Electron microscopic observations of FL cells infected with herpes simplex virus. III. Dense bodies.

FL cells infected with the -GCr Miyama strain of herpes simplex virus at an adsorbed multiplicity of approximately 10 were fixed at late stages of infection and examined by electron microscopy. Dense bodies containing electron-dense material and surrounded by a limiting membrane were occasionally observed in the perinuclear disternae and in intranuclear vacuoles. Budding of electron-dense material to the cisternae with acquisition of a limiting membrane at the inner nuclear membrane was also occasionally observed. These findings are in constrast with observations that in cells infected with cytomegalovirus numerous dense bodies and their budding process were observed only in the cytoplasmic area.     

Intramembrane changes occurring during maturation of herpes simplex virus type 1: freeze-fracture study.

During the maturation of two strains of herpes simplex virus type 1 (VR3 and Patton), intramembrane changes were detected with the freeze-fracture technique in the viral envelope and the infected cell plasma membrane, and these changes were compared with data obtained from thin sections. Regardless of the strain, the inner leaflet of the viral envelope of extracellular virions was characterized by a density of intramembrane particles (IMP) three times larger than the host nuclear and plasma membrane. Addition of IMP, which probably represent virus-coded proteins, was detected in the viral envelope only after budding from the nuclear membrane, whereas it occurred during envelopment of capsids at cytoplasmic vacuoles. Fused membranes also showed one of their fracture faces covered with a high density of IMP similar to that of the mature virion envelope. The internal side of the membrane leaflet bearing these numerous particles was always characterized by the presence of an electron-dense material in thin sections. In addition, the plasma membrane of fibroblasts and Vero cells showed strain-specific changes: patches of closely packed IMP were observed with the VR3 strain, whereas ridges almost devoid of IMP characterized the plasmalemma of cells infected with the Patton strain. These intramembrane changes, however, were not observed as early as herpes membrane antigens. Thus, application of the freeze-fracture technique to herpes simplex virus type 1-infected cells revealed striking structural differences between viral and uninfected cell membranes. These differences are probably related to insertion and clustering of virus-coded proteins in the hydrophobic part of the membrane bilayer.     

Ultrastructural characterization of an early, nonstructural polypeptide of herpes simplex virus type 1.

An immunoperoxidase procedure was employed to study the expression of a large-molecular-weight, virus-induced polypeptide (VP175; molecular weight, 175,000) at the light and electron microscopic levels in Vero cells infected with herpes simplex virus type 1 or with tsB2, a DNA-negative, temperature-sensitive mutant of herpes simplex virus type 1. In cells infected with herpes simplex virus type 1 and in cells infected with tsB2 at the permissive temperature (34 degrees C), VP175 was found within the nucleus. The protein was detected as early as 2 h postinfection and, by 3 h postinfection, was generally distributed in a marginated pattern contiguous with, and extending from, the inner lamella of the nuclear membrane. At 6 h postinfection, protein accumulations were dispersed throughout the nucleus, and, by 9 h postinfection, these accumulations tended to be localized in a marginated pattern near the nuclear membrane. It was also noted that, at 9 h postinfection, under permissive conditions, VP175 was not found in association with nucleocapsids or enveloped particles. In contrast, in cells infected with tsB2 at the nonpermissive temperature (39 degrees C) and harvested at 6 or 9 h postinfection, accumulations of VP175 were identified not only within the nucleus, but also within the cytoplasm in the form of annular or globular aggregates. These aggregates consisted of a granular matrix and were not bound by membranes.     

Herpesvirus Envelopment

The growth and envelopment processes of three representative herpesviruses, equine abortion, pseudorabies, and herpes simplex, were examined in baby hamster kidney (BHK 21/13) cells by bioassay (plaque-forming units) and electron microscopy. The envelopment process was identical for all three viruses. After assembly in the nucleus, the nucleocapsid acquired an envelope by budding from the inner nuclear membrane. This membrane was reduplicated as the enveloped particle was released so that the budding process did not result in disruption of the continuity of the nuclear membrane. That portion of the nuclear membrane which comprised the viral envelope was appreciably thicker than the remainder of the membrane and exhibited numerous projections on its surface. Once enveloped, the viral particles were seen in vesicles and vacuoles in the cell cytoplasm. These appeared to open at the cytoplasmic membrane, releasing the virus from the cell. There was no detectable difference in the size or appearance of enveloped particles in intra- or extracellular locations.     

Role of the membrane anchoring and cytoplasmic domains in intracellular transport and localization of viral glycoproteins.

