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PubMed1989

The structure of antiviral agents that inhibit uncoating when complexed with viral capsids.

The tertiary structure of most icosahedral viral capsid proteins consists of an eight-stranded antiparallel beta-barrel with a hydrophobic interior. In a group of picornaviruses, this hydrophobic pocket can be filled by suitable organic molecules, which stop viral uncoating after attachment and penetration into the host cell. The antiviral activity of these agents is probably due to increased rigidity of the capsid protein, thereby inhibiting disassembly. The hydrophobic pocket may be an essential functional component of the protein and may have been conserved in the evolution of many viruses from a common precursor. Since eight-stranded anti-parallel beta-barrels, with a topology as in viral capsid proteins, are not generally found in other proteins involved in cell metabolism, antiviral agents that bind in the interior of viral capsid proteins are likely to be more virus-specific and less cytotoxic. Furthermore, the greatest conservation of viral capsid proteins occurs within this pocket, whereas the least conserved part is the antigenic exterior. Thus, compounds that bind to such a pocket are likely to be effective against a broader group of serologically distinct viruses. Discovery of antiviral agents of this type depends on designing compounds that can enter and fit snugly into the hydrophobic pocket of a particular viral capsid protein.

Antiviral Agents↗
PubMed1996

The complexities of viral genome analysis: the primate lentiviruses.

Analysis of sequence information from RNA-based replication systems continues to challenge the computational molecular biology community. Recent sequence data from the study of primate lentiviruses indicate that extreme sequence heterogeneity, recombination, and cross-species transmissions are all observed in HIV evolution. These types of events will continue to make the development of effective anti-retroviral therapies difficult.

Animals↗
PubMed1998

Inter-RNA interaction of phage phi29 pRNA to form a hexameric complex for viral DNA transportation.

Ds-DNA viruses package their DNA into a preformed protein shell (procapsid) during maturation. Bacteriophage phi29 requires an RNA (pRNA) to package its genomic DNA into the procapsid. We report here that the pRNA upper and lower loops are involved in RNA/RNA interactions. Mutation in only one loop results in inactive pRNAs. However, mixing of two, three and six inactive mutant pRNAs restores DNA packaging activity as long as an interlocking hexameric ring can be predicted to form by base pairing of the mutated loops in separate RNA molecules. The stoichiometry of pRNA for the packaging of one viral DNA genome is six. Homogeneous pRNA purified from a single band in denaturing gels showed six bands when rerun in native gels. These results suggest that six pRNAs form a hexameric ring by the intermolecular interaction of two RNA loops to serve as part of the DNA transportation machinery.

Bacillus Phages↗
PubMed1992

In vitro reconstitution of active influenza virus ribonucleoprotein complexes using viral proteins purified from infected cells.

A procedure to obtain RNA-free preparations containing the nucleoprotein (NP) and polymerase (P) proteins from influenza virus-infected cell extracts has been developed. The influenza virus P proteins present in these preparations copied small synthetic RNA molecules derived from plasmid sequences. In addition, RNA molecules encapsidated with the NP and P proteins were amplified and packaged into virus particles when transfected into influenza virus-infected cells. Thus, the preparations of NP and P proteins display features similar to those isolated from purified influenza virions, and represent an alternative for the preparation of active influenza virus P protein.

Animals↗
PubMed1996

Recovery and characterization of a replicase complex in rotavirus-infected cells by using a monoclonal antibody against NSP2.

Replication of the rotavirus genome involves two steps: (i) transcription and extrusion of transcripts and (ii) minus-strand RNA synthesis in viral complexes containing plus-strand RNA. In this study, we showed evidence for the importance of the viral nonstructural protein of rotavirus, NSP2, in the replication of viral RNAs. RNA-binding properties of NSP2 were tested by UV cross-linking in vivo (in rotavirus-infected MA104 cells and recombinant baculovirus-expressing NSP2-infected Sf9 cells). In rotavirus-infected cells, NSP2 is bound to the 11 double-stranded RNA genomic segments of rotavirus. Quantitative analysis (using hydrolysis by RNase A) is consistent with NSP2 being directly bound to partially replicated viral RNA. Using various monoclonal antibodies and specific antisera against the structural (VP1, VP2, and VP6) and nonstructural (NSP1, NSP2, NSP3, and NSP5) proteins, we developed a solid-phase assay for the viral replicase. In this test, we recovered a viral RNA-protein complex with replicase activity only with a monoclonal antibody directed against NSP2. Our results indicated that these viral complexes contain the structural proteins VP1, VP2, and VP6 and the nonstructural protein NSP2. Our results show that NSP2 is closely associated in vivo with the viral replicase.

