naturally occurring empty particles

Triatoma virus (TrV) is a non-enveloped +ssRNA virus belonging to the insect virus family Dicistroviridae. Mass spectrometry (MS) and gel electrophoresis were used to detect the previously elusive capsid protein VP4. Its cleavage sites were established by sequencing the N-terminus of the protein precursor and MS, and its stoichiometry with respect to the other major capsid proteins (VP1–3) was found to be 1:1. We also characterized the polypeptides comprising the naturally occurring non-infectious empty capsids, i.e., RNA-free TrV particles. The empty particles were composed of VP0–VP3 plus at least seven additional polypeptides, which were identified as products of the capsid precursor polyprotein. We conclude that VP4 protein appears as a product of RNA encapsidation, and that defective processing of capsid proteins precludes genome encapsidation.

Elucidation of functional domains of Chandipura virus Nucleocapsid protein involved in oligomerization and RNA binding: Implication in viral genome encapsidation

Chandipura virus, a member of the vesiculovirus genera, has been recently recognized as an emerging human pathogen. Previously, we have shown that Chandipura virus Nucleocapsid protein N is capable of binding to both specific viral leader RNA as well as non-viral RNA sequences, albeit in distinct monomeric and oligomeric states, respectively. Here, we distinguish the regions of N involved in oligomerization and RNA binding using a panel of deletion mutants. We demonstrate that deletion in the N-terminal arm completely abrogates self-association of N protein. Monomer N specifically recognizes viral leader RNA using its C-terminal 102 residues, while oligomerization generates an additional RNA binding surface involving the N-terminal 320 amino acids of N overlapping with a protease resistant core that is capable of forming nucleocapsid like structure and also binding heterogeneous RNA sequences. Finally, we propose a model to explain the mechanism of genome encapsidation of this important human pathogen.

Nuclear Magnetic Resonance Structure of the Prohead RNA E-Loop Hairpin,

The Bacillus subtilis phage phi29 packaging motor requires prohead RNA for genome encapsidation. The nuclear magnetic resonance structure of the prohead RNA E-loop hairpin, r(5'AUUGAGUU), is presented and compared to predictions from MC-SYM. The prohead RNA E-loop hairpins contain sequences similar to rRNA hairpins. Comparison of predicted and experimentally determined prohead and ribosomal hairpin structures reveals that sequence similarity is a stronger determinant of hairpin structural similarity than grouping similar types of RNA. All the hairpins contain a U-turn motif but differ in the first noncanonical pair and backbone orientation. These structures provide benchmarks for further improvements in RNA structure predictions from sequence.

MicroRNA Antagonism of the Picornaviral Life Cycle: Alternative Mechanisms of Interference

In addition to modulating the function and stability of cellular mRNAs, microRNAs can profoundly affect the life cycles of viruses bearing sequence complementary targets, a finding recently exploited to ameliorate toxicities of vaccines and oncolytic viruses. To elucidate the mechanisms underlying microRNA-mediated antiviral activity, we modified the 3′ untranslated region (3′UTR) of Coxsackievirus A21 to incorporate targets with varying degrees of homology to endogenous microRNAs. We show that microRNAs can interrupt the picornavirus life-cycle at multiple levels, including catalytic degradation of the viral RNA genome, suppression of cap-independent mRNA translation, and interference with genome encapsidation. In addition, we have examined the extent to which endogenous microRNAs can suppress viral replication in vivo and how viruses can overcome this inhibition by microRNA saturation in mouse cancer models.

Molecular Dynamics and DFT Study on HIV-1 Nucleocapsid Protein-7 in Complex with Viral Genome

The HIV-1 nucleocapsid protein-7 (NCp7) is a highly basic, small zinc-binding protein involved in both deoxyribonucleic (DNA) and ribonucleic (RNA) acids annealing and in viral particle maturation including genome encapsidation, with an additional chaperoning activity toward reverse transcriptase by promoting the two obligatory strand transfers during reverse transcription. Because of its interaction with highly conserved sequences of the HIV-1 genome, NCp7 is being considered a new potential drug target, resistant to mutation, for antiviral activity. The high flexibility of this protein has, however, limited the identification of structural determinants involved in the interaction with stranded sequences of DNA and RNA. Here, we provide a quantum mechanics (density functional theory) study of the zinc-binding motifs and a molecular dynamics simulation of the protein in complex with RNA and DNA, starting from available nuclear magnetic resonance (NMR) structures. Results show that the interaction between the NCp7 and the viral genome is probably based on electrostatic interactions due to a cluster of basic residues, which is reinforced by the exploitation of nonelectrostatic contacts that further stabilize the complexes. Moreover, a possible mechanism for DNA destabilization that involves amino acids T24 and R26 is also hypothesized. Finally, a network of hydrophobic and hydrogen-bond interactions for the stabilization of complexes with DNA and, especially, with RNA is described here for the first time. The complexes between NCp7 and both DNA and RNA, resulting from computer simulations, showed structural properties that are in agreement with most of the currently available molecular biology evidence and could be considered as reliable models (better than NMR structures currently available) for subsequent structure-based ligand design approaches.

