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PubMed2002

Knotting probability of DNA molecules confined in restricted volumes: DNA knotting in phage capsids.

When linear double-stranded DNA is packed inside bacteriophage capsids, it becomes highly compacted. However, the phage is believed to be fully effective only if the DNA is not entangled. Nevertheless, when DNA is extracted from a tailless mutant of the P4 phage, DNA is found to be cyclic and knotted (probability of 0.95). The knot spectrum is very complex, and most of the knots have a large number of crossings. We quantified the frequency and crossing numbers of these knots and concluded that, for the P4 tailless mutant, at least half the knotted molecules are formed while the DNA is still inside the viral capsid rather than during extraction. To analyze the origin of the knots formed inside the capsid, we compared our experimental results to Monte Carlo simulations of random knotting of equilateral polygons in confined volumes. These simulations showed that confinement of closed chains to tightly restricted volumes results in high knotting probabilities and the formation of knots with large crossing numbers. We conclude that the formation of the knots inside the viral capsid is driven mainly by the effects of confinement.

Bacteriophages↗

PubMed2005

DNA knots reveal a chiral organization of DNA in phage capsids.

Icosahedral bacteriophages pack their double-stranded DNA genomes to near-crystalline density and achieve one of the highest levels of DNA condensation found in nature. Despite numerous studies, some essential properties of the packaging geometry of the DNA inside the phage capsid are still unknown. We present a different approach to the problems of randomness and chirality of the packed DNA. We recently showed that most DNA molecules extracted from bacteriophage P4 are highly knotted because of the cyclization of the linear DNA molecule confined in the phage capsid. Here, we show that these knots provide information about the global arrangement of the DNA inside the capsid. First, we analyze the distribution of the viral DNA knots by high-resolution gel electrophoresis. Next, we perform Monte Carlo computer simulations of random knotting for freely jointed polygons confined to spherical volumes. Comparison of the knot distributions obtained by both techniques produces a topological proof of nonrandom packaging of the viral DNA. Moreover, our simulations show that the scarcity of the achiral knot 4(1) and the predominance of the torus knot 5(1) over the twist knot 5(2) observed in the viral distribution of DNA knots cannot be obtained by confinement alone but must include writhe bias in the conformation sampling. These results indicate that the packaging geometry of the DNA inside the viral capsid is writhe-directed.

Bacteriophages↗

PubMed2005

Parvoviral virions deploy a capsid-tethered lipolytic enzyme to breach the endosomal membrane during cell entry.

Enveloped viruses deliver their virions into the host cell by fusion with the cellular plasma or endosomal membrane, thus creating topological continuity between the cytosol and the inside of the viral envelope. Nonenveloped viruses are, by their very nature, denied this strategy and must employ alternative methods to breach their host cell's delimiting membrane. We show here that the compact icosahedral parvoviral virion gains entry by deploying a lipolytic enzyme, phospholipase A(2) (PLA(2)), that is expressed at the N terminus of VP1, the minor coat protein. This region of VP1 is normally sequestered within the viral shell but is extruded during the entry process as a capsid-tethered domain. A single amino acid substitution in the active site of the VP1 PLA(2) inactivates enzymatic activity and abrogates infectivity. We have used transencapsidation of a vector expressing green fluorescent protein to show that infection by this PLA(2)-defective mutant can be complemented by coinfection with wild-type or mutant full virions, provided they can express a functional PLA(2). Even though wild-type empty capsids contain an active form of the enzyme, it is not externalized under physiological conditions, and such capsids are not able to complement the PLA(2) mutant. Significantly, highly efficient rescue can be achieved by polyethyleneimine-induced endosome rupture or by coinfection with adenovirus as long as uptake of the two viruses is simultaneous and the adenovirus is capable of deploying pVI, a capsid protein with endosomolytic activity. Together, these results demonstrate a previously unrecognized enzymatic mechanism for nonenveloped virus penetration.

Amino Acid Sequence↗

PubMed1989

Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1.

The effects on human immunodeficiency virus type 1 virion morphogenesis and on virus replication of mutations that affect posttranslational processing of the capsid precursor protein are described. A change in the glycine residue at position two from the N terminus abolishes the myristoylation of the precursor proteins and also prevents virus particle release. Mutations in the viral protease gene abolish proteolytic cleavage of the capsid precursor but do not prevent the formation and budding of virion particles of immature appearance. Mutations that alter the sequence of the sites normally used for cleavage of the major capsid protein p24 from the capsid precursor alter virion morphogenesis and prevent virus replication.

Animals↗

PubMed1991

Identification of an immunodominant epitope within the capsid protein of hepatitis C virus.