Animals↗
PubMed2001

The major core protein P4a (A10L gene) of vaccinia virus is essential for correct assembly of viral DNA into the nucleoprotein complex to form immature viral particles.

The vaccinia virus (VV) A10L gene codes for a major core protein, P4a. This polypeptide is synthesized at late times during viral infection and is proteolytically cleaved during virion assembly. To investigate the role of P4a in the virus life cycle and morphogenesis, we have generated an inducer-dependent conditional mutant (VVindA10L) in which expression of the A10L gene is under the control of the Escherichia coli lacI operator/repressor system. Repression of the A10L gene severely impairs virus growth, as observed by both the inability of the virus to form plaques and the 2-log reduction of viral yields. This defect can be partially overcome by addition of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). Synthesis of viral proteins other than P4a occurred, although early shutoff of host protein synthesis and expression of viral late polypeptides are clearly delayed, both in the absence and in the presence of IPTG, compared with cells infected with the parental virus. Viral DNA replication and concatemer resolution appeared to proceed normally in the absence of the A10L gene product. In cells infected with VVindA10L in the absence of the inducer virion assembly is blocked, as defined by electron microscopy. Numerous spherical immature viral particles that appear devoid of dense viroplasmic material together with highly electron-dense regular structures are abundant in VVindA10L-infected cells. These regularly spaced structures can be specifically labeled with anti-DNA antibodies as well as with a DNase-gold conjugate, indicating that they contain DNA. Some images suggest that these DNA structures enter into spherical immature viral particles. In this regard, although it has not been firmly established, it has been suggested that DNA uptake occurs after formation of spherical immature particles. Overall, our results showed that P4a and/or its cleaved products are essential for the correct assembly of the nucleoprotein complex within immature viral particles.

Animals↗
PubMed2001

Human immunodeficiency virus type 1 Vif protein is packaged into the nucleoprotein complex through an interaction with viral genomic RNA.

The human immunodeficiency virus type 1 (HIV-1) Vif protein plays a critical role in the production of infectious virions. Previous studies have demonstrated the presence of small amounts of Vif in virus particles. However, Vif packaging was assumed to be nonspecific, and its functional significance has been questioned. We now report that packaging of Vif is dependent on the packaging of viral genomic RNA in both permissive and restrictive HIV-1 target cells. Mutations in the nucleocapsid zinc finger domains that abrogate packaging of viral genomic RNA abolished packaging of Vif. Additionally, an RNA packaging-defective virus exhibited significantly reduced packaging of Vif. Finally, deletion of a putative RNA-interacting domain in Vif abolished packaging of Vif into virions. Virion-associated Vif was resistant to detergent extraction and copurified with components of the viral nucleoprotein complex and functional reverse transcription complexes. Thus, Vif is specifically packaged into virions as a component of the viral nucleoprotein complex. Our data suggest that the specific association of Vif with the viral nucleoprotein complex might be functionally significant and could be a critical requirement for infectivity of viruses produced from restrictive host cells.

Gene Products, vif↗
PubMed1979

Histone modifications in simian virus 40 and in nucleoprotein complexes containing supercoiled viral DNA.

Simian virus (SV40) nucleoprotein complexes containing circular supercoiled viral DNA were extracted from infected cells and purified by differential centrifugation. The protein content of these complexes was compared by electrophoresis on 15% acrylamide gels with the protein content of purified SV40 virions and with histones from virus-infected cells. The electrophoretic patterns of histones from each of the sources revealed several major differences. SV40 virions contained histones H3, H2B, H2A, and H4 but not H1. Nucleoprotein complexes and host cells contained all five major histone groups. Relative to cellular histones, virion and nucleoprotein complex histones were enriched 15 to 40% in histones H3 and H4. In addition to the major classes of histones, several subfractions of histones H1, H3, and H4 were observed in acrylamide gels of proteins from SV40 virions and viral nucleoprotein complexes. Acetate labeling experiments indicated that each subfraction of histones H3 and H4 had a different level of acetylation. The histones from SV40 virions and nucleoprotein complexes were acetylated to significantly higher levels than those of infected host cells. No apparent differences in phosphorylation of the major histone groups were observed.

Acetylation↗
PubMed2006

Viral determinants of integration site preferences of simian immunodeficiency virus-based vectors.

Preferential integration into transcriptionally active regions of genomes has been observed for retroviral vectors based on gamma-retroviruses and lentiviruses. However, differences in the integration site preferences were detected, which might be explained by differences in viral components of the preintegration complexes. Viral determinants of integration site preferences have not been defined. Therefore, integration sites of simian immunodeficiency virus (SIV)-based vectors produced in the absence of accessory genes or lacking promoter and enhancer elements were compared. Similar integration patterns for the different SIV vectors indicate that vif, vpr, vpx, nef, env, and promoter or enhancer elements are not required for preferential integration of SIV into transcriptionally active regions of genomes.

