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The human cytomegalovirus (HCMV) virion envelope contains a complex consisting of glycoproteins gH and gL plus proteins encoded by the UL128 locus (UL128L): pUL128, pUL130, and pUL131A. UL128L is necessary for efficient infection of myeloid, epithelial, and endothelial cells but limits replication in fibroblasts. Consequently, disrupting mutations in UL128L are rapidly selected when clinical isolates are cultured in fibroblasts. In contrast, bacterial artificial chromosome (BAC)-cloned strains TB40-BAC4, FIX, and TR do not contain overt disruptions in UL128L, yet no virus reconstituted from them has been reported to acquire mutations in UL128L in vitro. We performed BAC mutagenesis and reconstitution experiments to test the hypothesis that these strains contain subtle mutations in UL128L that were acquired during passage prior to BAC cloning. Compared to strain Merlin containing wild-type UL128L, all three strains produced higher yields of cell-free virus. Moreover, TB40-BAC4 and FIX spread cell to cell more rapidly than wild-type Merlin in fibroblasts but more slowly in epithelial cells. The differential growth properties of TB40-BAC4 and FIX (but not TR) were mapped to single-nucleotide substitutions in UL128L. The substitution in TB40-BAC4 reduced the splicing efficiency of UL128, and that in FIX resulted in an amino acid substitution in UL130. Introduction of these substitutions into Merlin dramatically increased yields of cell-free virus and increased cell-to-cell spread in fibroblasts but reduced the abundance of pUL128 in the virion and the efficiency of epithelial cell infection. These substitutions appear to represent mutations in UL128L that permit virus to be propagated in fibroblasts while retaining epithelial cell tropism.

Broad cell tropism contributes to the pathogenesis of human cytomegalovirus (HCMV), but the extent to which cell type influences HCMV gene expression is unclear. A bespoke HCMV DNA microarray was used to monitor the transcriptome activity of the low passage Merlin strain of HCMV at 12, 24, 48 and 72 h post-infection, during a single round of replication in human fetal foreskin fibroblast cells (HFFF-2s), human retinal pigmented epithelial cells (RPE-1s) and human astrocytoma cells (U373MGs). In order to correlate transcriptome activity with concurrent biological responses, viral cytopathic effect, growth kinetics and genomic loads were examined in the three cell types. The temporal expression pattern of viral genes was broadly similar in HFFF-2s and RPE-1s, but dramatically different in U373MGs. Of the 165 known HCMV protein-coding genes, 41 and 48 were differentially regulated in RPE-1s and U373MGs, respectively, compared with HFFF-2s, and 22 of these were differentially regulated in both RPE-1s and U373MGs. In RPE-1s, all differentially regulated genes were downregulated, but, in U373MGs, some were down- and others upregulated. Differentially regulated genes were identified among the immediate-early, early, early late and true-late viral gene classes. Grouping of downregulated genes according to function at landmark stages of the replication cycle led to the identification of potential bottleneck stages (genome replication, virion assembly, and virion maturation and release) that may account for cell type-dependent viral growth kinetics. The possibility that cell type-specific differences in expressed cellular factors are responsible for modulation of viral gene expression is discussed.

Deep sequencing was used to bring high resolution to the human cytomegalovirus (HCMV) transcriptome at the stage when infectious virion production is under way, and major findings were confirmed by extensive experimentation using conventional techniques. The majority (65.1%) of polyadenylated viral RNA transcription is committed to producing four noncoding transcripts (RNA2.7, RNA1.2, RNA4.9, and RNA5.0) that do not substantially overlap designated protein-coding regions. Additional noncoding RNAs that are transcribed antisense to protein-coding regions map throughout the genome and account for 8.7% of transcription from these regions. RNA splicing is more common than recognized previously, which was evidenced by the identification of 229 potential donor and 132 acceptor sites, and it affects 58 protein-coding genes. The great majority (94) of 96 splice junctions most abundantly represented in the deep-sequencing data was confirmed by RT-PCR or RACE or supported by involvement in alternative splicing. Alternative splicing is frequent and particularly evident in four genes (RL8A, UL74A, UL124, and UL150A) that are transcribed by splicing from any one of many upstream exons. The analysis also resulted in the annotation of four previously unrecognized protein-coding regions (RL8A, RL9A, UL150A, and US33A), and expression of the UL150A protein was shown in the context of HCMV infection. The overall conclusion, that HCMV transcription is complex and multifaceted, has implications for the potential sophistication of virus functionality during infection. The study also illustrates the key contribution that deep sequencing can make to the genomics of nuclear DNA viruses.