We have isolated cDNA clones from the 5' end of the Hutchinson strain of hepatitis C virus. Sequences encoding various segments of the HCV structural region were fused to the gene for glutathione S-transferase and analyzed for the expression of hepatitis C virus-capsid fusion proteins. With a set of these fusion proteins, both human and chimpanzee immune responses to capsid were studied. An immunodominant epitope was located within the amino-terminal portion of capsid that is preferentially recognized by antibodies in both human and chimpanzee hepatitis C virus-positive sera. In addition, analyses of sequential serum samples taken from humans and chimpanzees with either chronic or apparently self-limited infections revealed that a strong anti-capsid response develops rapidly after onset of infection.

Amino Acid Sequence↗

PubMed1995

Single-site cleavage in the 5'-untranslated region of Leishmaniavirus RNA is mediated by the viral capsid protein.

Leishmaniavirus (LRV) is a double-stranded RNA virus that persistently infects the protozoan parasite Leishmania. LRV produces a short RNA transcript, corresponding to the 5' end of positive-sense viral RNA, both in vivo and in in vitro polymerase assays. The short transcript is generated by a single site-specific cleavage event in the 5' untranslated region of the 5.3-kb genome. This cleavage event can be reproduced in vitro with purified viral particles and a substrate RNA transcript possessing the viral cleavage site. A region of nucleotides required for cleavage was identified by analyzing the cleavage sites yielding the short transcripts of various LRV isolates. A 6-nt deletion at this cleavage site completely abolished RNA processing. In an in vitro cleavage assay, baculovirus-expressed capsid protein possessed an endonuclease activity identical to that of native virions, showing that the viral capsid protein is the RNA endonuclease. Identification of the LRV capsid protein as an RNA endonuclease is unprecedented among known viral capsid proteins.

Animals↗

PubMed1996

Association with capsid proteins promotes nuclear targeting of simian virus 40 DNA.

All animal DNA viruses except pox virus utilize the cell nucleus as the site for virus reproduction. Yet, a critical viral infection process, nuclear targeting of the viral genome, is poorly understood. The role of capsid proteins in nuclear targeting of simian virus 40 (SV40) DNA, which is assessed by the nuclear accumulation of large tumor (T) antigen, the initial sign of the infectious process, was tested by two independent approaches: antibody interception experiments and reconstitution experiments. When antibody against viral capsid protein Vp1 or Vp3 was introduced into the cytoplasm, the nuclear accumulation of T antigen was not observed in cells either infected or cytoplasmically injected with virion. Nuclearly introduced anti-Vp3 IgG also showed the inhibitory effect. In the reconstitution experiments, SV40 DNA was allowed to interact with protein components of the virus, either empty particles or histones, and the resulting complexes were tested for the capability of protein components to target the DNA to the nucleus from cytoplasm as effectively as the targeting of DNA in the mature virion. In cells injected with empty particle-DNA, but not in minichromosome-injected cells, T antigen was observed as effectively as in SV40-injected cells. These results demonstrate that SV40 capsid proteins can facilitate transport of SV40 DNA into the nucleus and indicate that Vp3, one of the capsid proteins, accompanies SV40 DNA as it enters the nucleus during virus infection.

Animals↗

PubMed1998

Localization of the N terminus of hepatitis B virus capsid protein by peptide-based difference mapping from cryoelectron microscopy.

Recently, cryoelectron microscopy of isolated macromolecular complexes has advanced to resolutions below 10 A, enabling direct visualization of alpha-helical secondary structure. To help correlate such density maps with the amino acid sequences of the component proteins, we advocate peptide-based difference mapping, i. e., insertion of peptides, approximately 10 residues long, at targeted points in the sequence and visualization of these peptides as bulk labels in cryoelectron microscopy-derived difference maps. As proof of principle, we have appended an extraneous octapeptide at the N terminus of hepatitis B virus capsid protein and determined its location on the capsid surface by difference imaging at 11 A resolution. Hepatitis B virus capsids are icosahedral particles, approximately 300 A in diameter, made up of T-shaped dimers (subunit Mr, 16-21 kDa, depending on construct). The stems of the Ts protrude outward as spikes, whereas the crosspieces pack to form the contiguous shell. The two N termini per dimer reside on either side of the spike-stem, at the level at which it enters the shell. This location is consistent with formation of the known intramolecular disulfide bond between the cysteines at positions 61 and -7 (in the residual propeptide) in the "e-antigen" form of the capsid protein and has implications for why this clinically important antigen remains unassembled in vivo.

Amino Acid Sequence↗

PubMed2002

Nuclear import strategies of high risk HPV16 L1 major capsid protein.

