Full Capsids Research Articles

Efficient Capsid Antigen Presentation From Adeno-Associated Virus Empty Virions In Vivo.

Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials for hemophilic patients. Although promising, the clinical results suggest that the capsid-specific CD8+T cell response has a negative effect on therapeutic success. In an in vitro analysis using an engineered AAV virus carrying immune-dominant SIINFEKL peptide in the capsid backbone, we have previously demonstrated that capsid antigen presentation from full (genome containing) AAV capsids requires endosome escape and is proteasome dependent and that no capsid antigen presentation is induced from empty virions. In the present study, we examined capsid antigen presentation from administration of empty virions in animal models. In wild-type mice, similar to AAV full particles, capsid antigen presentation from AAV empty virion infection was dose dependent, and the kinetics studies showed that antigen presentation was detected from 2 to 40 days after AAV empty virion administration. In the transporter associated with antigen processing 1 deficient (TAP−/−) mice, capsid antigen presentation was inhibited from both AAV full and empty virions, but higher inhibition was achieved from AAV full particle administration than that from empty virions. This indicates that the pathway of capsid antigen presentation from AAV transduction is dependent on proteasome-mediated degradation of AAV capsids (mainly for full particles) and that the endosomal pathway may also play a role in antigen presentation from empty particles but not full virions. The capsid antigen presentation efficiency from AAV preparations was positively correlated with the amount of empty virions contaminated with full particles. Collectively, the results indicate that contamination of AAV empty virions induces efficient antigen presentation in vivo and the mechanism of capsid antigen presentation from empty virions involves both endosomal and proteasomal pathways. The elucidation of capsid antigen presentation from AAV empty virions may allow us to rationally design effective strategies to prevent elimination of AAV transduced target cells by capsid specific CD8+ T cells.

Impact of Inverted Terminal Repeat Integrity on rAAV8 Production Using the Baculovirus/Sf9 Cells System.

Adeno-associated virus (AAV) inverted terminal repeats (ITRs) are key elements of AAV. These guanine-cytosine-rich structures are involved in the replication and encapsidation of the AAV genome, along with its integration in and excision from the host genome. These sequences are the only AAV-derived DNA sequences conserved in recombinant AAV (rAAV), as they allow its replication, encapsidation, and long-term maintenance and expression in target cells. Due to the original vector design, plasmids containing the gene of interest flanked by ITRs and used for rAAV production often present incomplete, truncated, or imperfect ITR sequences. For example, pSUB201 and its derivatives harbor a truncated (14 nt missing on the external part of the ITR), flop-orientated ITR plus 46 bp of non-ITR viral DNA at each end of the rAAV genome. It has been shown that rAAV genomes can be replicated, even with incomplete, truncated, or imperfect ITR sequences, leading to the production of rAAV vectors in transfection experiments. Nonetheless, it was hypothesized that unmodified wild-type (WT) ITR sequences could lead to a higher yield of rAAV, with less non-rAAV encapsidated DNA originating from the production cells and/or baculovirus shuttle vector genomes. This work studied the impact of imperfect ITRs on the level of encapsidated rAAV genomes and baculovirus-derived DNA sequences using the baculovirus/Sf9 cells production system. Replacement of truncated ITRs with WT and additional wtAAV2 sequences has an impact on the two major features of rAAV production: (1) a rise from 10% to 40% of full capsids obtained, and (2) up to a 10-fold reduction in non-rAAV encapsidated DNA. Furthermore, this study considered the impact on these major parameters of additional ITR elements and ITRs coupled with various regulatory elements of different origins. Implementation of the use of complete ITRs in the frame of the baculovirus-based rAAV expression system is one step that will be required to optimize the quality of rAAV-based gene therapy drugs.

Resolving Adeno-Associated Viral Particle Diversity With Charge Detection Mass Spectrometry.

