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Thioxothiazolo[3,4-a]quinazoline derivatives inhibit the human cytomegalovirus alkaline nuclease

Human cytomegalovirus (HCMV, human herpesvirus 5) is an opportunistic pathogen responsible for serious disease in immunocompromised patients. Current antiviral therapies rely predominantly on drugs interfering with viral DNA replication and packaging. However, the serious side effects of existing drugs and the emergence of drug resistance indicate the need for new targets for anti-HCMV therapy. One such target is the viral alkaline nuclease (AN), an enzyme highly conserved among the Herpesviridae. In this study, we validated the HCMV AN, encoded by the viral UL98 open reading frame, as a drug target by demonstrating that a UL98-deficient HCMV mutant is severely attenuated and shows a reduced ability to spread in cell culture. We established a fluorescence-based enzyme assay suitable for high-throughput screening and used it on a small-molecule compound library. The most promising hit, a thioxothiazolo[3,4-a]quinazoline derivative, blocked AN activity in vitro and inhibited HCMV replication in plaque reduction (PRA) and fluorescence reduction assays (FRA). Several derivatives of the hit compound were tested, some of which had similar or better inhibitory activities. The most potent derivative of hit scaffold A, compound AD-51, inhibited HCMV replication with a 50% effective concentrations (EC50) of 0.9 μM in the FRA and 1.1 μM in the PRA. AD-51 was also active against ganciclovir, foscarnet, and letermovir-resistant HCMVs. Moreover, it inhibited herpes simplex virus, Kaposi's sarcoma-associated herpesvirus, and murine CMV, a mouse virus serving as a model for HCMV. These results suggest that thioxothiazolo[3,4-a]quinazoline derivatives are a new class of herpesvirus inhibitors targeting the viral AN.

Porcine deltacoronavirus resists antibody neutralization through cell-to-cell transmission

ABSTRACT Porcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus that has been reported to infect a variety of animals and even humans. Cell–cell fusion has been identified as an alternative pathway for the cell-to-cell transmission of certain viruses, but the ability of PDCoV to exploit this transmission model, and the relevant mechanisms, have not been fully elucidated. Herein, we provide evidence that cell-to-cell transmission is the main mechanism supporting PDCoV spread in cell culture and that this efficient spread model is mediated by spike glycoprotein-driven cell–cell fusion. We found that PDCoV efficiently spread to non-susceptible cells via cell-to-cell transmission, and demonstrated that functional receptor porcine aminopeptidase N and cathepsins in endosomes are involved in the cell-to-cell transmission of PDCoV. Most importantly, compared with non-cell-to-cell infection, the cell-to-cell transmission of PDCoV was resistant to neutralizing antibodies and immune sera that potently neutralized free viruses. Taken together, our study revealed key characteristics of the cell-to-cell transmission of PDCoV and provided new insights into the mechanism of PDCoV infection.

Evaluation of SARS-CoV-2 ORF7a Deletions from COVID-19-Positive Individuals and Its Impact on Virus Spread in Cell Culture.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the COVID-19 outbreak, posed a primary concern of public health worldwide. The most common changes in SARS-CoV-2 are single nucleotide substitutions, also reported insertions and deletions. This work investigates the presence of SARS-CoV-2 ORF7a deletions identified in COVID-19-positive individuals. Sequencing of SARS-CoV-2 complete genomes showed three different ORF7a size deletions (190-nt, 339-nt and 365-nt). Deletions were confirmed through Sanger sequencing. The ORF7a∆190 was detected in a group of five relatives with mild symptoms of COVID-19, and the ORF7a∆339 and ORF7a∆365 in a couple of co-workers. These deletions did not affect subgenomic RNAs (sgRNA) production downstream of ORF7a. Still, fragments associated with sgRNA of genes upstream of ORF7a showed a decrease in size when corresponding to samples with deletions. In silico analysis suggests that the deletions impair protein proper function; however, isolated viruses with partial deletion of ORF7a can replicate in culture cells similarly to wild-type viruses at 24 hpi, but with less infectious particles after 48 hpi. These findings on deleted ORF7a accessory protein gene, contribute to understanding SARS-CoV-2 phenotypes such as replication, immune evasion and evolutionary fitness as well insights into the role of SARS-CoV-2_ORF7a in the mechanism of virus-host interactions.