During the late phase of human papillomavirus (HPV) infection, the L1 major capsid proteins enter the nuclei of host epithelial cells and, together with the L2 minor capsid proteins, assemble the replicated viral DNA into virions. We investigated the nuclear import of the L1 major capsid protein of high risk HPV16. When digitonin-permeabilized HeLa cells were incubated with HPV16 L1 capsomeres, the L1 protein was imported into the nucleus in a receptor-mediated manner. HPV16 L1 capsomeres formed complexes with Kap alpha2beta1 heterodimers via interaction with Kap alpha2. Accordingly, nuclear import of HPV16 L1 capsomeres was mediated by Kap alpha2beta1 heterodimers, required RanGDP and free GTP, and was independent of GTP hydrolysis. Remarkably, HPV16 L1 capsomeres also interacted with Kap beta2 and binding of RanGTP to Kap beta2 did not dissociate the HPV16 L1.Kap beta2 complex. Significantly, HPV16 L1 capsomeres inhibited the nuclear import of Kap beta2 and of a Kap beta2-specific M9-containing cargo. These data suggest that, during the productive stage of infection, while the HPV16 L1 major capsid protein enters the nucleus via the Kap alpha2beta1-mediated pathway to assemble the virions, it also inhibits the Kap beta2-mediated nuclear import of host hnRNP A1 protein and, in this way, favors virion formation.

Active Transport, Cell Nucleus↗

PubMed2004

Mutational analysis of essential interactions involved in the assembly of hepatitis E virus capsid.

The hepatitis E virus (HEV) capsid consists of a single structural protein, a portion of which is engaged in isosahedral contact to form a basal shell, and another portion in dimeric contact to form the homodimers protruding from the shell. Previous studies revealed that homodimers of the truncated HEV capsid proteins, E2 (amino acids 394-606) and p239 (amino acids 368-606), model dominant antigenic determinants of HEV. Immunization with these proteins protected rhesus monkeys against the virus, and three monoclonal antibodies against the homodimers could neutralize HEV infectivity and/or immune-capture of the virus. Furthermore, homodimers of p239 further interact to form particles of 23 nm diameter, rendering it an efficacious candidate vaccine. In light of this we postulate that the interactions involved in the formation of the homodimers and particles might be similar to those involved in assembly of the virus capsid. Presently, mutational analysis was carried out to identify these sites of interactions. The site of dimeric interactions was located to a cluster of six hydrophobic amino acids residues, Ala597, Val598, Ala599, Leu601, and Ala602; furthermore, the site involved in particle formation was located at amino acids 368-394. The possibility that these sites are also involved in assembly of the virus capsid is supported by the fact that they are located at two major and highly conserved hydrophobic regions of the HEV structural protein.

Capsid Proteins↗

PubMed1983

Simian virus 40 chromatin interaction with the capsid proteins.

It has been established that both in virions and in infected cells, the cellular core histones fold the SV40 DNA into nucleosomes to form the SV40 chromosome or chromatin. We and others have begun to examine how the capsid proteins assemble the SV40 chromatin into virions and to investigate whether these proteins interact with the encapsidated chromatin. To follow the pathway of virus assembly, we have analyzed the nucleoproteins which accumulate in cells infected with the SV40 mutants temperature-sensitive in assembly: tsC, tsBC, and tsB. (The temperature-sensitivity of these mutants result from alterations in the amino acid sequence of the major capsid protein VP1). We have found that mutants belonging to the same class accumulate similar types of nucleoproteins at the nonpermissive temperature (40 degrees C) and thus, share characteristics in common. For example, the tsC mutants accumulate only the 75 S chromatin. Both tsBC and tsB mutants produce in addition to chromatin, nucleoprotein complexes which sediment broadly from 100-160 S and contain all the three capsid proteins VP1, VP2, and VP3. These nucleoproteins can be distinguished morphologically, however. Under the electron microscope, the tsBC 100-160 S nucleoproteins appear as chromatin to which a small cluster of the capsid proteins is attached; the tsB nucleoproteins appear as partially assembled virions. In addition, we find that the 220 S virions are assembled in cells coinfected with tsB and tsC mutants at 40 degrees C, in agreement with genetic analysis. Our observations favor the hypothesis that the VP1 protein contains three discrete domains. We speculate that each domain may play a specific function in SV40 assembly. To gain more insight into VP1-VP1 interactions, we have examined the nucleoproteins which result from treatment of the mature wild-type virions with increasing concentrations of the reducing agent DTT. In the presence of as low a concentration of DTT as 0.1 mM, the virion shell can be penetrated by micrococcal nuclease, which then cleaves the viral DNA. This result indicates that some of the disulfide bonds bridging the VP1 proteins are on the virion surface.