Human cytomegalovirus (HCMV) in clinical material cannot replicate efficiently in vitro until it has adapted by mutation. Consequently, wild-type HCMV differ fundamentally from the passaged strains used for research. To generate a genetically intact source of HCMV, we cloned strain Merlin into a self-excising BAC. The Merlin BAC clone had mutations in the RL13 gene and UL128 locus that were acquired during limited replication in vitro prior to cloning. The complete wild-type HCMV gene complement was reconstructed by reference to the original clinical sample. Characterization of viruses generated from repaired BACs revealed that RL13 efficiently repressed HCMV replication in multiple cell types; moreover, RL13 mutants rapidly and reproducibly emerged in transfectants. Virus also acquired mutations in genes UL128, UL130, or UL131A, which inhibited virus growth specifically in fibroblast cells in wild-type form. We further report that RL13 encodes a highly glycosylated virion envelope protein and thus has the potential to modulate tropism. To overcome rapid emergence of mutations in genetically intact HCMV, we developed a system in which RL13 and UL131A were conditionally repressed during virus propagation. This technological advance now permits studies to be undertaken with a clonal, characterized HCMV strain containing the complete wild-type gene complement and promises to enhance the clinical relevance of fundamental research on HCMV.

Mutations that occurred during adaptation of human cytomegalovirus to cell culture were monitored by isolating four strains from clinical samples, passaging them in various cell types and sequencing ten complete virus genomes from the final passages. Mutational dynamics were assessed by targeted sequencing of intermediate passages and the original clinical samples. Gene RL13 and the UL128 locus (UL128L, consisting of genes UL128, UL130 and UL131A) mutated in all strains. Mutations in RL13 occurred in fibroblast, epithelial and endothelial cells, whereas those in UL128L were limited to fibroblasts and detected later than those in RL13. In addition, a region containing genes UL145, UL144, UL142, UL141 and UL140 mutated in three strains. All strains exhibited numerous mutations in other regions of the genome, with a preponderance in parts of the inverted repeats. An investigation was carried out on the kinetic growth yields of viruses derived from selected passages that were predominantly non-mutated in RL13 and UL128L (RL13+UL128L+), or that were largely mutated in RL13 (RL13-UL128L+) or both RL13 and UL128L (RL13-UL128L-). RL13-UL128L- viruses produced greater yields of infectious progeny than RL13-UL128L+ viruses, and RL13-UL128L+ viruses produced greater yields than RL13+UL128L+ viruses. These results suggest strongly that RL13 and UL128L exert at least partially independent suppressive effects on growth in fibroblasts. As all isolates proved genetically unstable in all cell types tested, caution is advised in choosing and monitoring strains for experimental studies of vulnerable functions, particularly those involved in cell tropism, immune evasion or growth temperance.

We have assessed two approaches to sequencing complete human cytomegalovirus (HCMV) genomes (236 kbp) in DNA extracted from infected cell cultures (strains 3157, HAN13, HAN20 and HAN38) or clinical specimens (strains JP and 3301). The first approach involved amplifying genomes from the DNA samples as overlapping PCR products, sequencing these by the Sanger method, acquiring reads from a capillary instrument and assembling these using the Staden programs. The second approach involved generating sequence data from the DNA samples by using an Illumina Genome Analyzer (IGA), processing the filtered reads by reference-independent (de novo) assembly, utilizing the resulting sequence to direct reference-dependent assembly of the same data and finishing by limited PCR sequencing. Both approaches were successful. In particular, the investigation demonstrated the utility of IGA data for efficiently sequencing genomes from clinical samples containing as little as 3 % HCMV DNA. Analysis of the genome sequences obtained showed that each of the strains grown in cell culture was a mutant. Certain of the mutations were shared among strains from independent clinical sources, thus suggesting that they may have arisen in a common ancestor during natural infection. Moreover, one of the strains (JP) sequenced directly from a clinical specimen was mutated in two genes, one of which encodes a proposed immune-evasion function, viral interleukin-10. These observations imply that HCMV mutants exist in human infections.

The genomes of commonly used variants of human cytomegalovirus (HCMV) strains Towne and AD169 each contain a substantial mutation in which a region (U(L)/b') at the right end of the long unique region has been replaced by an inverted duplication of a region from the left end of the genome. Using high-throughput technology, we have sequenced HCMV strain Towne (ATCC VR-977) and confirmed the presence of two variants, one exhibiting the replacement in U(L)/b' and the other intact in this region. Both variants are mutated in genes RL13, UL1, UL40, UL130, US1 and US9. We have also sequenced a novel AD169 variant (varUC) that is intact in U(L)/b' except for a small deletion that affects genes UL144, UL142, UL141 and UL140. Like other AD169 variants, varUC is mutated in genes RL5A, RL13, UL36 and UL131A. A subpopulation of varUC contains an additional deletion affecting genes IRS1, US1 and US2.