A Novel Small Molecule Inhibits Hepatitis C Virus Propagation in Cell Culture.

ABSTRACTHepatitis C virus (HCV) can cause acute and chronic infection that is associated with considerable liver-related morbidity and mortality. In recent years, there has been a shift in the treatment paradigm with the discovery and approval of agents that target specific proteins vital for viral replication. We employed a cell culture-adapted strain of HCV and human hepatoma-derived cells lines to test the effects of our novel small-molecule compound (AO13) on HCV. Virus inhibition was tested by analyzing RNA replication, protein expression, and virus production in virus-infected cells treated with AO13. Treatment with AO13 inhibited virus spread in cell culture and showed a 100-fold reduction in the levels of infectious virus production. AO13 significantly reduced the level of viral RNA contained within cell culture fluids and reduced the cellular levels of HCV core protein, suggesting that the compound might act on a late step in the viral life cycle. Finally, we observed that AO13 did not affect the release of infectious virus from infected cells. Docking studies and molecular dynamics analyses suggested that AO13 might target the NS5B RNA polymerase, however, real-time RT-PCR analyses of cellular levels of HCV RNA showed only an ∼2-fold reduction in viral RNA levels in the presence of AO13. Taken together, this study revealed that AO13 showed consistent, but low-level antiviral effect against HCV, although the mechanism of action remains unclear.IMPORTANCE The discovery of curative antiviral drugs for a chronic disease such as HCV infection has encouraged drug discovery in the context of other viruses for which no curative drugs currently exist. Since we currently face a novel virus that has caused a pandemic, the need for new antiviral agents is more apparent than ever. We describe here a novel compound that shows a modest antiviral effect against HCV that could serve as a lead compound for future drug development against other important viruses such as SARS-CoV-2.

SARS-CoV-2 Production in a Scalable High Cell Density Bioreactor.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has demonstrated the value of pursuing different vaccine strategies. Vaccines based on whole viruses, a widely used vaccine technology, depend on efficient virus production. This study aimed to establish SARS-CoV-2 production in the scalable packed-bed CelCradleTM 500-AP bioreactor. CelCradleTM 500-AP bottles with 0.5 L working volume and 5.5 g BioNOC™ II carriers were seeded with 1.5 × 108 Vero (WHO) cells, approved for vaccine production, in animal component-free medium and infected at a multiplicity of infection of 0.006 at a total cell number of 2.2–2.5 × 109 cells/bottle seven days post cell seeding. Among several tested conditions, two harvests per day and a virus production temperature of 33 °C resulted in the highest virus yield with a peak SARS-CoV-2 infectivity titer of 7.3 log10 50% tissue culture infectious dose (TCID50)/mL at 72 h post-infection. Six harvests had titers of ≥6.5 log10 TCID50/mL, and a total of 10.5 log10 TCID50 were produced in ~5 L. While trypsin was reported to enhance virus spread in cell culture, addition of 0.5% recombinant trypsin after infection did not improve virus yields. Overall, we demonstrated successful animal component-free production of SARS-CoV-2 in well-characterized Vero (WHO) cells in a scalable packed-bed bioreactor.

Induction of Rod-Shaped Structures by Herpes Simplex Virus Glycoprotein I.