Recombinant adeno-associated viruses (AAVs) are promising vectors for human gene therapy. However, current methods for evaluating AAV particle populations and vector purity are inefficient and low resolution. Here, we show that charge detection mass spectrometry (CDMS) can resolve capsids that contain the entire vector genome from those that contain partial genomes and from empty capsids. Measurements were performed for both single-stranded and self-complementary genomes. The self-complementary AAV vector preparation appears to contain particles with partially truncated genomes averaging at half the genome length. Comparison to results from electron microscopy with manual particle counting shows that CDMS has no significant mass discrimination in the relevant mass range (after a correction for the ion velocity is taken into account). Empty AAV capsids are intrinsically heterogeneous, and capsids from different sources have slightly different masses. However, the average masses of both the empty and full capsids are in close agreement with expected values. Mass differences between the empty and full capsids for both single-stranded and self-complementary AAV vectors indicate that the genomes are largely packaged without counterions.

Late Maturation Steps Preceding Selective Nuclear Export and Egress of Progeny Parvovirus.

Although the mechanism is not well understood, growing evidence indicates that the nonenveloped parvovirus minute virus of mice (MVM) may actively egress before passive release through cell lysis. We have dissected the late maturation steps of the intranuclear progeny with the aims of confirming the existence of active prelytic egress and identifying critical capsid rearrangements required to initiate the process. By performing anion-exchange chromatography (AEX), we separated intranuclear progeny particles by their net surface charges. Apart from empty capsids (EC), two distinct populations of full capsids (FC) arose in the nuclei of infected cells. The earliest population of FC to appear was infectious but, like EC, could not be actively exported from the nucleus. Further maturation of this early population, involving the phosphorylation of surface residues, gave rise to a second, late population with nuclear export potential. While capsid surface phosphorylation was strictly associated with nuclear export capacity, mutational analysis revealed that the phosphoserine-rich N terminus of VP2 (N-VP2) was dispensable, although it contributed to passive release. The reverse situation was observed for the incoming particles, which were dephosphorylated in the endosomes. Our results confirm the existence of active prelytic egress and reveal a late phosphorylation event occurring in the nucleus as a selective factor for initiating the process. In general, the process of egress of enveloped viruses is active and involves host cell membranes. However, the release of nonenveloped viruses seems to rely more on cell lysis. At least for some nonenveloped viruses, an active process before passive release by cell lysis has been reported, although the underlying mechanism remains poorly understood. By using the nonenveloped model parvovirus minute virus of mice, we could confirm the existence of an active process of nuclear export and further characterize the associated capsid maturation steps. Following DNA packaging in the nucleus, capsids required further modifications, involving the phosphorylation of surface residues, to acquire nuclear export potential. Inversely, those surface residues were dephosphorylated on entering capsids. These spatially controlled phosphorylation-dephosphorylation events concurred with the nuclear export-import potential required to complete the infectious cycle.

The structure of Sinorhizobium meliloti phage ΦM12, which has a novel T=19l triangulation number and is the founder of a new group of T4-superfamily phages

ΦM12 is the first example of a T=19l geometry capsid, encapsulating the recently sequenced genome. Here, we present structures determined by cryo-EM of full and empty capsids. The structure reveals the pattern for assembly of 1140 HK97-like capsid proteins, pointing to interactions at the pseudo 3-fold symmetry axes that hold together the asymmetric unit. The particular smooth surface of the capsid, along with a lack of accessory coat proteins encoded by the genome, suggest that this interface is the primary mechanism for capsid assembly. Two-dimensional averages of the tail, including the neck and baseplate, reveal that ΦM12 has a relatively narrow neck that attaches the tail to the capsid, as well as a three-layer baseplate. When free from DNA, the icosahedral edges expand by about 5nm, while the vertices stay at the same position, forming a similarly smooth, but bowed, T=19l icosahedral capsid.