The role of the transmembrane and the cytoplasmic regions of viral glycoproteins namely, the envelope glycoprotein gD of herpes simplex virus and the integral membrane glycoprotein E3-11.6 K of the nonenveloped adenovirus that are localized in the nuclear envelope has been studied. Chimeras of the cell surface glycoprotein G of vesicular stomatitis virus containing the transmembrane and (or) the cytoplasmic-tail domains of either herpes simplex virus gD or adenovirus E3-11.6 K protein were examined for their intracellular transport and localization. The results show that hybrids containing the membrane anchoring and (or) the cytoplasmic tail domains of either herpes simplex virus gD or adenovirus E3-11.6 K glycoprotein were localized in the nuclear envelope as well as in the endoplasmic reticulum and the Golgi complex. These results suggest that the membrane anchoring and the cytoplasmic domains of herpes simplex virus glycoproteins gD, as well as the adenovirus integral membrane protein E3-11.6 K, were necessary for localization in the nuclear envelope and could influence retention in the endoplasmic reticulum and the Golgi complex.     

Deoxythymidine kinase induced in HeLa TK- cells by herpes simplex virus type I and type II. II. Purification and characterization.

Deoxythymidine kinase activities were induced in HeLa TK- (deoxythymidine kinase-deficient) cells infected with either herpes simplex virus type I or herpes simplex virus type II. The herpes simplex virus type I-induced enzyme was found in the cytoplasmic and nuclear fractions of the infected cells, whereas the herpes simplex type II-induced deoxythymidine kinase could only be found in the cytoplasm. Herpes simplex virus type I and II specific deoxythymidine kinases were purified by affinity column chromatography. Both purified deoxythymidine kinases retained the deoxycytidine kinase activity present in the crude preparation. The purified herpes simplex virus type I deoxythymidine kinase had a different mobility on electrophoresis, but the same sedimentation rate on a glycerol gradient as the corresponding unpurified enzyme, whereas the purified herpes simplex virus type II deoxythymidine kinase had the same mobility and sedimentation rate as the corresponding unpurified enzyme. In the presence of Mg2+ATP and dithiothreitol, herpes simplex virus type II deoxythymidine kinase was more stable than herpes simplex virus type I deoxythymidine kinase at both 45 degrees and 4 degrees. The deoxycytidine kinase activity present in the purified preparations was inactivated at the same rate as the deoxythymidine kinase activity. In the presence of the other substrate, deoxythymidine, herpes simplex virus type I deoxythymidine kinase was more stable than herpes simplex virus type II kinase. The purified herpes simplex virus type I and II deoxythymidine kinase had different activation energies when Mg2+ATP and deoxythymidine were used as substrates, but showed the same sensitivity toward ammonium sulfate inhibition.     

Intracellular transport of herpes simplex virus gD occurs more rapidly in uninfected cells than in infected cells.

A mouse L cell line which expresses the herpex simplex virus type 1 immediate-early polypeptides ICP4 and ICP47 was cotransfected with a cloned copy of the BglII L fragment of herpes simplex virus type 2, which includes the gene for gD, and the plasmid pSV2neo, which contains the aminoglycosyl 3'-phosphotransferase (agpt) gene conferring resistance to the antibiotic G418. A G418-resistant transformed cell line was isolated which expressed herpes simplex virus type 2 gD at higher levels than were found in infected cells. The intracellular transport and processing of gD was compared in transformed and infected cells. In the transformed Z4/6 cells gD was rapidly processed and transported to the cell surface; in contrast, the processing and cell surface appearance of gD in infected parental Z4 cells occurred at a much slower rate, and gD accumulated in nuclear membrane to a greater extent. Thus, the movement of HSV-2 gD to the cell surface in infected cells is retarded as viral glycoproteins accumulate in the nuclear envelope, probably because they interact with other viral structural components.     

Similarities and Differences in the Development of Laboratory Strains and Freshly Isolated Strains of Herpes Simplex Virus in HEp-2 Cells: Electron Microscopy

HEp-2 cells infected with two laboratory strains (mP and MP) and two freshly isolated strains (F and G) of herpes simplex virus were fixed at intervals between 4 and 50 hr postinfection and sectioned, and were then examined with the electron microscope. These studies revealed the following. (i) All four strains caused identical segregation of nucleoli and aggregation of host chromosomes at the nuclear membrane. (ii) The development of MP virus could not be differentiated from that of its parent mP strain. (iii) There were quantitative differences between laboratory (mP) and freshly isolated (F) type 1 strains. Thus, cells infected with F contained numerous nuclear crystals of nucleocapsids and relatively few cytoplasmic structures containing enveloped nucleocapsids. Conversely, cells infected with mP or with MP virus contained numerous cytoplasmic structures with enveloped nucleocapsids and relatively few nuclear crystals of nucleocapsids. (iv) There were qualitative differences between type 2 strain (G) isolated from genital lesions and type 1 strains. Thus, cells infected with the G strain contain numerous filaments in nuclei and unenveloped and partially enveloped nucleocapsids in the cytoplasm. Of particular interest is the finding that cytoplasmic membranes in apposition to nucleocapsids were thickened and bent as if they were enveloping the particle. The significance of the qualitative differences in the development of the four strains is discussed.     