The envelope glycoprotein I (gI) of herpes simplex virus 1 (HSV-1) is a critical mediator of virus-induced cell-to-cell spread and cell-cell fusion. Here, we report a previously unrecognized property of this molecule. In transfected cells, the HSV-1 gI was discovered to induce rod-shaped structures that were uniform in width but variable in length. Moreover, the gI within these structures was conformationally different from the typical form of gI, as a previously used monoclonal antibody mAb3104 and a newly made peptide antibody to the gI extracellular domain (ECD) (amino acids [aa] 110 to 202) both failed to stain the long rod-shaped structures, suggesting the formation of a higher-order form. Consistent with this observation, we found that gI could self-interact and that the rod-shaped structures failed to recognize glycoprotein E, the well-known binding partner of gI. Further analyses by deletion mutagenesis and construction of chimeric mutants between gI and gD revealed that the gI ECD is the critical determinant, whereas the transmembrane domain served merely as an anchor. The critical amino acids were subsequently mapped to proline residues 184 and 188 within a conserved PXXXP motif. Reverse genetics analyses showed that the ability to induce a rod-shaped structure was not required for viral replication and spread in cell culture but rather correlated positively with the capability of the virus to induce cell fusion in the UL24syn background. Together, this work discovered a novel feature of HSV-1 gI that may have important implications in understanding gI function in viral spread and pathogenesis.IMPORTANCE The HSV-1 gI is required for viral cell-to-cell spread within the host, but the molecular mechanisms of how gI exactly works have remained poorly understood. Here, we report a novel property of this molecule, namely, induction of rod-shaped structures, which appeared to represent a higher-order form of gI. We further mapped the critical residues and showed that the ability of gI to induce rod-shaped structures correlated well with the capability of HSV-1 to induce cell fusion in the UL24syn background, suggesting that the two events may have an intrinsic link. Our results shed light on the biological properties of HSV-1 gI and may have important implications in understanding viral pathogenesis.

Cell-to-Cell Transmission Is the Main Mechanism Supporting Bovine Viral Diarrhea Virus Spread in Cell Culture.

After initiation of an infective cycle, spread of virus infection can occur in two fundamentally different ways: (i) viral particles can be released into the external environment and diffuse through the extracellular space until they interact with a new host cell, and (ii) virions can remain associated with infected cells, promoting the direct passage between infected and uninfected cells that is referred to as direct cell-to-cell transmission. Although evidence of cell-associated transmission has accumulated for many different viruses, the ability of members of the genus Pestivirus to use this mode of transmission has not been reported. In the present study, we used a novel recombinant virus expressing the envelope glycoprotein E2 fused to mCherry fluorescent protein to monitor the spreading of bovine viral diarrhea virus (BVDV) (the type member of the pestiviruses) infection. To demonstrate direct cell-to-cell transmission of BVDV, we developed a cell coculture system that allowed us to prove direct transmission from infected to uninfected cells in the presence of neutralizing antibodies. This mode of transmission requires cell-cell contacts and clathrin-mediated receptor-dependent endocytosis. Notably, it overcomes antibody blocking of the BVDV receptor CD46, indicating that cell-to-cell transmission of the virus involves the engagement of coreceptors on the target cell.IMPORTANCE BVDV causes one of the most economically important viral infections for the cattle industry. The virus is able to cross the placenta and infect the fetus, leading to the birth of persistently infected animals, which are reservoirs for the spread of BVDV. The occurrence of persistent infection has hampered the efficacy of vaccination because it requires eliciting levels of protection close to sterilizing immunity to prevent fetal infections. While vaccination prevents disease, BVDV can be detected if animals with neutralizing antibodies are challenged with the virus. Virus cell-to-cell transmission allows the virus to overcome barriers to free virus dissemination, such as antibodies or epithelial barriers. Here we show that BVDV exploits cell-cell contacts to propagate infection in a process that is resistant to antibody neutralization. Our results provide new insights into the mechanisms underlying the pathogenesis of BVDV infection and can aid in the design of effective control strategies.

Full-length infectious clone of an Iranian isolate of chicken anemia virus.