Most human cytomegalovirus (HCMV) genes are highly conserved in sequence among strains, but some exhibit a substantial degree of variation. Two of these genes are UL146, which encodes a CXC chemokine, and UL139, which is predicted to encode a membrane glycoprotein. The sequences of these genes were determined from a collection of 184 HCMV samples obtained from Africa, Australia, Asia, Europe, and North America. UL146 is hypervariable throughout, whereas variation in UL139 is concentrated in a sequence encoding a potentially highly glycosylated region. The UL146 sequences fell into 14 genotypes, as did all previously reported sequences. The UL139 sequences grouped into 8 genotypes, and all previously reported sequences fell into a subset of these. There were minor differences among continents in genotypic frequencies for UL146 and UL139, but no clear geographical separation, and identical nucleotide sequences were represented among communities distant from each other. The frequent detection of multiple genotypes indicated that mixed infections are common. For both genes, the degree of divergence was sufficient to preclude reliable sequence alignments between genotypes in the most variable regions, and the mode of evolution involved in generating the genotypes could not be discerned. Within genotypes, constraint appears to have been the predominant mode, and positive selection was detected marginally at best. No evidence was found for linkage disequilibrium. The emerging scenario is that the HCMV genotypes developed in early human populations (or even earlier), becoming established via founder or bottleneck effects, and have spread, recombined and mixed worldwide in more recent times.

The genetic content of wild-type human cytomegalovirus was investigated by sequencing the 235 645 bp genome of a low passage strain (Merlin). Substantial regions of the genome (genes RL1-UL11, UL105-UL112 and UL120-UL150) were also sequenced in several other strains, including two that had not been passaged in cell culture. Comparative analyses, which employed the published genome sequence of a high passage strain (AD169), indicated that Merlin accurately reflects the wild-type complement of 165 genes, containing no obvious mutations other than a single nucleotide substitution that truncates gene UL128. A sizeable subset of genes exhibits unusually high variation between strains, and comprises many, but not all, of those that encode proteins known or predicted to be secreted or membrane-associated. In contrast to unpassaged strains, all of the passaged strains analysed have visibly disabling mutations in one or both of two groups of genes that may influence cell tropism. One comprises UL128, UL130 and UL131A, which putatively encode secreted proteins, and the other contains RL5A, RL13 and UL9, which are members of the RL11 glycoprotein gene family. The case in support of a lack of protein-coding potential in the region between UL105 and UL111A was also strengthened.

Two novel spliced genes (UL131A and UL128) flanking UL130 were predicted from sequence comparisons between human cytomegalovirus (HCMV) and its closest known relative, chimpanzee cytomegalovirus (CCMV), and the splicing patterns were confirmed by mRNA mapping experiments. Both genes were transcribed with late kinetics and shared a polyadenylation site. Comparisons with wild-type HCMV in infected human tissues showed that three of five isolates passaged in cell culture contained disruptions of UL128, one was frameshifted in UL131A and one exhibited a deletion affecting UL131A and UL130. CCMV and the Colburn strain of simian cytomegalovirus, which have been passaged in cell culture, also exhibit disruptions of UL128. These observations indicate that expression of either one of UL128 and UL131A is deleterious to growth of primate cytomegaloviruses in cell culture. Although the functions of these genes are unknown, sequence comparisons suggest that UL128 encodes a beta-chemokine.

A significant proportion of the human cytomegalovirus (HCMV) genome comprises 12 multigene families that probably arose by gene duplication. One, the RL11 family, contains 12 members, most of which are predicted to encode membrane glycoproteins. Comparisons of sequences near the left end of the genome in several HCMV strains revealed two adjacent open reading frames that potentially encode related proteins: RL6, which is hypervariable, and RL5A, which has not been recognized previously. These genes potentially encode a domain that is the hallmark of proteins encoded by the RL11 family, and thus constitute two new members. A homologous domain is also present in a subset of human adenovirus E3 membrane glycoproteins. Evolution of genes specifying the shared domain in cytomegaloviruses and adenoviruses is characterized by extensive divergence, gene duplication and selective sequence loss. These features prompt speculation about the roles of these genes in the two virus families.

The gene complement of wild-type human cytomegalovirus (HCMV) is incompletely understood, on account of the size and complexity of the viral genome and because laboratory strains have undergone deletions and rearrangements during adaptation to growth in culture. We have determined the sequence (241 087 bp) of chimpanzee cytomegalovirus (CCMV) and have compared it with published HCMV sequences from the laboratory strains AD169 and Toledo, with the aim of clarifying the gene content of wild-type HCMV. The HCMV and CCMV genomes are moderately diverged and essentially collinear. On the basis of conservation of potential protein-coding regions and other sequence features, we have discounted 51 previously proposed HCMV ORFs, modified the interpretations for 24 (including assignments of multiple exons) and proposed ten novel genes. Several errors were detected in the published HCMV sequences. We presently recognize 165 genes in CCMV and 145 in AD169; this compares with an estimate of 189 unique genes for AD169 made in 1990. Our best estimate for the complement of wild-type HCMV is 164 to 167 genes.