Evidence for the Failure of Adeno-associated Virus Serotype 5 to Package a Viral Genome ≥8.2 kb

Limited packaging capacity hinders adeno-associated virus (AAV) gene therapy. A recent study seems to have provided a solution to this problem. Allocca et al. reported that AAV-5 could package an 8.9 kb vector genome. Here we tested whether this approach can be used to deliver a large genome for Duchenne muscular dystrophy (DMD) gene therapy. We first evaluated AAV-5 packaging of an 8.2 kb genome. This vector carries two independent reporter gene cassettes, one for alkaline phosphatase (AP) and another for LacZ. Viral yield was log-fold lower than that of a regular AAV-5. Nevertheless, both AP and LacZ genes were detected in purified virus. Injection to dystrophic muscle resulted in both AP and LacZ expression. On electron microscopy, virion structure appeared normal. Surprisingly, we did not find the full-length single-stranded viral genome by alkaline gel electrophoresis. Neither did we see the full-length double-stranded replication forms in adenovirus coinfected cells. We suspect that AP and LacZ expression may have come from partially packaged 5' or 3'-half of the genome. Additional studies revealed failure of AAV-5 to package and express an 8.7 kb minidystrophin gene cassette. In summary, our results do not support the extraordinary packaging capacity of AAV-5.

The relationship between mutation frequency and replication strategy in positive-sense single-stranded RNA viruses.

For positive-sense single-stranded RNA virus genomes, there is a trade-off between the mutually exclusive tasks of transcription, translation and encapsidation. The replication strategy that maximizes the intracellular growth rate of the virus requires iterative genome transcription from positive to negative, and back to positive sense. However, RNA viruses experience high mutation rates, and the proportion of genomes with lethal mutations increases with the number of replication cycles. Thus, intracellular mutant frequency will depend on the replication strategy. Introducing apparently realistic mutation rates into a model of viral replication demonstrates that strategies that maximize viral growth rate could result in an average of 26 mutations per genome by the time plausible numbers of positive strands have been generated, and that virus viability could be as low as 0.1 per cent. At high mutation rates or when a high proportion of mutations are deleterious, the optimal strategy shifts towards synthesizing more negative strands per positive strand, and in extremis towards a 'stamping-machine' replication mode where all the encapsidated genomes come from only two transcriptional steps. We conclude that if viral mutation rates are as high as current estimates suggest, either mutation frequency must be considerably higher than generally anticipated and the proportion of viable viruses produced extremely small, or replication strategies cannot be optimized to maximize viral growth rate. Mechanistic models linking mutation frequency to replication mechanisms coupled with data generated through new deep-sequencing technologies could play an important role in improving the estimates of viral mutation rate.

Dengue Virus Capsid Protein Usurps Lipid Droplets for Viral Particle Formation

Dengue virus is responsible for the highest rates of disease and mortality among the members of the Flavivirus genus. Dengue epidemics are still occurring around the world, indicating an urgent need of prophylactic vaccines and antivirals. In recent years, a great deal has been learned about the mechanisms of dengue virus genome amplification. However, little is known about the process by which the capsid protein recruits the viral genome during encapsidation. Here, we found that the mature capsid protein in the cytoplasm of dengue virus infected cells accumulates on the surface of ER-derived organelles named lipid droplets. Mutagenesis analysis using infectious dengue virus clones has identified specific hydrophobic amino acids, located in the center of the capsid protein, as key elements for lipid droplet association. Substitutions of amino acid L50 or L54 in the capsid protein disrupted lipid droplet targeting and impaired viral particle formation. We also report that dengue virus infection increases the number of lipid droplets per cell, suggesting a link between lipid droplet metabolism and viral replication. In this regard, we found that pharmacological manipulation of the amount of lipid droplets in the cell can be a means to control dengue virus replication. In addition, we developed a novel genetic system to dissociate cis-acting RNA replication elements from the capsid coding sequence. Using this system, we found that mislocalization of a mutated capsid protein decreased viral RNA amplification. We propose that lipid droplets play multiple roles during the viral life cycle; they could sequester the viral capsid protein early during infection and provide a scaffold for genome encapsidation.