An Iranian field strain of chicken anemia virus (CAV), designated IR CAV, was isolated in the Marek's disease virus-transformed lymphoblastoid cell line MDCC-MSB1 (MSB1) culture for the first time. The full-length CAV DNA of this strain was cloned in the bacterial plasmid pTZ57R/T to create the molecular clone pTZ-CAV. The nucleotide and deduced amino acid sequences of viral proteins of IR CAV were compared with those of representative CAV sequences including reference and commercial vaccine strains. IR CAV was not related to vaccine strains and also found to have glutamine at positions 139 and 144 confirming previous studies in which such mutations were associated with a slow rate of virus spread in cell culture. pTZ-CAV was digested with PstI to release IR CAV DNA and then transfected into MSB1 cell by electroporation. The transfected cells showed cytopathic effect similar to virion-initiated infection. One-day old specific pathogen-free chicks were inoculated with the regenerated virus, which had been obtained from transfected MSB1 cells, and compared with the chicks inoculated with IR CAV. Gross lesions in the birds inoculated with the regenerated virus illustrated the infectious nature of the regenerated virus from the cloned IR CAV DNA.

Impaired antiviral response of adenovirus-transformed cell lines supports virus replication.

Activation of the innate immune response represents one of the most important cellular mechanisms to limit virus replication and spread in cell culture. Here, we examined the effect of adenoviral gene expression on the antiviral response in adenovirus-transformed cell lines; HEK293, HEK293SF and AGE1.HN. We demonstrate that the expression of the early region protein 1A in these cell lines impairs their ability to activate antiviral genes by the IFN pathway. This property may help in the isolation of newly emerging viruses and the propagation of interferon-sensitive virus strains.

JC polyomavirus attachment, entry, and trafficking: unlocking the keys to a fatal infection.

The human JC polyomavirus (JCPyV) causes a lifelong persistent infection in the reno-urinary tract in the majority of the adult population worldwide. In healthy individuals, infection is asymptomatic, while in immunocompromised individuals, the virus can spread to the central nervous system and cause a fatal demyelinating disease known as progressive multifocal leukoencephalopathy (PML). There are currently very few treatment options for this rapidly progressing and devastating disease. Understanding the basic biology of JCPyV-host cell interactions is critical for the development of therapeutic strategies to prevent or treat PML. Research in our laboratory has focused on gaining a detailed mechanistic understanding of the initial steps in the JCPyV life cycle in order to define how JCPyV selectively targets cells in the kidney and brain. JCPyV requires sialic acids to attach to host cells and initiate infection, and JCPyV demonstrates specificity for the oligosaccharide lactoseries tetrasaccharide c (LSTc) with an α2,6-linked sialic acid. Following viral attachment, JCPyV entry is facilitated by the 5-hydroxytryptamine (5-HT)2 family of serotonin receptors via clathrin-dependent endocytosis. JCPyV then undergoes retrograde transport to the endoplasmic reticulum (ER) where viral disassembly begins. A novel retrograde transport inhibitor termed Retro-2(cycl) prevents trafficking of JCPyV to the ER and inhibits both initial virus infection and infectious spread in cell culture. Understanding the molecular mechanisms by which JCPyV establishes infection will open up new avenues for the prevention or treatment of virus-induced disease.

Inhibition of cytomegalovirus infection and photothermolysis of infected cells using bioconjugated gold nanoparticles.

Human cytomegalovirus (CMV) is a herpesvirus that causes major health problems in neonates as well as in immunocompromised individuals1. At present, a vaccine is not available for CMV infection and the available antiviral drugs suffer from toxicity, poor efficacy and resistance12. Here, we chemically conjugated a monoclonal antibody raised against CMV surface glycoprotein (gB) with gold nanoparticles (GNP) and characterized the potential of this gB-GNP conjugate for antiviral activity against CMV. The gB-GNP blocks viral replication, virus-induced cytopathogenic effects and virus spread in cell culture without inducing cytotoxicity. High concentrations of gB-GNP that coat the surface of virus particles block virus entry, whereas lower concentrations block a later stage of virus life cycle. Also, cells treated with gB-GNP gain resistance to CMV infection. In addition, infected cells when bound to gB-GNP can be selectively lysed after exposing them to specific wavelength of laser (nanophotothermolysis). Thus, we have not only designed a potential antiviral strategy that specifically blocks CMV infection at multiple stages of virus life cycle, but we have also characterized a technique that can potentially be useful in eliminating CMV infected cells from donor tissue during transplant or transfusion.