Metabolism and mode of action of (R)-9-(3,4-dihydroxybutyl)guanine in herpes simplex virus-infected vero cells

The metabolism and mode of action of the anti-herpes compound buciclovir [R)-9-(3,4-dihydroxybutyl)-guanine, BCV) has been studied in herpes simplex virus-infected and uninfected Vero cells. In uninfected cells, a low and constant concentration of intracellular BCV was found, while in herpes simplex virus-infected cells, an increasing concentration of BCV phosphates was found due to metabolic trapping. The major phosphorylation product was BCV triphosphate (BCVTP) which was 92% of the total amount of BCV phosphates. BCV phosphates were accumulated to the same extent in cells infected with either a herpes simplex virus type 1 or a herpes simplex virus type 2 strain while thymidine kinase-deficient mutants of herpes simplex virus type 1 were 10 times less efficient in accumulating BCV phosphates. In uninfected Vero cells, the concentration of the phosphorylated forms of BCV was less than 1% of that found in herpes simplex virus-infected cells. The BCVTP formed in herpes simplex virus-infected cells was highly stable, as 80% of the amount of BCVTP was still present even 17 h after removal of extracellular BCV. BCV was a good substrate for herpes simplex virus type 1- and type 2-induced thymidine kinases but not for the cellular cytosol or mitochondrial thymidine kinases. BCV monophosphate could be phosphorylated by cellular guanylate kinase to BCV diphosphate. BCVTP was a selective and competitive inhibitor to deoxyguanosine triphosphate of the purified herpes simplex virus type 1- and type 2-induced DNA polymerases. BCVTP could neither act as an alternative substrate in the herpes simplex virus type 2 or cellular DNA polymerase reactions, nor could [3H]BCV monophosphate be detected in DNA formed by herpes simplex virus type 2 DNA polymerase, or be detected in nucleic acids extracted from herpes simplex virus type 1-infected cells. These data indicate that BCVTP may inhibit the herpes simplex virus-induced DNA polymerase without being incorporated into DNA.     

Positively charged DNA-binding proteins cause apparent cell membrane translocation

Several positively charged DNA-binding proteins such as the human immunodeficiency virus Tat protein, the Antennapedia (Antp) homeobox protein, and the herpes simplex virus VP22 protein have been reported to translocate across cell membranes and accumulate in cell nuclei. The import occurs by a poorly understood mechanism that appears to be receptor- and energy-independent. We showed that both VP22 and the positively charged histone H1 adhered to the cell membrane of living cells and were not removed by extensive washing. However, after fixation the proteins relocated to the cell nucleus. The nuclear accumulation of VP22 and histone H1 after fixation shows that positively charged proteins may appear to translocate across the cell membrane because of a fixation artifact. The majority of studies on "membrane permeable" proteins and peptides have been performed using fixation techniques, and our study shows that influx of these proteins may occur during fixation rather than in living cells.     

Replication of herpes simplex virus: egress of progeny virus at specialized cell membrane sites

In the final stages of the herpes simplex virus 1 (HSV-1) life cycle, a viral nucleocapsid buds into a vesicle of trans-Golgi network (TGN)/endosome origin, acquiring an envelope and an outer vesicular membrane. The virus-containing vesicle then traffics to the plasma membrane where it fuses, exposing a mature virion. Although the process of directed egress has been studied in polarized epithelial cell lines, less work has been done in nonpolarized cell types. In this report, we describe a study of HSV-1 egress as it occurs in nonpolarized cells. The examination of infected Vero cells by electron, confocal, and total internal reflection fluorescence (TIRF) microscopy revealed that HSV-1 was released at specific pocket-like areas of the plasma membrane that were found along the substrate-adherent surface and cell-cell-adherent contacts. Both the membrane composition and cytoskeletal structure of egress sites were found to be modified by infection. The plasma membrane at virion release sites was heavily enriched in viral glycoproteins. Small glycoprotein patches formed early in infection, and virus became associated with these areas as they expanded. Glycoprotein-rich areas formed independently from virion trafficking as confirmed by the use of a UL25 mutant with a defect in capsid nuclear egress. The depolymerization of the cytoskeleton indicated that microtubules were important for the trafficking of virions and glycoproteins to release sites. In addition, the actin cytoskeleton was found to be necessary for maintaining the integrity of egress sites. When actin was depolymerized, the glycoprotein concentrations dispersed across the membrane, as did the surface-associated virus. Lastly, viral glycoprotein E appeared to function in a different manner in nonpolarized cells compared to previous studies of egress in polarized epithelial cells; the total amount of virus released at egress sites was slightly increased in infected Vero cells when gE was absent. However, gE was important for egress site formation, as Vero cells infected with gE deletion mutants formed glycoprotein patches that were significantly reduced in size. The results of this study are interpreted to indicate that the egress of HSV-1 in Vero cells is directed to virally induced, specialized egress sites that form along specific areas of the cell membrane.     