Cidofovir Inhibits Genome Encapsidation and Affects Morphogenesis during the Replication of Vaccinia Virus

Cidofovir (CDV) is one of the most effective antiorthopoxvirus drugs, and it is widely accepted that viral DNA replication is the main target of its activity. In the present study, we report a detailed analysis of CDV effects on the replicative cycles of distinct vaccinia virus (VACV) strains: Cantagalo virus, VACV-IOC, and VACV-WR. We show that despite the approximately 90% inhibition of production of virus progeny, virus DNA accumulation was reduced only 30%, and late gene expression and genome resolution were unaltered. The level of proteolytic cleavage of the major core proteins was diminished in CDV-treated cells. Electron microscopic analysis of virus-infected cells in the presence of CDV revealed reductions as great as 3.5-fold in the number of mature forms of virus particles, along with a 3.2-fold increase in the number of spherical immature particles. A detailed analysis of purified virions recovered from CDV-treated cells demonstrated the accumulation of unprocessed p4a and p4b and nearly 67% inhibition of DNA encapsidation. However, these effects of CDV on virus morphogenesis resulted from a primary effect on virus DNA synthesis, which led to later defects in genome encapsidation and virus assembly. Analysis of virus DNA by atomic force microscopy revealed that viral cytoplasmic DNA synthesized in the presence of CDV had an altered structure, forming aggregates with increased strand overlapping not observed in the absence of the drug. These aberrant DNA aggregations were not encapsidated into virus particles.

Insight into Poliovirus Genome Replication and Encapsidation Obtained from Studies of 3B-3C Cleavage Site Mutants

A poliovirus (PV) mutant (termed GG), which is incapable of producing 3AB, VPg, and 3CD proteins due to a defective cleavage site between the 3B and 3C proteins, replicated, producing 3BC-linked RNA rather than the VPg-linked RNA produced by the wild type (WT). GG PV RNA is quasi-infectious. The yield of infectious GG PV relative to replicated RNA is reduced by almost 5 logs relative to that of WT PV. Proteolytic activity required for polyprotein processing is normal for the GG mutant. 3BC-linked RNA can be encapsidated as efficiently as VPg-linked RNA. However, a step after genome replication but preceding virus assembly that is dependent on 3CD and/or 3AB proteins limits production of infectious GG PV. This step may involve release of replicated genomes from replication complexes. A pseudorevertant (termed EG) partially restored cleavage at the 3B-3C cleavage site. The reduced rate of formation of 3AB and 3CD caused corresponding reductions in the observed rate of genome replication and infectious virus production by EG PV without impacting the final yield of replicated RNA or infectious virus relative to that of WT PV. Using EG PV, we showed that genome replication and encapsidation were distinct steps in the multiplication cycle. Ectopic expression of 3CD protein reversed the genome replication phenotype without alleviating the infectious-virus production phenotype. This is the first report of a trans-complementable function for 3CD for any picornavirus. This observation supports an interaction between 3CD protein and viral and/or host factors that is critical for genome replication, perhaps formation of replication complexes.

Identification of Functional Domains in Reovirus Replication Proteins μNS and μ2

Mammalian reoviruses are nonenveloped particles containing a genome of 10 double-stranded RNA (dsRNA) gene segments. Reovirus replication occurs within viral inclusions, which are specialized nonmembranous cytoplasmic organelles formed by viral nonstructural and structural proteins. Although these structures serve as sites for several major events in the reovirus life cycle, including dsRNA synthesis, gene segment assortment, and genome encapsidation, biochemical mechanisms of virion morphogenesis within inclusions have not been elucidated because much remains unknown about inclusion anatomy and functional organization. To better understand how inclusions support viral replication, we have used RNA interference (RNAi) and reverse genetics to define functional domains in two inclusion-associated proteins, muNS and mu2, which are interacting partners essential for inclusion development and viral replication. Removal of muNS N-terminal sequences required for association with mu2 or another muNS-binding protein, sigmaNS, prevented the capacity of muNS to support viral replication without affecting inclusion formation, indicating that muNS-mu2 and muNS-sigmaNS interactions are necessary for inclusion function but not establishment. In contrast, introduction of changes into the muNS C-terminal region, including sequences that form a putative oligomerization domain, precluded inclusion formation as well as viral replication. Mutational analysis of mu2 revealed a critical dependence of viral replication on an intact nucleotide/RNA triphosphatase domain and an N-terminal cluster of basic amino acid residues conforming to a nuclear localization motif. Another domain in mu2 governs the capacity of viral inclusions to affiliate with microtubules and thereby modulates inclusion morphology, either globular or filamentous. However, viral variants altered in inclusion morphology displayed equivalent replication efficiency. These studies reveal a modular functional organization of inclusion proteins muNS and mu2, define the importance of specific amino acid sequences and motifs in these proteins for viral replication, and demonstrate the utility of complementary RNAi-based and reverse genetic approaches for studies of reovirus replication proteins.