Melittin-loaded immunoliposomes against viral surface proteins, a new approach to antiviral therapy

In this study, melittin, a well-characterized pore-forming lytic amphiphilic peptide susceptible to be vehiculized in lipid membranes, has been utilized to study their antiviral properties. For this purpose, an assay based on melittin loaded-immunoliposomes previously described by our group was adapted to antiviral purposes by means of monoclonal antibodies targeting the surface G glycoprotein of the fish viral haemorrhagic septicemia rhabdovirus (VHSV). We also studied the antiviral action of these immunoliposomes in vitro and the results showed that they are capable of inhibiting the VHSV infectivity by 95.2% via direct inactivation of the virus. Furthermore, the inhibition of the infectivity when treatments were added at different times post-infection and the analysis of the infection foci sizes suggested altogether that they also act by reducing the VHSV spread in cell culture and by killing the infected cells which express the G glycoprotein in their plasmatic membranes.

Identification and functional analysis of the small membrane-associated protein pUL11 of avian infectious laryngotracheitis virus

pUL11 is a highly conserved, small, acylated, membrane-associated tegument protein of herpesviruses. It is involved in final envelopment of nascent virions in the cytoplasm, although the precise mechanism is still unknown. By screening of mouse monoclonal antibodies (mAb) raised against purified particles of infectious laryngotracheitis virus (ILTV) of chickens (Veits et al., 2003a), we identified two mAb recognizing the 15kDa UL11 protein (pUL11) of this avian alphaherpesvirus. These mAb permitted detection and precise localization of pUL11 in mature ILT virions, as well as in the cytoplasm of infected chicken cells by Western blot analyses, indirect immunofluorescence tests, and immunoelectron microscopy. For investigation of gene function UL11-deleted ILTV recombinants were generated. Like its homologues in several other alphaherpesviruses, ILTV-pUL11 was shown to be nonessential for productive virus replication. However, compared to wild-type and UL11 rescued ILTV the deletion mutants exhibited significantly reduced virus yields and moderately impaired spread in cell culture. In the absence of pUL11, electron microscopy of infected cells revealed accumulations of tegument proteins with nucleocapsids, and marked distortions of Golgi membranes in the cytoplasm, which obviously inhibited the formation of mature, enveloped virus particles. Taken together, our results demonstrate that pUL11 is relevant for secondary envelopment of ILTV, and confirm functional conservation of this protein in herpesviruses. The now available unique pUL11-specific mAb will help to further analyze this function, which is presumably mediated by physical interactions with other viral gene products, in cultured cells and in the natural animal host of ILTV.

Self-Assembling Peptide Nanotubes with Antiviral Activity against Hepatitis C Virus

Hepatitis C virus (HCV) infects chronically 3% of the world population and the current therapy against this pathogen is only partially effective. With the aim of developing novel antiviral strategies against HCV, we screened a D,L-α-peptide library using anunbiased methodology based on a cell culture infection system for HCV. We found a family of highlyactive amphiphilic eight-residue cyclic D,L-α-peptides that specifically blocked entry of all tested HCV genotypes into target cells at a postbinding step without affecting infection by other enveloped RNA viruses. Structure-activity relationship studies indicate that antiviral activity was dependent on cyclic D,L-α-peptide self-assembly processes and that, although they possess a net neutral charge,they display a characteristic charge distribution. Our results indicate that supramolecular amphiphilic peptide structures constitute a class of highly selective HCV entry inhibitors.