Evolutionary studies have a large theoretical component and will not directly provide therapies for herpesvirus infections. However, they do provide a conceptual framework within which we can evaluate the origins of the various systems that contribute to viral lifestyle. An evolutionary context allows ancient systems that are fundamental to the replication of all herpesviruses to be distinguished from those that have developed relatively recently in order to tailor viruses to particular biological niches. Both categories are in principle accessible to intervention, either to prevent basic replicative capabilities or to reduce the advantages that the virus has in its interactions with the host. Phylogenetic data provide estimates of evolutionary rate for herpesviruses that are only between one and two orders of magnitude greater than those of their hosts. However, it is becoming apparent that certain genes have evolved much faster under selection pressures and by mechanisms that are not well understood. Nonetheless, the mutation rates of even the most highly conserved genes are sufficient to permit herpesviruses to escape from antiviral therapy. Greater understanding of the origins and functions of herpesvirus genes may lead to new insights into the determinants of pathogenesis and hence to new diagnostic and therapeutic targets.

We have investigated the human cytomegalovirus (HCMV) US22 gene family members UL23, UL24, UL43 and US22. Specific antibodies were generated to identify pUL23 (33 kDa), pUL24 (40 kDa) and pUL43 (48 kDa), while pUS22 was identified by monoclonal antibody HWLF1. A C-terminally truncated UL43 product (pUL43t; 21 kDa) produced by a deletion mutant was also investigated. The UL24 and UL43 genes were expressed with early-late (gamma1) and true-late (gamma2) kinetics, respectively. Immunoblot and immuno-EM studies demonstrated that pUL23, pUL24, pUL43 and pUS22 were virion tegument components. Immunofluorescence and immuno-EM studies showed that pUL23, pUL24, pUL43 and pUL43t were located in cytoplasmic protein aggregates, manifesting two forms: complex juxtanuclear structures and smaller, membrane-bound aggregates resembling dense bodies. The complex-type aggregate is a putative site of particle maturation. Because pUL43t was present in protein aggregates, but under-represented in virus particles compared to pUL43, it was concluded that N-terminal sequences target pUL43 to protein aggregates and that C-terminal sequences are important for incorporation into particles. Since three other US22 family products (pUL36, pTRS1 and pIRS1) are documented tegument components, at least seven of the twelve US22 family genes encode tegument proteins, suggesting that the products of the remaining five genes might be similarly located. These findings demonstrate a common biological feature among most, if not all, US22 family proteins and implicate the family in events occurring immediately after virus penetration.

All members of the herpesvirus family have a characteristic virion structure, comprising a DNA containing, icosahedral capsid, embedded in a proteinaceous layer (tegument) and surrounded by a lipid envelope. Human cytomegalovirus (HCMV, the prototypic beta-herpesvirus) has a genome that is significantly larger (>50 %) than that of the alpha-herpesvirus HSV-1. Although the internal volume of the HCMV capsid is approximately 17 % larger than that of HSV-1, this slight increase in volume does not provide adequate space to encapsidate the full length HCMV genome at the same packing density as HSV-1. We have investigated the nature of DNA packing in HCMV and HSV-1 virions by electron-cryomicroscopy and image processing. Radial density profiles calculated from projection images of HCMV and HSV-1 capsids suggest that there is no increase in the volume of the HCMV capsid upon DNA packaging. Packing density of the viral DNA was assessed for both HCMV and HSV-1 by image analysis of both full and empty particles. Our results for packing density in HSV-1 are in good agreement with previously published measurements, showing an average inter-layer spacing of approximately 26 A. Measurements taken from our HCMV images, however, suggest that the viral genomic DNA is more densely packed, with an average inter-layer spacing of approximately 23 A. We propose therefore, that the combination of greater volume in HCMV capsids and increased packing density of viral DNA accounts for its ability to encapsidate a large genome., (Copyright 2000 Academic Press.)

The three-dimensional structure of B capsids of the beta-herpesvirus human cytomegalovirus (HCMV) was investigated at a resolution of 3.5 nm from electron cryomicrographs by image processing and compared with the structure obtained for the alpha-herpesvirus herpes simplex virus type 1 (HSV-1). The main architectural features of the HSV-1 and HCMV capsids are similar: the T = 16 icosahedral lattice consists of 162 capsomers, composed of two distinct morphological units, 12 pentamers and 150 hexamers, with triplex structures linking adjacent capsomers at positions of local threefold symmetry. The main differences in the HSV-1 and HCMV capsids are found in the diameter of the capsids (125 and 130 nm, respectively); the hexamer spacing and relative tilt (center-to-center hexon spacing at outer, edge, 17.9 and 15.8 nm, respectively); the morphology of the tips of the hexons (similar in length but 33% thinner in HCMV); and the average diameter of the scaffold (44 and 76 nm, respectively). By analogy with HSV-1, the mass on the HCMV hexon tip is attributed to the smallest capsid protein (HCMV gene UL48/49). The differences in capsid structure are discussed in relation to the ability of the HCMV structure to package a genome some 60% larger than that of HSV-1., (Copyright 1998 Academic Press.)