Capsid↗

PubMed2003

The role of capsid-endothelial interactions in the innate immune response to adenovirus vectors.

Adenovirus (Ad) vectors can produce inflammatory responses at high doses. Intravenous administration of an Ad vector expressing green fluorescent protein (AdGFP) to naive mice induced a biphasic pattern of liver cytokine/chemokine gene expression over 7 days. Tumor necrosis factor alpha (TNF-alpha), macrophage inflammatory protein 2 (MIP-2), and interferon gamma-inducible protein 10 (IP-10) genes were upregulated, with two distinct peaks of mRNA expression occurring at 6 hr and 5 days. The administration of transcription-defective AdGFP particles induced the early but not the late peak of chemokine/cytokine gene expression, confirming that Ad vector-induced inflammation is capsid dependent in the early phase and transcription dependent in the late phase. To determine the role of adenoviral capsid motifs in the early phase, capsid-modified Ad vectors were employed. The intravenous administration of the RGD-deleted Ad vector AdL.PB*, the fiber mutant AdL.F*, or the double mutant AdL.F*PB* induced similar levels of cytokine/chemokine expression compared with the wild-type vector AdLuc. Kupffer cell blockade significantly reduced liver TNF-alpha, MIP-2, and IP-10 gene expression and liver inflammation after the administration of AdL.PB* or AdL.F*PB*. Fluorescence microscopy of AdLuc- and AdL.PB*-transduced liver at 1 hr revealed localization of Ad vectors to liver sinusoids in Kupffer cell-depleted mice. AdL.PB* induced less E-selectin and VCAM-1 gene expression in liver, confirming reduced endothelial activation in mice receiving RGD-deleted Ad vectors. In vitro studies of endothelial cells demonstrated reduced transduction and endothelial activation by AdL.PB* compared with AdLuc. These results demonstrate that adenovirus capsid RGD motifs are required for efficient transduction and endothelial cell activation. Altering vector tropism represents a feasible strategy to modulate the innate response to Ad vectors in nontargeted tissues.

Adenoviridae↗

PubMed2001

Electron cryo-microscopy and image reconstruction of adeno-associated virus type 2 empty capsids.

Adeno-associated virus type 2 empty capsids are composed of three proteins, VP1, VP2 and VP3, which have relative molecular masses of 87, 72 and 62 kDa, respectively, and differ in their N-terminal amino acid sequences. They have a likely molar ratio of 1:1:8 and occupy symmetrical equivalent positions in an icosahedrally arranged protein shell. We have investigated empty capsids of adeno-associated virus type 2 by electron cryo-microscopy and icosahedral image reconstruction. The three-dimensional map at 1.05 nm resolution showed sets of three elongated spikes surrounding the three-fold symmetry axes and narrow empty channels at the five-fold axes. The inside of the capsid superimposed with the previously determined structure of the canine parvovirus (Q. Xie and M.S. Chapman, 1996, J. Mol. Biol., 264, 497-520), whereas the outer surface showed clear discrepancies. Globular structures at the inner surface of the capsid at the two-fold symmetry axes were identified as possible positions for the N-terminal extensions of VP1 and VP2.

Blotting, Western↗

PubMed2001

DNA helicase-mediated packaging of adeno-associated virus type 2 genomes into preformed capsids.

Helicases not only catalyse the disruption of hydrogen bonding between complementary regions of nucleic acids, but also move along nucleic acid strands in a polar fashion. Here we show that the Rep52 and Rep40 proteins of adeno-associated virus type 2 (AAV-2) are required to translocate capsid-associated, single-stranded DNA genomes into preformed empty AAV-2 capsids, and that the DNA helicase function of Rep52/40 is essential for this process. Furthermore, DNase protection experiments suggest that insertion of AAV-2 genomes proceeds from the 3' end, which correlates with the 3'-->5' processivity demonstrated for the Rep52/40 helicase. A model is proposed in which capsid-immobilized helicase complexes act as molecular motors to 'pump' single-stranded DNA across the capsid boundary.

Capsid↗

PubMed1986

Relation between genomic and capsid structures in RNA viruses.

We described a new computer program for calculation of RNA secondary structure. Calculation of 20 viral RNAs with this program showed that genomes of the icosahedral capsid viruses had higher folding probabilities than those of the helical capsid viruses. As this explains virus assembly quite well, the information of capsid structure must be imprinted not only in the capsid protein structures but also in the base sequence of the whole genome. We compared folding probability of the original sequence with that of the random sequence in which base composition was the same as the original. All the actual genomes of RNA viruses were more folded than the corresponding random sequences, even though most transcripts of chromosomal genes tended to be less folded. The data can be related to encapsidation of viral genomes. It was thus suggested that there exists a relation between actual sequences and random sequences with the same base ratios, and that the base ratio itself has some evolutional meaning.