Packaging of viral RNAs in virions of adenoviruses

Earlier, we detected viral RNAs packaged in the porcine adenovirus (PAdV) -3 virions. Using Southern blot analysis, we further demonstrated that the viral RNAs were predominantly packaged in CsCl purified mature capsids (containing viral genome) than empty/intermediate capsids. Some of the packaged viral RNAs appear to be polyadenylated. Real-time reverse transcription (RT)-PCR analysis indicated that the copy number of the tested viral mRNAs encoding E1Bsmall and fiber proteins was less than one per full capsid. Moreover, detection of viral RNA packaged in CsCl purified human adenovirus (HAdV) -5 virions indicates that the viral RNA packaging might be a common phenomenon in members of Adenoviridae family. Further quantitative analysis of viral protein, DNA, and RNA in CsCl purified mature and empty/intermediate capsids of recombinant HAdV-5 expressing enhanced green fluorescent protein indicated that the traceable viral RNA detected in empty/intermediate capsids seems associated with the presence of traceable viral genomic DNA. Taken together, our data suggest that the viral RNAs may be passively packaged in adenovirus virion during encapsidation of viral genomic DNA in cell nuclei. Thus, viral RNA packaging may be a characteristic feature of adenoviral genomic DNA encapsidation.

Mechanism of Reduction in Titers from Lentivirus Vectors Carrying Chromatin Insulator Elements in the 3′ LTR

Random integration of viral vectors can result in undesirable activation of surrounding genes by enhancers in the vectors (vector genotoxicity), or result in variable expression due to effects of surrounding chromatin on the vector transgene (chromatin position effects). Vector genotoxicity has become an area of intense study since the occurrence of gene therapy related leukemias in 5 patients in the French X-SCID trial. Additionally, we find consistent and therefore 2–3 fold higher expression from vectors insulated by the chicken b-globin hypersensitive site (cHS4) insulator; and these vectors achieve therapeutic correction in human b0-thalassemia major, where a very high transgene expression is necessary. However, vectors that insulate the correcting transgene from position effects and genotoxicity significantly compromise viral titers by an order of magnitude. In order to define the mechanism by which this occurs and improve the titers of insulated vector systems, we placed the 1.2Kb cHS4 insulator or different regions of cHS4 that may have insulator activity and/or inert DNA spacers in the 3′LTR of self-inactivating lentiviruses carrying a large b-globin transgene and regulatory elements. We also lengthened the β-globin lentivirus vector by an additional 1.2Kb, by insertion of an internal transgene cassette. We found that addition of 1.2Kb transgene internally to a large “globin” vector did not reduce vector infectious titers. However, when cHS4 sequences or inert DNA spacers of increasing size were placed in the 3′LTR, infectious titers decreased proportional to the length of the insert. This effect occurred regardless of the type of sequence inserted in the 3′ LTR. Vectors carrying the1.2Kb cHS4 or l-DNA spacer in the 3′ LTR had the lowest titers. We next examined the stage of the vector life-cycle affected by large LTR inserts in packaging cells, the quantity and quality of the virus particles generated, and post-entry viral steps in target cells. Equal amounts of full-length viral genomic transcripts were produced in the packaging cells with vectors with or without the 1.2Kb insulator insert. All insertion in the 3′ LTR are placed in the U3 enhancer deleted region, proximal to the viral polyadenylation signal. Also, self-inactivating vectors have a U3 enhancer deletion that also deletes enhancers of polyadenylation. However, despite the insertion of cHS4 elements in this region, no increase in viral readthrough transcription (as measured by northern blot analysis and an enzyme-based assay) occurred with vectors carrying the large 1.2Kb insert. Packaging efficiency was also identical with insulated and uninsulated vectors. Similar degree of viral genome encapsidation occurred, as measured by p24 ELISA, virus associated reverse transcriptase and viral RNA analysis, demonstrating that similar amounts of intact viral particles were produced with insulated and uninsulated vector plasmids. However, lentiviruses carrying the 1.2Kb insert in the 3′LTR were inefficiently processed following target-cell entry, with reduced reverse transcription and integration efficiency. This primarily occurred from increased homologous recombination resulting in increased 1-LTR circles of the insulated vector viral DNA; resulting in reduced vector integration and hence lower transduction/infectious titers. Thus, we found that large inserts in the viral 3′ LTR are packaged efficiently, but have inefficient post-entry viral mRNA processing. In a parallel study, we also did a structure-function analysis of cHS4 fragments and identified key elements necessary for optimal insulation. Vectors constructed with this minimized 650bp cHS4 sequences had a minimal reduction in titers, yet retained full insulator activity. These studies have important implications in the design of gamma-retrovirus or lentivirus vectors with insulator, transgenes or enhancer inserts into the 3′ LTR. [FU and PA contributed equally to this work]