Cells, Cultured↗
PubMed2006

Photochemically enhanced transduction of polymer-complexed adenovirus targeted to the epidermal growth factor receptor.

BACKGROUND: The development of methods for specific delivery of genes into target tissues is an important issue for the further progress of gene therapy. Biological and physical targeting techniques may be combined to redirect gene therapy vectors to specific cells and enhance gene transfer. METHODS: The polymer poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA) was conjugated with avidin or poly(ethylene glycol) (PEG) and complexed with adenovirus serotype 5 (Ad5). Targeting of polymer-coated Ad5 to the epidermal growth factor receptor (EGFR) was accomplished by the binding of biotin-EGF to pDMAEMA-avidin. A photochemical treatment procedure using photosensitizer and light was applied to increase transduction with EGFR-targeted viral complexes. RESULTS: pDMAEMA-avidin efficiently enhanced transduction through unspecific viral uptake into cells, while pDMAEMA-PEG provided charge shielding of the complexes and increased the specificity to EGFR when biotin-EGF ligands were used. Transduction of PEG-containing, EGFR-targeted viral complexes was inhibited by 66% in coxsackie and adenovirus receptor (CAR)-deficient RD cells and by 47% in CAR-expressing DU 145 cells in receptor antibody experiments. The photochemical treatment had a substantial effect on transduction, enhancing the percentage of reporter gene positive cells from 20% to 75% of the total viable RD cell population and from 10% to 70% in DU 145 cells. CONCLUSION: Photochemical treatment of cells infected with targeted viral vectors exhibiting a neutral surface charge is a potent method for enhancing transgene expression.

Adenoviridae↗
PubMed2001

Population genetic analysis of the protease locus of human immunodeficiency virus type 1 quasispecies undergoing drug selection, using a denaturing gradient-heteroduplex tracking assay.

Monitoring the evolution of human immunodeficiency virus type 1 (HIV-1) drug resistance requires measuring the frequency of closely related genetic variants making up the complex viral quasispecies found in vivo. In order to resolve both major and minor (>/=2%) protease gene variants differing by one or more nucleotide substitutions, we analyzed PCR products derived from plasma viral quasispecies by using a combination of denaturing gradient gel electrophoresis and DNA heteroduplex tracking assays. Correct population sampling of the high level of genetic diversity present within viral quasispecies could be documented by parallel analysis of duplicate, independently generated PCR products. The composition of genetically complex protease gene quasispecies remained constant over short periods of time in the absence of treatment and while plasma viremia fell >100-fold following the initiation of protease inhibitor ritonavir monotherapy. Within a month of initiating therapy, a strong reduction in the genetic diversity of plasma viral populations at the selected protease locus was associated with rising plasma viremia and the emergence of drug resistance. The high levels of protease genetic diversity seen before treatment reemerged only months later. In one patient, reduction in genetic diversity at the protease gene was observed concomitantly with an increase in diversity at the envelope gene (E. L. Delwart, P. Heng, A. Neumann, and M. Markowitz, J. Virol. 72:2416-2421, 1998), indicating that opposite population genetic changes can take place in different HIV-1 loci. The rapid emergence of drug-resistant HIV-1 was therefore associated with a strong, although only transient, reduction in genetic diversity at the selected locus. The denaturing gradient-heteroduplex tracking assay is a simple method for the separation and quantitation of very closely related, low-frequency, genetic variants within complex viral populations.

Biological Evolution↗
PubMed2000

When two into one won't go: fitting in the presence of steric hindrance and partial occupancy.

Combining structural data from cryo-electron microscopy (cryo-EM) and X-ray crystallography to give pseudo-atomic models of large molecular complexes has proved particularly suitable for studying viruses and viral complexes. Several groups are developing programs to fit X-ray data to EM data. These programs are in general tailored to particular problems with regard to size, symmetry, number of rigid bodies, resolution etc. Here, two approaches are described to fitting X-ray data to EM data in the presence of steric interference and their relative merits and limitations are indicated. These fitting techniques are applied to the case of the rotavirus double-layered particle (DLP) in complex with antibodies which inhibit the transcription of mRNA by the DLP. This is a particularly good test case, as the cryo-electron microscopy map of the DLP-Fab complex, the X-ray structure of the viral protein (VP6) and also that of the VP6-Fab complex are available. The estimation of partial occupancy is also considered.

Animals↗