Animals↗

PubMed1996

Electron microscopic observations of aberrant capsids of pseudorabies virus.

Interactions between four strains of pseudorabies virus (PrV) and seven kinds of cell lines were examined. Three kinds of cells (SKL, CPK and PK-15) were especially infected with PrV at an MOI of about 10 PFC/cell. At sequential intervals after infection, cells of these types were collected for electron microscopic observations and the infectious doses of culture fluids were assayed. Developmental features of PrV were found to be very similar to those of herpes simplex virus (HSV), with the slight difference that PrV developed a little earlier and more vigorously than did HSV. At the late stage of infection, aberrant capsids of PrV were observed frequently in the nucleus of SKL but rarely in the nuclei of CPK and PK-15. The titer of infectious virus produced by SKL was much larger than that produced by CPK and PK-15. Immuno-electron microscopic examination using a monoclonal antibody against the major capsid protein of PrV clearly demonstrated that the complete and aberrant capsids have a common epitope. The mechanism of the formation of aberrant capsids is discussed.

Animals↗

PubMed2005

Transduction of pancreatic islets with pseudotyped adeno-associated virus: effect of viral capsid and genome conversion.

BACKGROUND: Recombinant adeno-associated viral (rAAV) vectors currently show promise for islet gene therapy. In the presence of complementing AAV2 Rep proteins, AAV2 genomes can be packaged with other serotype capsids to assemble infectious virions. During transduction, the ssDNA to dsDNA conversion is one of the major rate-limiting steps that contribute to the slow onset of transgene expression. METHODS: Using pseudotyping strategy, we produced double-stranded (dsAAV) and single-stranded (ssAAV) rAAV2 genomes carrying the GFP reporter gene packaged into AAV1, AAV2, and AAV5 capsids. The ability of cross-packaged AAV1, AAV2, and AAV5 at the same genome containing particle (gcp) concentration to transduce murine and human pancreatic islets was evaluated by GFP positive cell percentage. Transgenic expression was also determined by transplant transduced human islet into SCID mice. RESULTS: Pseudotyped rAAV2/1 based vectors transduced murine islets at greater efficiency than either rAAV2/2 or rAAV2/5 vectors. For human islets transduction, the rAAV2/2 vector was more efficient than rAAV2/1 or rAAV2/5 vectors. rAAV2/2 transduced human islets more efficiently than murine islets, while rAAV2/1 transducted murine islets more efficiently than human islets. dsAAV, which do not require second strand synthesis and thus are potentially more efficient, evidenced 5 fold higher transduction ability than ssAAV vectors. Pseudotyped rAAV transduced islet grafts maintained normal function, expressed transgenic product persistently in vivo, and reversed diabetes. CONCLUSIONS: The transduction efficiency of rAAV vectors was dependent on the cross-packaged capsid. The vector capsids permit species-specific transduction. For human islets, dsAAV2/2 vectors may be the most efficient vector for clinical development.

Adenoviridae↗

PubMed1976

Structure and assembly of the capsid of bacteriophage P22.

Identification of the genes and proteins involved in phage P22 formation has permitted a detailed analysis of particle assembly, revealing some unexpected aspects. The polymerization of the major coat protein (gene 5 product) into an organized capsid is directed by a scaffolding protein (gene 8 product) which is absent from mature phage. The resulting capsid structure (prohead) is the precursor for DNA encapsidation. All of the scaffolding protein exits from the prohead in association with DNA packaging. These molecules then recycle, directing further rounds of prohead assembly. The structure of the prohead has been studied by electron microscopy of thin sections of phage infected cells, and by low angle X-ray scattering of concentrated particles. The results show that the prohead is a double shell structure, or a ball within a shell. The inner ball or shell is composed of the scaffolding protein while the outer shell is composed of coat protein. The conversion from prohead to mature capsid is associated with an expansion of the coat protein shell. It is possible that the scaffolding protein molecules exit through the capsid lattice. When DNA encapsidation within infected cells is blocked by mutation, scaffolding protein is trapped in proheads and cannot recycle. Under these conditions, the rate of synthesis of gp8 increases, so that normal proheads continue to form. These results suggest that free scaffolding protein negatively regulates its own further synthesis, providing a coupling between protein synthesis and protein assembly.

Capsid↗