Molecular Characterization of Chicken Infectious Anemia Virus Circulating in Argentina During 2007

Chicken infectious anemia virus (CAV) is a worldwide-distributed infectious agent that affects commercial poultry. Although this agent was first detected in Argentina in 1994, no further studies on CAV in this country were reported after that. The recent increased occurrence of clinical cases of immunosuppression that could be caused by CAV has prompted this study. Our results confirmed that CAV is still circulating in commercial flocks in Argentina. Phylogenetic analysis focusing on the VP1 nucleotide sequence showed that all Argentinean isolates grouped together in a cluster, sharing a high similarity (> 97%) with genotype B reference strains. However, Argentinean isolates were distantly related to other strains commonly used for vaccination in this country, such as Del-Ros and Cux-1. Sequence analysis of predicted VP1 peptides showed that most of the Argentinean isolates have a glutamine residue at positions 139 and 144, suggesting that these isolates might have a reduced spread in cell culture compared with Cux-1. In addition, a particular amino acid substitution at position 290 is present in all studied Argentinean isolates, as well as in several VP1 sequences from Malaysia, Australia, and Japan isolates. Our results indicate that it is possible to typify CAV strains by comparison of VPI nucleotide sequences alone because the same tree topology was obtained when using the whole genome sequence. The molecular analysis of native strains sheds light into the epidemiology of CAV in Argentinean flocks. In addition, this analysis could be considered in future control strategies focused not only on breeders but on broilers and layer flocks.

Varicella-Zoster Virus Glycoprotein M Homolog Is Glycosylated, Is Expressed on the Viral Envelope, and Functions in Virus Cell-to-Cell Spread

Although envelope glycoprotein M (gM) is highly conserved among herpesviruses, the varicella-zoster virus (VZV) gM homolog has never been investigated. Here we characterized the VZV gM homolog and analyzed its function in VZV-infected cells. The VZV gM homolog was expressed on virions as a glycoprotein modified with a complex N-linked oligosaccharide and localized mainly to the Golgi apparatus and the trans-Golgi network in infected cells. To analyze its function, a gM deletion mutant was generated using the bacterial artificial chromosome system in Escherichia coli, and the virus was reconstituted in MRC-5 cells. VZV is highly cell associated, and infection proceeds mostly by cell-to-cell spread. Compared with wild-type VZV, the gM deletion mutant showed a 90% reduction in plaque size and 50% of the cell-to-cell spread in MRC-5 cells. The analysis of infected cells by electron microscopy revealed numerous aberrant vacuoles containing electron-dense materials in cells infected with the deletion mutant virus but not in those infected with wild-type virus. However, enveloped immature particles termed L particles were found at the same level on the surfaces of cells infected with either type of virus, indicating that envelopment without a capsid might not be impaired. These results showed that VZV gM is important for efficient cell-to-cell virus spread in cell culture, although it is not essential for virus growth.

Functional Characterization of the Major and Minor Phosphorylation Sites of the P Protein of Borna Disease Virus

The phosphoprotein P of Borna disease virus (BDV) is an essential cofactor of the viral RNA-dependent RNA polymerase. It is preferentially phosphorylated at serine residues 26 and 28 by protein kinase C epsilon (PKCepsilon) and, to a lesser extent, at serine residues 70 and 86 by casein kinase II (CKII). To determine whether P phosphorylation is required for viral polymerase activity, we generated P mutants lacking either the PKCepsilon or the CKII phosphate acceptor sites by replacing the corresponding serine residues with alanine (A). Alternatively, these sites were replaced by aspartic acid (D) to mimic phosphorylation. Functional characterization of the various mutants in the BDV minireplicon assay revealed that D substitutions at the CKII sites inhibited the polymerase-supporting activity of P, while A substitutions maintained wild-type activity. Likewise, D substitutions at the PKC sites did not impair the cofactor function of BDV-P, whereas A substitutions at these sites led to increased activity. Interestingly, recombinant viruses could be rescued only when P mutants with modified PKCepsilon sites were used but not when both CKII sites were altered. PKCepsilon mutant viruses showed a reduced capacity to spread in cell culture, while viral RNA and protein expression levels in persistently infected cells were almost normal. Further mutational analyses revealed that substitutions at individual CKII sites were, with the exception of a substitution of A for S86, detrimental for viral rescue. These data demonstrate that, in contrast to other viral P proteins, the cofactor activity of BDV-P is negatively regulated by phosphorylation.