Effect of Envelope Proteins on the Mechanical Properties of Influenza Virus

The envelope of the influenza virus undergoes extensive structural change during the viral life cycle. However, it is unknown how lipid and protein components of the viral envelope contribute to its mechanical properties. Using atomic force microscopy, here we show that the lipid envelope of spherical influenza virions is ∼10 times softer (∼0.05 nanonewton nm(-1)) than a viral protein-capsid coat and sustains deformations of one-third of the virion's diameter. Compared with phosphatidylcholine liposomes, it is twice as stiff, due to membrane-attached protein components. We found that virus indentation resulted in a biphasic force-indentation response. We propose that the first phase, including a stepwise reduction in stiffness at ∼10-nm indentation and ∼100 piconewtons of force, is due to mobilization of membrane proteins by the indenting atomic force microscope tip, consistent with the glycoprotein ectodomains protruding ∼13 nm from the bilayer surface. This phase was obliterated for bromelain-treated virions with the ectodomains removed. Following pH 5 treatment, virions were as soft as pure liposomes, consistent with reinforcing proteins detaching from the lipid bilayer. We propose that the soft, pH-dependent mechanical properties of the envelope are critical for the pH-regulated life cycle and support the persistence of the virus inside and outside the host.

Adenovirus Virion Stability and the Viral Genome: Size Matters

Ongoing investigations into the fundamental aspects of virus biology continue to provide crucial insights for the development of more effective virus-based therapeutics. Within the field of gene therapy, these discoveries have translated into virus vectors with improved tissue tropism, prolonged transgene expression, and enhanced safety. However, radical modifications to the viral genome or capsid constituents can have marked effects on virion stability. In particular, recent insights into the role of viral genomes in contributing to the physical stability of the virion highlight the need to reconsider this heretofore underappreciated aspect of vector design. Here we discuss the impact of the genome on virion stability, with particular emphasis on the development of adenovirus (Ad)-based vectors.

Interaction of Parvovirus B19 with Human Erythrocytes Alters Virus Structure and Cell Membrane Integrity

The unique region of the capsid protein VP1 (VP1u) of B19 virus (B19V) elicits a dominant immune response and has a phospholipase A(2) (PLA(2)) activity required for the infection. Despite these properties, we have observed that the VP1u-PLA(2) motif occupies an internal position in the capsid. However, brief exposure to increasing temperatures induced a progressive accessibility of the PLA(2) motif as well as a proportional increase of the PLA(2) activity. Similarly, upon binding on human red blood cells (RBCs), a proportion of the capsids externalized the VP1u-PLA(2) motif. Incubation of B19V with RBCs from 17 healthy donors resulted in extensive virus attachment ranging between 3,000 and 30,000 virions per cell. B19V empty capsids represent an important fraction of the viral particles circulating in the blood (30 to 40%) and bind to RBCs in the same way as full capsids. The extensive B19V binding to RBCs did not cause direct hemolysis but an increased osmotic fragility of the cells by a mechanism involving the PLA(2) activity of the exposed VP1u. Analysis of a blood sample from an individual with a recent B19V infection revealed that, at this particular moment of the infection, the virions circulating in the blood were mostly associated to the RBC fraction. However, the RBC-bound B19V was not able to infect susceptible cells. These observations indicate that RBCs play a significant role during B19V infection by triggering the exposure of the immunodominant VP1u including its PLA(2) constituent. On the other hand, the early exposure of VP1u might facilitate viral internalization and/or uncoating in target cells.

Deciphering the Kinetic Mechanism of Spontaneous Self-Assembly of Icosahedral Capsids

Self-assembly of viral proteins into icosahedral capsids is an interesting yet poorly understood phenomenon of which elucidation may aid the exploration of beneficial applications of capsids in materials science and medicine. Using molecular dynamics simulations of coarse-grained models for capsid proteins, we show that the competition between the formation of full capsids and nonidealized structures is strongly dependent upon the protein concentration and temperature, occurring kinetically as a cascade of elementary reactions in which free monomers are added to the growing oligomers on a downhill free-energy landscape. However, the insertion of the final subunits is the rate-limiting, energetically unfavorable step in viral capsid assembly. A phase diagram has been constructed to show the regions where capsids or nonidealized structures are stable at each concentration and temperature. We anticipate that our findings will provide guidance in identifying suitable conditions required for in vitro viral capsid assembly experiments.