First step in characterization of cis-acting sequences involved in fowl adenovirus 1 (CELO) packaging and its effect on the development of a helper-dependent vector strategy

Adenovirus-based vectors are widely developed for potential utilization as vectors in vaccine and gene therapy strategies. We focused on developing a helper-dependent adenoviral (HD-Ad) vector for the potential use of CELO, a member of the Aviadenovirus genus, in avian species vaccination. Our aim was to localize sequences which could play an essential role in CELO genome encapsidation and, when deleted, was unable to produce viruses to develop a helper CELO virus. A panel of 6 mutants with deletions between nt 80 and 350 of the CELO genome was constructed and characterized for its ability to produce viable virus. To develop a helper-dependent adenoviral vector derived from CELO, a helper virus was developed by inserting loxP sequences around the region containing the identified putative packaging sequences. A LMH (Leghorn Male Hepatocarcinoma) cell line expressing Cre recombinase was developed to allow the excision of this region. We demonstrated that the region from nt 200 to 250 was important and the region from nt 250 to 300 at the left end of the CELO genome was essential for virus encapsidation. We also showed that the loxP-flanked region was efficiently removed in a Cre expressing cell line to produce a candidate helper virus.

Inhibition of HIV‐1 by a Peptide Ligand of the Genomic RNA Packaging Signal Ψ

The interaction of the nucleocapsid NCp7 of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein with the RNA packaging signal Psi ensures specific encapsidation of the dimeric full length viral genome into nascent virus particles. Being an essential step in the HIV-1 replication cycle, specific genome encapsidation represents a promising target for therapeutic intervention. We previously selected peptides binding to HIV-1 Psi-RNA or stem loops (SL) thereof by phage display. Herein, we describe synthesis of peptide variants of the consensus HWWPWW motif on membrane supports to optimize Psi-RNA binding. The optimized peptide, psi-pepB, was characterized in detail with respect to its conformation and binding properties for the SL3 of the Psi packaging signal by NMR and tryptophan fluorescence quenching. Functional analysis revealed that psi-pepB caused a strong reduction of virus release by infected cells as monitored by reduced transduction efficiencies, capsid p24 antigen levels, and electron microscopy. Thus, this peptide shows antiviral activity and could serve as a lead compound to develop new drugs targeting HIV-1.

Specific targeting of hepatitis C virus core protein by an intracellular single-chain antibody of human origin

The hepatitis C virus (HCV) core protein is essential for viral genome encapsidation and plays an important role in steatosis, immune evasion, and hepatocellular carcinoma. It may thus represent a promising therapeutic target to interfere with the HCV life-cycle and related pathogenesis. In this study, we used phage display to generate single-chain variable domain antibody fragments (scFv) to the core protein from bone marrow plasma cells of patients with chronic hepatitis C. An antibody with high-affinity binding (scFv42C) was thus identified, and the binding site was mapped to the PLXG motif (residues 84-87) of the core protein conserved among different genotypes. Whereas scFv42C displayed diffuse cytoplasmic fluorescence when expressed alone in the Huh7 human hepatoma cell line, cotransfection with the core gene shifted its subcellular distribution into that of core protein. The intracellular association of scFv42C with its target core protein was independently demonstrated by the fluorescence resonance energy transfer technique. Interestingly, expression of the single-chain antibody reduced core protein levels intracellularly, particularly in the context of full HCV replication. Moreover, cell proliferation as induced by the core protein could be reversed by scFv4C coexpression. Therefore, scFv42C may represent a novel anti-HCV agent, which acts by sequestering core protein and attenuating core protein-mediated pathogenesis.