Herpes simplex virus (HSV) is a human pathogen that causes diseases ranging in medical importance from herpes labialis, through genital herpes and herpes keratitis, to herpes encephalitis-a life-threatening disease. HSV types 1 and 2 have the ability to enter a latent phase during in vivo infection, during which time the virus is able to evade immune surveillance, and from which state it is able to escape from time to time to cause disease, especially in immunocompromised individuals. This characteristic makes antiviral chemotherapy an indispensable weapon in the management of recurrent herpesvirus infection.

Comparison of the herpes simplex virus type 1 (HSV-1) DNA sequence with that of other alpha, beta and gamma-herpesviruses, allied with molecular genetic studies have greatly increased understanding of the HSV genome and the functions encoded by individual virus genes and has facilitated the development of rational antiviral strategies. Here we review the coding content of the HSV-1 genome and identify: genes encoding structural components of the capsid, tegument or envelope; genes whose products are essential for growth in tissue culture; and genes that are conserved between members of the alpha, beta and gamma-herpesvirinae. The HSV lifecycle and the main regulation cascade is discussed and genes that present targets for antiviral intervention identified. The protein content of the infectious virion particle is reviewed and compared with that of two additional non-infectious HSV-related particles species (L-particles and pre-DNA replication particles (PREPs)). The potential of HSV-1 L particles and PREP particles as DNA-free HSV-1 vaccine candidates and the desirability of deleting specific gene products from live HSV vaccines is discussed.

Herpes simplex virus type 1(HSV-1) L-particles are known to be composed mainly of envelope and tegument proteins, to lack the nucleocapsid, and to be noninfectious. Thus L-particles represent interesting vaccine candidates. L-particles at > 1000/cell interfered with HSV-1 virion adsorption and penetration While L-particles did not affect HSV-1 growth kinetics in resting or nonresting BHK cultures infected with purified virions, treatment with L-particles before, or after, transfection with HSV-1 DNA resulted in a progressive increase in plaque numbers (five- to sixfold at 1000 L-particles/cell). Transfection assays using HSV-1 ts mutant DNA (ts 1201) revealed that enhancement was due to induction of otherwise nonreplicating genomes. The enhancement obtained with L-particles produced by WT HSV-1 or by mutants that are either deleted, or defective, in certain gene products was compared. Most important were the Vmw110 (ICP0) and Vmw65 (alpha-TIF) proteins, but VP11/12, VP13/14, and vhs also have a role. The L-particle-associated Vmw175 (ICP 4) protein did not appear be involved. The effect of homologous and heterologous combinations of pseudorabies virus, equineherpesvirus-1, and HSV-1 DNA's and L-particles was investigated in transfection assays. The L-particles of each virus, to varying extent, enhanced the plaquing efficiency of their own DNA but were also effective in heterologous combinations.

Compared with the published DNA sequence (M. S. Chee, et al. Curr. Top. Microbiol. Immunol. 154:125-170, 1990), most isolates of human cytomegalovirus strain AD169 contain an additional 929 bp after nucleotide 54612. This results in a changed reading frame for the 5'-terminal 50 codons of gene UL42 and expansion of gene UL43 (a US22 family member) from 187 (3'-truncated) to 423 (full-length) codons. The UL42 and UL43 gene products are nonessential for growth in culture.

We have investigated the ability of monkey kidney cell lines (SupD3 and SupD12) inducibly expressing an amber suppressor tRNA(ser) to suppress amber nonsense mutations in three genes of herpes simplex virus type 1 (HSV-1). HSV-1 mutant TK4, which contains a nonsense mutation in the non-essential viral thymidine kinase (TK) gene, synthesized a full-length TK polypeptide at about 30% of the wild-type (wt) level in induced SupD3 cells but not in the parental non-suppressor (Sup0) cells. Using complementing cells, we constructed HSV-1 mutants carrying nonsense mutations in an essential gene, UL8, encoding a protein essential for viral DNA replication (ambUL8) or in a partially dispensable gene, UL12, encoding alkaline nuclease (ambUL12). The growth of the mutants in Vero or Sup0 cells was either totally (ambUL8) or severely (ambUL12) impaired, whereas in cells expressing suppressor tRNA the mutants produced infectious virus. However, the yields were much lower than obtained with wt HSV-1. In Vero or Sup0 cells the mutants ambUL8 and ambUL12 failed to synthesize full-length UL8 and UL12 protein products, respectively. Western immunoblotting showed that the virus ambUL12 produced full-length UL12 protein in SupD12 cells which yielded a level of 25.9% of the alkaline nuclease activity of the wt HSV-1 control. Our results show that the levels of suppression of the nonsense mutations in ambUL8 and ambUL12 are insufficient to allow their continuing propagation in the available Sup+ cells. Possible reasons are discussed.