Proteolytic Mapping of the Adeno-associated Virus Capsid

The three-dimensional structures of the viral capsid of three AAV serotypes have previously been determined by X-ray crystallography or cryoelectron microscopy. These studies of AAV and similar studies of autonomous parvoviruses have yielded important structural information about the virions in a low-energy conformation. However, there is little information on the structural properties of AAV virions in solution under physiological conditions. We demonstrate that proteolytic digestion of AAV2 virions with trypsin results in cleavage at a specific site on the capsid surface while the capsid remains intact. The products of digestion were mapped using unique antibodies, protein sequencing, mass spectroscopy, and 3D structure modeling to a region on a surface loop that is common to all three AAV2 structural proteins. Empty AAV2 capsids could be distinguished from full (DNA-containing) capsids, having an increased susceptibility of VP2 to trypsin and being digested more rapidly by chymotrypsin. Proteolytic analysis utilizing trypsin or chymotrypsin was also capable of distinguishing AAV2 from AAV1 and AAV5, as seen by differential susceptibility and unique fragment patterns. These data demonstrate a novel approach for studying the structure of AAV capsids in solution and should be valuable in the testing and engineering of AAV vectors for gene transfer.

Nanoindentation studies of full and empty viral capsids and the effects of capsid protein mutations on elasticity and strength

The elastic properties of capsids of the cowpea chlorotic mottle virus have been examined at pH 4.8 by nanoindentation measurements with an atomic force microscope. Studies have been carried out on WT capsids, both empty and containing the RNA genome, and on full capsids of a salt-stable mutant and empty capsids of the subE mutant. Full capsids resisted indentation more than empty capsids, but all of the capsids were highly elastic. There was an initial reversible linear regime that persisted up to indentations varying between 20% and 30% of the diameter and applied forces of 0.6-1.0 nN; it was followed by a steep drop in force that is associated with irreversible deformation. A single point mutation in the capsid protein increased the capsid stiffness. The experiments are compared with calculations by finite element analysis of the deformation of a homogeneous elastic thick shell. These calculations capture the features of the reversible indentation region and allow Young's moduli and relative strengths to be estimated for the empty capsids.

Quantitation of adenovirus type 5 empty capsids

Adenovirus empty capsids are immature intermediates that lack DNA and viral core proteins. Highly purified preparations of empty and full capsids were generated by subjecting purified adenovirus preparations to repeated cesium chloride gradient separations. PAGE results revealed that empty capsids contain at least five bands that correspond to proteins absent from the mature virus proteome. Peptide mapping by matrix-assisted laser desorption/ionization time-of-flight MS revealed that three of these bands correspond to varying forms of L1 52/55 kDa, a protein involved in the encapsidation of the viral DNA. One band at around 31 kDa was found to include precursors to proteins VI and VIII. These precursors correspond to proteins that have not been cleaved by the adenovirus-encoded protease and are not present in the mature full capsids. The precursor to protein VIII (pVIII), a capsid cement protein, is used in this study as a marker in reverse-phased HPLC (RP-HPLC) analyses of adenovirus for the quantitation of empty capsids. A novel calculation method applied to the integration of RP-HPLC chromatograms allowed for the generation of a percentage empty capsid value in a given adenovirus preparation. The percentage empty capsid values generated to date by this method show a high degree of precision and good agreement with a cesium chloride gradient/SDS–PAGE quantitation method of empty capsids. The advantage of this method lies in the accurate, precise, and rapid generation of the percentage of empty capsids in a given purified virus preparation without relying on tedious and time-consuming cesium chloride gradient separations and extractions.

Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to heparan sulfate proteoglycan binding.

The adeno-associated virus type 2 (AAV2) uses heparan sulfate proteoglycan (HSPG) as its primary cellular receptor. In order to identify amino acids within the capsid of AAV2 that contribute to HSPG association, we used biochemical information about heparin and heparin sulfate, AAV serotype protein sequence alignments, and data from previous capsid studies to select residues for mutagenesis. Charged-to-alanine substitution mutagenesis was performed on individual residues and combinations of basic residues for the production and purification of recombinant viruses that contained a green fluorescent protein (GFP) reporter gene cassette. Intact capsids were assayed for their ability to bind to heparin-agarose in vitro, and virions that packaged DNA were assayed for their ability to transduce normally permissive cell lines. We found that mutation of arginine residues at position 585 or 588 eliminated binding to heparin-agarose. Mutation of residues R484, R487, and K532 showed partial binding to heparin-agarose. We observed a general correlation between heparin-agarose binding and infectivity as measured by GFP transduction; however, a subset of mutants that partially bound heparin-agarose (R484A and K532A) were completely noninfectious, suggesting that they had additional blocks to infectivity that were unrelated to heparin binding. Conservative mutation of positions R585 and R588 to lysine slightly reduced heparin-agarose binding and had comparable effects on infectivity. Substitution of AAV2 residues 585 through 590 into a location predicted to be structurally equivalent in AAV5 generated a hybrid virus that bound to heparin-agarose efficiently and was able to package DNA but was noninfectious. Taken together, our results suggest that residues R585 and R588 are primarily responsible for heparin sulfate binding and that mutation of these residues has little effect on other aspects of the viral life cycle. Interactive computer graphics examination of the AAV2 VP3 atomic coordinates revealed that residues which contribute to heparin binding formed a cluster of five basic amino acids that presented toward the icosahedral threefold axis from the surrounding spike protrusion. Three other kinds of mutants were identified. Mutants R459A, H509A, and H526A/K527A bound heparin at levels comparable to that of wild-type virus but were defective for transduction. Another mutant, H358A, was defective for capsid assembly. Finally, an R459A mutant produced significantly lower levels of full capsids, suggesting a packaging defect.

The Canine Minute Virus (Minute Virus of Canines) Is a Distinct Parvovirus That Is Most Similar to Bovine Parvovirus

We characterized the genome and proteins of the canine minute virus (the minute virus of canines (MVC)). The genome sequence showed MVC to be an autonomous parvovirus encoding a large nonstructural protein 1 gene, a smaller nonstructural protein, and overlapping VP1 and VP2 protein genes. The virus was most closely related to bovine parvovirus (BPV), with which it was 43% identical at the DNA sequence level, while the NS1 and VP1 proteins were 33.6 and 41.4% identical to those of BPV, respectively. Spliced messages of the NS1 gene transcripts were detected by RT-PCR. VP1 and VP2 proteins were detected in purified capsids, as were modified versions of each protein, and VP3 was also found in full capsids.

Canine Parvovirus Capsid Assembly and Differences in Mammalian and Insect Cells

We examined the assembly processes of the capsid proteins of canine parvovirus (CPV) in mammalian and insect cells. In CPV-infected cells empty capsids assembled within 15 min, and then continued to form over the following 1 h, while full (DNA-containing) capsids were detected only after 60 min, and those accumulated slowly over several hours. In cells expressing VP1 and VP2 or only VP2, empty capsid formation was also efficient, but was slightly slower than that in infected cells. Small amounts of trimer forms of VP2 were detected in cells expressing wild type capsid proteins, but were not seen for mutants containing changes that prevented capsid assembly. CPV capsids accumulated in the cell nucleus, but mutant VP1 and VP2 proteins that did not assemble became distributed throughout the nucleus and the cytoplasm, irrespective of whether they were expressed as VP1 and VP2, or as VP2 only. Urea or pH treatment of empty capsids released dimer, trimer, or pentamer capsid protein combinations, while treatment of full capsids consistently released trimer and, in some cases, pentamer forms. When wild type or assembly-defective VP2 genes were expressed from recombinant baculoviruses in insect cells, most of the protein was recovered as noncapsid aggregates, and only a small proportion assembled into capsids. Both the assembled capsids and the noncapsid aggregates were seen primarily in the cytoplasm of the insect cells. The VP2 expressed in insect cells that was recovered in aggregates had an isoelectric point of about pH 6.3, while that recovered from assembled capsids had a pI of about 5.2, similar to that seen for the VP2 of capsids recovered from mammalian cells.