Identification of selective inhibitors of VHSV from biased combinatorial libraries of N, N′-disubstituted 2,5-piperazinediones

A combinatorial strategy has been used to design and identify inhibitors of viral haemorragic septicemia virus (VHSV), a salmonid rhabdovirus of economic importance. Two libraries of N, N′-disubstituted 2,5-piperazinediones (DKP), DKP-I and DKP-II were screened for inhibition of VHSV infectivity. Among the 98 DKP-derivatives (R1-DKP-R2) screened, a novel class of VHSV in vitro inhibitors was identified. Evidences are presented showing that the selected DKP-derivatives cause dose-dependent inhibition of VHSV infectivity in the absence of cellular toxicity. Preliminary characterization of its inhibition mechanism ruled out direct inactivation of the virus (virucidal effect) or interference with early viral replication steps. Furthermore, analysis of infection foci sizes, virus titers, viral protein accumulation and presence of cell free virus derived from VHSV-infected cell cultures in the presence of DKP-derivates suggested that virus assembly/release was impaired leading to a reduced virus spread in cell culture. New DKP-derivatives with a significant higher specific activity need to be developed to start testing its possible practical use but the selected DKP-derivatives described here may contribute to their further development as well as being tools to improve our knowledge on the fish rhabdovirus infection cycle.

Ribozyme therapy for HIV infection

Within the past few years encouraging progress has been made in the treatment of HIV-1 infection, largely due to the combined use of HIV-1 protease inhibitors with nucleoside and non-nucleoside reverse transcriptase inhibitors. Despite this, HIV-1 infection is still a major global problem and the emergence of a drug resistant virus is ever present. There is a continuing need to develop new therapeutic strategies as well as improve upon all forms of existing therapies for the treatment of this viral infection. It has now been almost a decade since the first demonstration that ribozymes can effectively inhibit HIV-1 infectious spread in cell culture. Since then, ribozymes have progressed into human clinical trials primarily through gene therapy approaches. This progression brings ribozymes into the forefront as an important addition to the growing arsenal of anti-HIV-1 weapons. The following review covers the developments in anti-HIV-1 ribozyme usage over the past decade and summarizes the current state of ribozyme development for the purpose of inhibiting HIV-1 infection.

Functional cDNA Clones of The Flaviviridae: Strategies and Applications

Publisher Summary This chapter summarizes the trials and tribulations of constructing the functional clones for Flaviviridae viruses and discusses some recent advances in the field. The family Flaviviridae is a remarkably diverse and widespread group of human and animal pathogens. These viruses are enveloped with single-stranded messenger-sense genome RNAs. Three genera are recognized: the classical flaviviruses, the pestiviruses, and the hepaciviruses. For members of each genus, genetic manipulation of functional complementary DNA (cDNA) clones is now yielding insights into viral replication and pathogenesis and providing new avenues for vaccine development. Functional cDNA clones now exist for representative members of all three genera of the Flaviviridae. Highly infectious RNA transcripts, efficient transfection conditions, rapid replication and spread in cell culture, and tractable animal models provide a solid foundation for the state-of-the-art genetic studies on flaviviruses and pestiviruses. For Hepatitis C virus (HCV), challenges remain, but the availability of functional clones should expedite studies on HCV proteins and RNA elements, and on virus evolution and host immune responses, and contribute to the establishment of cell culture replication assays and alternative animal models.