Herpes simplex virus (HSV)-infected cells produce not only infectious nucleocapsid-containing virions but also virion-related noninfectious light particles (L-particles) composed of the envelope and tegument components of the virus particle (J. F. Szilágyi and C. Cunningham, J. Gen. Virol. 62:661-668, 1991). We show that BHK and MeWO cells infected either with wild-type (WT) HSV type 1 (HSV-1) in the presence of viral DNA replication inhibitors (cytosine-beta-D-arabinofuranoside, phosphonoacetic acid, and acycloguanosine) or with a viral DNA replication-defective mutant of HSV-1 (ambUL8) synthesize a new type of virus-related particle that is morphologically similar to an L-particle but differs in its relative protein composition. These novel particles we term pre-viral DNA replication enveloped particles (PREPs). The numbers of PREPs released into the culture medium were of the same order as those of L-particles from control cultures. The particle/PFU ratios of different PREP stocks ranged from 6 x 10(5) to 3.8 x 10(8), compared with ratios of 3 x 10(3) to 1 x 10(4) for WT L-particle stocks. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analyses revealed that true late proteins, such as 273K (VP1-2), 82/81K (VP13/14), and gC (VP8), were greatly reduced or absent in PREPs and that gD (VP17) and 40K proteins were also underrepresented. In contrast, the amounts of proteins 175K (VP4; IE3), 92/91K (VP11/12), 38K (VP22), and gE (with BHK cells) were increased. The actual protein composition of PREPs showed some cell line-dependent differences, particularly in the amount of gE. PREPs were biologically competent and delivered functional Vmw65 (VP16; alpha TIF) to target cells, but the efficiency of complementation of the HSV-1 (strain 17) mutant in1814 was 10 to 30% of that of WT L-particles.

The effect of cicloxolone sodium (CCX) on the replication of vesicular stomatitis virus (VSV) was investigated. The drug was active during all stages of the virus replication cycle, indicating that it does not operate by the specific inhibition of any single essential virus gene product. The drug reduced the number of VSV particles assembled and released by 100- to 1000-fold. Infectious virus yield was reduced 1000- to 10000-fold, giving a 10-fold or greater increase in the particle/p.f.u. ratio. The reduced number of virus particles produced in the presence of CCX results from two superimposed effects: suppression of VSV secondary transcription and viral protein synthesis, and perturbation of virion assembly. The inhibition of VSV assembly is due to impairment of a Golgi apparatus function related to transport of VSV glycoprotein G to the cell surface, and is characterized by accumulation of viral G and M proteins within the cell. Incubation of VSV-infected cells in the presence of two glycosylation inhibitors, tunicamycin and monensin, similarly leads to intracellular accumulation of G and M proteins, suggesting a common mechanism of action affecting VSV virion assembly. The differential effect of CCX concentration on intracellular levels of the L, N and NS proteins was analysed. CCX also possesses a virucidal effect on mature infectious VSV particles in suspension, 300 microM reducing the VSV titre about 10-fold in 24 h at 4 degrees C or 37 degrees C. The mode of antiviral activity against VSV is compared with that against herpes simplex virus.

The effect of cicloxolone sodium (CCX) on the replication of typical representatives of different virus families [adenovirus type 5 (Ad-5), reovirus type 3 (Reo-3), Bunyamwera and Germiston viruses, poliovirus type 1 (Polio-1) and Semliki Forest virus (SFV)] in tissue culture was investigated. The Golgi apparatus inhibitor monensin (Mon) and CCX were shown to have analogous effects on some aspects of virus replication. Although the Mon-like effect of CCX played no role in the antiviral activity against Ad-5, Reo-3 or Polio-1, it could entirely account for the antiviral activity against the Bunyamwera and Germiston viruses, for which inhibition of glycoprotein processing was responsible for the antiviral activity. In the case of SFV, the Mon-like activity of CCX caused cytoplasmic assembly of fully infectious SFV within vacuoles and thus impaired virus release without altering total infectious virus yield. Fewer Ad-5 and Reo-3 progeny were produced in the presence of the drug. CCX had a dose-dependent biphasic effect on the particle:p.f.u. ratio of the Reo-3 yield. At low CCX concentration (less than 50 microM) the virus yield contained poor quality, non-infectious virus, but at higher CCX concentration (greater than or equal to 100 microM) low quality virus could no longer be successfully assembled. We conclude that the antiviral effect can be manifested in three ways: (i) by a reduction in the virus particle yield produced; (ii) by a loss of quality (relative infectivity); (iii) by a virucidal effect of the drug. We have previously defined three CCX sensitivity classes. Mechanisms (i), (ii) and (iii) operate against viruses belonging to class CCXs-1 [herpes simplex virus (HSV) type 1, HSV-2 and vesicular stomatitis virus], but essentially only (i) and (ii) affect Reo-3 (CCXs-2), whereas (i) and possibly (iii) affect Ad-5 (CCXs-2). In the case of SFV (CCXs-3) none of these mechanisms operate, but relocation of assembled virus is found.