Cytoplasmic trafficking of the canine parvovirus capsid and its role in infection and nuclear transport.

To begin a successful infection, viruses must first cross the host cell plasma membrane, either by direct fusion with the membrane or by receptor-mediated endocytosis. After release into the cytoplasm those viruses that replicate in the nucleus must target their genome to that location. We examined the role of cytoplasmic transport of the canine parvovirus (CPV) capsid in productive infection by microinjecting two antibodies that recognize the intact CPV capsid into the cytoplasm of cells and also by using intracellular expression of variable domains of a neutralizing antibody fused to green fluorescence protein. The two antibodies tested and the expressed scFv all efficiently blocked virus infection, probably by binding to virus particles while they were in the cytoplasm and before entering the nucleus. The injected antibodies were able to block most infections even when injected 8 h after virus inoculation. In control studies, microinjected capsid antibodies did not interfere with CPV replication when they were coinjected with an infectious plasmid clone of CPV. Cytoplasmically injected full and empty capsids were able to move through the cytosol towards the nuclear membrane in a process that could be blocked by nocodazole treatment of the cells. Nuclear transport of the capsids was slow, with significant amounts being found in the nucleus only 3 to 6 h after injection.

Assaying for Structural Variation in the Parvovirus Capsid and Its Role in Infection

The capsid of canine parvovirus (CPV) was assayed for susceptibility to proteases and for structural variation. The natural cleavage of VP2 to VP3 in CPV full (DNA containing) particles recovered from tissue culture occurred within the sequence Arg-Asn-Glu-Arg↓Ala-Thr. Trypsin, chymotrypsin, bromelain, and cathepsin B all cleaved >90% of the VP2 to VP3 in full but not in empty capsids and did not digest the capsid further. Digestion with proteinase K, Pronase, papain, or subtilisin cleaved the VP2 to VP3 and also cleaved at additional internal sites, causing particle disintegration and protein degradation. Several partial digestion products produced by proteinase K or subtilisin were ∼31–32.5 kDa, indicating cleavage within loop 3 of the capsid protein as well as other sites. Protease treatment of capsids at pH 5.5 or 7.5 did not significantly alter their susceptibility to digestion. The isoelectric point of CPV empty capsids was pH 5.3, and full capsids were 0.3 pH more acidic, but after proteolysis of VP2 to VP3, the pI of the full capsids became the same as that of the empty capsids. Antibodies against various capsid protein sequences showed the amino termini of most VP2 molecules were on the outside of full but not empty particles, that the VP1-unique sequence was internal, and that the capsid could be disintegrated by heat or urea treatment to expose the internal sequences. Capsids added to cells were localized within the cell cytoplasm in vesicles that appeared to be lysosomes. Microinjected capsids remained primarily in the cytoplasm, although a small proportion was observed to be in the nucleus after 2 h. After CPV capsids labeled with [35S]methionine were bound to cells at 0°C and the cells warmed, little cleavage of VP1 or VP2 was observed even after prolonged incubation. Inoculation of cells with virus in the presence of proteinase inhibitors did not significantly reduce the infection.

The null mutant of the U(L)31 gene of herpes simplex virus 1: construction and phenotype in infected cells.