Cicloxolone sodium (CCX) is a broad spectrum antiviral agent which has a largely non-specific and complex mode of antiviral action. However, the experimental finding that herpes simplex virus type 2 (HSV-2) (strain HG52) is consistently more sensitive to inhibition by CCX than HSV-1 (117 syn+) additionally implies the specific involvement of HSV genes. HSV-1/HSV-2 intertypic recombinants have been utilized to investigate this genetic difference by comparing their CCX ED50 concentrations. No short stretch of HSV-2 DNA was associated with its greater sensitivity to CCX, implying that two or more non-contiguously located HSV genes are involved. Correlating CCX sensitivity with recombinant virus genome structures allowed separate evaluation of the gene regions encoding glycoproteins gB, gC, gD, gE, gG, gH and gI and this suggests that the gene locations encoding gH and gC determine the CCX sensitivity difference. The selective inhibitor of Golgi apparatus glycosylation, monensin, gave results analogous to those obtained with CCX.

The related triterpenoid compounds carbenoxolone sodium (CBX) and cicloxolone sodium (CCX) have been investigated in clinical trials for treatment of herpes simplex virus (HSV) infections. When the drugs were tested in vitro, two dose-related effects on BHK cells became apparent: the rate of cell growth was reduced and the drugs exhibited cytotoxicity at high concentrations. Flow 2002 cells, in contrast, were apparently unaffected by all drug concentrations tested. The effect of up to 3 days incubation with 100 microM-CCX on BHK cells was reversible. The presence of 500 microM-CBX or 300 microM-CCX during the HSV replication cycle reduced the infectious virus yield to less than 0.01%: CCX is the more potent anti-herpes agent. The contribution made by cytotoxicity to the overall antiviral effect (measured by 24 h yield) was negligible in Flow 2002 cells, and was relatively unimportant in BHK cells. The amount of HSV-1 or HSV-2 adsorbing to pretreated BHK cells was reduced by 20% and 40% respectively at the highest drug concentrations. Neither 500 microM-CBX nor 300 microM-CCX treatment for 24 h completely inhibited HSV-1 replication, but HSV-2 replication was abolished. The drugs appear to be continuously active throughout the infectious cycle. Infectious HSV particles appeared to become inactivated during or soon after egress from the cell. The two triterpenoid drugs lowered the number of virus particles made, and to a much greater extent reduced the infectious virus yield; thus, the progeny virus quality is greatly diminished. HSV-2 infections were more readily inhibited by either CCX or CBX than were HSV-1 infections.

Dose-response experiments show that the presence of 300 microM cicloxolone sodium (CCX) or 500 microM carbenoxolone sodium (CBX) during the HSV replication cycle reduced the infectious virus yield by 10,000- to 100,000-fold: CCX is the more potent anti-herpes agent. HSV-2 replication was consistently more severely restricted by either drug than was that of HSV-1. The ED50 values obtained for either drug against HSV-1 or HSV-2 correlate well with data from dose-response curves. CCX, and to a lesser extent CBX, can be cytotoxic but the degree of cytotoxicity varies between cell lines and is also affected by the physiological state of the cells. Triterpenoid drugs exhibit some activity against virus particles in suspension but the effect is small and contributes little to the overall antiviral effect. The drugs appear to be active throughout the replication cycle. In contrast to all other anti-herpesvirus agents in clinical use the triterpenoid compounds do not appear to act directly to block virus DNA synthesis. HSV mutants resistant to ACG and PAA, or lacking a thymidine kinase gene, appear as sensitive as wild-type virus to CCX inhibition. HSV growth in the presence of the drugs resulted in a lower number of assembled virus particles but reduced to a much greater extent the infectious virus yield: thus the progeny virus quality is greatly diminished. Thermolability of progeny virus correlated well with this diminution of quality in increasing CCX concentration. SDS PAGE analysis of the structural proteins of virus particles made in cells treated with 300 microM CCX revealed numerous differences in the relative intensities of protein bands, which is in keeping with the changed quality of the drug-produced virus. SDS PAGE analysis of the polypeptides induced in drug treated infected cells revealed two effects; some polypeptides were synthesised in reduced amounts and the nuclear/cytoplasmic distribution of certain proteins was affected. Post-translational processing by glycosylation and sulphation of both cellular and HSV induced proteins was strongly inhibited by the triterpenoid drugs, while phosphorylation of only a few polypeptides appeared to be affected.

Electron microscope studies been made of mock-infected or herpes simplex virus (HSV) type 1 (strain 17)- or HSV-2(strain HG52)-infected Flow 2002 cells grown in the absence or presence of various concentrations of cicloxolone sodium (CCX). Fifty non-serial, thin sections of mock-infected or HSV-infected cells, which contained a portion of the nucleus, were examined by transmission electron microscopy. With increasing drug concentration, counts of capsid structures both in the nucleus and cytoplasm showed a decrease in the number of virions per cell section, an increase in the ratio of nuclear to cytoplasmic virus, a relative reduction in the percentage of cytoplasmic enveloped virus particles, but no effect on the ratio of empty to core-containing capsid structures. The high particle to p.f.u. ratio induced by CCX treatment is thus not explained through failure to assemble morphologically mature core-containing capsids. In part it can be explained by non-envelopment, but in addition, specific effects on other virion proteins (tegument and envelope) must be involved. The extracellular virus particle yield was unaffected, indicating that CCX treatment enhances the egress of HSV. In the presence of CCX the encapsidation of HSV DNA into DNase-resistant structures was unaffected.

The isolation of spontaneous non-temperature-sensitive revertants of herpes simplex virus type 1 (HSV-1) (strain 17) ts mutants, following a single round of selection at non-permissive temperature, is reported. The distinction between total and incomplete reversion was assessed by the wild-type performance of non-ts revertants in three aspects of HSV infection: (i) virus growth, (ii) induction of HSV-specified enzyme activities (where appropriate) and (iii) the gel profile of infected cell polypeptides. Wild-type behaviour was taken to indicate total reversion, while less than wild-type performance in any test indicated incomplete reversion. Only five of 19 HSV-1 mutants (ts L, U, X, R and G syn) examined for reversion of the ts lesion failed to yield non-ts revertants; these mutants may well contain multiple mutations. Total reversion was obtained with mutants ts K, F, E, S, M, N and I and incomplete reversion with the mutants ts D, T, B, P, A, W, F, H and possibly K. Thus, both total and incomplete revertants of ts F (and possibly ts K) were obtained. Some conclusions concerning the relative importance of individual polypeptide bands have been drawn from these studies. Within our sample in the revertant profiles Vmw 175, 155, 114, 65/64, 43, 40, 39, 30, 28, 21 and 16.5K always returned to wild-type levels; Vmw 273, 100, 67 and 38/37K almost always regained wild-type intensity but polypeptides Vmw 122, 117, 82/81, 57, 51, 27 and 12.5K were frequently not made in wild-type amounts.

The effect of the triterpenoid drugs carbenoxolone sodium (CBX) and cicloxolone sodium (CCX) on DNA and protein synthesis in uninfected and herpes simplex virus (HSV)-infected BHK-21 or Flow 2002 cells has been studied. No consistent effect of 500 microM-CBX or 300 microM-CCX on HSV DNA synthesis was identified. With 300 microM-CCX, some inhibition of cellular DNA synthesis was observed, but this was relatively slight. The restriction enzyme digest profiles of HSV DNA from drug-treated cells appeared normal. The synthesis of several HSV-specified polypeptides was much reduced by treatment with CBX or CCX and the transport of certain proteins between the nuclear and cytoplasmic compartments of infected cells was also affected. CBX or CCX treatment strongly inhibited post-translational glycosylation and sulphation of both host- and HSV-specified proteins, but the phosphorylation of only a few proteins appeared affected. The drugs induced quantitative changes in the synthesis of some BHK cell polypeptides, but these were not considered important. CCX treatment of Flow 2002 cells, however, induced the synthesis of several new polypeptides, some of which had the same apparent molecular weights as identified Flow 2002 cell stress proteins. When treated with concentrations greater than 50 microM-CCX, the plasma membranes of both uninfected and HSV-infected cells became increasingly leaky. The SDS-PAGE polypeptide profile of purified virus particles made in BHK cells treated with 300 microM-CCX differed markedly from that synthesized under drug-free conditions. The greatly reduced amount of infectious virus made in cells treated with 300 microM-CCX was more thermolabile at 42 degrees C than virus produced in the absence of the drug. Our results indicate that the antiviral activity of the triterpenoid drugs is non-specific and operates by interfering with or changing the normal function of cell membranes so that cells, although retaining their viability, can no longer assemble the virus components efficiently into infectious particles. As a consequence, the population of virus particles made is of inferior quality. While we have no evidence that there is also a specific anti-HSV effect, this possibility cannot yet be ruled out.