Earlier studies have shown that the U(L)31 protein is homogeneously distributed throughout the nucleus and cofractionates with nuclear matrix. We report the construction from an appropriate cosmid library a deletion mutant which replicates in rabbit skin cells carrying the U(L)31 gene under a late (gamma1) viral promoter. The mutant virus exhibits cytopathic effects and yields 0.01 to 0.1% of the yield of wild-type parent virus in noncomplementing cells but amounts of virus 10- to 1,000-fold higher than those recovered from the same cells 3 h after infection. Electron microscopic studies indicate the presence of small numbers of full capsids but a lack of enveloped virions. Viral DNA extracted from the cytoplasm of infected cells exhibits free termini indicating cleavage/packaging of viral DNA from concatemers for packaging into virions, but analyses of viral DNAs by pulsed-field electrophoresis indicate that at 16 h after infection, both the yields of viral DNA and cleavage of viral DNA for packaging are decreased. The repaired virus cannot be differentiated from the wild-type parent. These results suggest the possibility that U(L)31 protein forms a network to enable the anchorage of viral products for the synthesis and/or packaging of viral DNA into virions.

Structure of L-A virus: a specialized compartment for the transcription and replication of double-stranded RNA.

The genomes of double-stranded (ds)RNA viruses are never exposed to the cytoplasm but are confined to and replicated from a specialized protein-bound compartment-the viral capsid. We have used cryoelectron microscopy and three-dimensional image reconstruction to study this compartment in the case of L-A, a yeast virus whose capsid consists of 60 asymmetric dimers of Gag protein (76 kD). At 16-A resolution, we distinguish multiple domains in the elongated Gag subunits, whose nonequivalent packing is reflected in subtly different morphologies of the two protomers. Small holes, 10-15 A across, perforate the capsid wall, which functions as a molecular sieve, allowing the exit of transcripts and the influx of metabolites, while retaining dsRNA and excluding degradative enzymes. Scanning transmission electron microscope measurements of mass-per-unit length suggest that L-A RNA is an A-form duplex, and that RNA filaments emanating from disrupted virions often consist of two or more closely associated duplexes. Nuclease protection experiments confirm that the genome is entirely sequestered inside full capsids, but it is packed relatively loosely; in L-A, the center-to-center spacing between duplexes is 40-45 A, compared with 25-30 A in other double-stranded viruses. The looser packing of L-A RNA allows for maneuverability in the crowded capsid interior, in which the genome (in both replication and transcription) must be translocated sequentially past the polymerase immobilized on the inner capsid wall.

The role of the gene 71 product in the life cycle of equine herpesvirus 1.

Equine herpesvirus type 1 (EHV-1) gene 71 encodes a heavily O-glycosylated 192 kDa protein with no identified herpesvirus homologue. Isolation of a deletion mutant in gene 71 (ED71) demonstrated that its protein product is not essential in vitro. To investigate the role of the gene 71 protein in the virus life cycle, ED71 has been characterized in vitro in terms of cellular adsorption, penetration, egress and transmission compared to wild-type and revertant virus. ED71 virions adsorbed to cells less efficiently than wild-type and revertant virus with a consequential effect on virus penetration; virus egress was significantly impaired and the timing of release was also delayed. The percentage of both full and empty capsids accumulating in the nuclei of ED71-infected cells was significantly higher than in wild-type virus-infected cells but the most notable differences were the low number of particles and the low ratio of enveloped to unenveloped capsids in the cytoplasm. The primary mode of transmission of the mutant virus is by direct cell-to-cell spread and the fact that a neutralizing antiserum did not reduce ED71 plaque size, supported the conclusion that deletion of gene 71 impairs the ability of virus to spread via release and readsorption to uninfected cells. Thus, deletion of EHV-1 gene 71 results in a defect in virus maturation and capsid envelopment. Progeny virus is consequently impaired in adsorption/penetration presumably due to the particles lacking the glycoprotein spikes predicted to be encoded by this gene and hence spreads by direct cell-to-cell contact.