Evidence for post-translational glycosylation of a nonglycosylated precursor protein of herpes simplex virus type 1.

Incubation of herpes simplex virus type 1-infected Vero and HEp-2 cells at a reduced temperature (34 degrees C) enhanced the detection of the nonglycosylated precursors (pgB97 and pgC75) to the gB and gC glycoproteins in the cytoplasmic and nuclear fractions. Relative to the fully glycosylated and high-mannose forms detected, the nonglycosylated precursors were the predominant components associated with the nuclear fraction of infected cells. Furthermore, addition of protease inhibitors to the fractionation buffers did not affect the distribution or abundance of the nonglycosylated precursors, suggesting that the presence of pgB97 and pgC75 was not the result of proteolysis. When infected Vero or HEp-2 cells were harvested at various times postinfection, the nonglycosylated precursors were detected after the initial appearance of the high mannose components (pgB110 and pgC105). In Vero cells, pgB97 and pgC75 were detected simultaneously at 8 h postinfection, whereas detection was not apparent in HEp-2 cells until 20 h postinfection. Conditions which favored detection of appreciable amounts of nonglycosylated precursors provided an unique approach to probe possible post-translational modifications in the absence of inhibitors of glycosylation. In nuclear fractions isolated from cycloheximide-treated HEp-2 or Vero cells, numerous discrete gC-immunoreactive bands migrating with decreased electrophoretic mobility relative to the nonglycosylated precursor pgC75 were observed. This series of one to four additional bands was eliminated by digestion with endoglycosidase H, and the appearance of these bands was blocked by the addition of tunicamycin. Collectively, the data suggest that high-mannose core oligosaccharides may be added to the nonglycosylated precursor of the gC glycoprotein of herpes simplex virus type 1 in a post-translational fashion.     

Membrane phenotype of murine effector and suppressor T cells involved in delayed hypersensitivity and protective immunity to herpes simplex virus

The membrane phenotype of T cells involved in delayed hypersensitivity (DH), protective immunity, and suppression of delayed hypersensitivity to herpes simplex virus (HSV) has been determined. T cells from immune lymph nodes transferring DH and antiviral immunity to normal recipients were characterized as Lyt 1+2-. There appeared to be no detectable antiviral role for Lyt 1-2+ cells in the transferred cell suspension. Splenic T cells suppressing the induction of DH to HSV were characterized as being both Lyt 1+2- and Lyt 1-2+ 4 weeks after their induction. At earlier times, i.e., after 7 days, the suppression was mediated solely by the Lyt 1+2- population. Thereafter, a progressive increase in the contribution of the Lyt 1-2+ suppressor was observed. Both the early and later phases of suppression were due to I-J positive cells. The nature of the two suppressor cell types is discussed in relation to suppressor cell "cascades" and to the pathogenesis of herpes simplex virus infection.     

Identification and expression of a human cytomegalovirus glycoprotein with homology to the Epstein-Barr virus BXLF2 product, varicella-zoster virus gpIII, and herpes simplex virus type 1 glycoprotein H.

An open reading frame with the characteristics of a glycoprotein-coding sequence was identified by nucleotide sequencing of human cytomegalovirus (HCMV) genomic DNA. The predicted amino acid sequence was homologous with glycoprotein H of herpes simplex virus type 1 and the homologous protein of Epstein-Barr virus (BXLF2 gene product) and varicella-zoster virus (gpIII). Recombinant vaccinia viruses that expressed this gene were constructed. A glycoprotein of approximately 86 kilodaltons was immunoprecipitated from cells infected with the recombinant viruses and from HCMV-infected cells with a monoclonal antibody that efficiently neutralized HCMV infectivity. In HCMV-infected MRC5 cells, this glycoprotein was present on nuclear and cytoplasmic membranes, but in recombinant vaccinia virus-infected cells it accumulated predominantly on the nuclear membrane.     

Differential feulgen-deoxyribonucleic acid hydrolysis patterns of Herpes simplex virus type 1 and type 2 infected cells.

Infection of human embryonic lung cells with herpes simplex virus type 1 (HSV-1) and herpes simplex type 1 (HSV-2) resulted in: (a) qualitative (nuclear cytopathologic) alterations and quantitative (nuclear area) differences in infected compared to control nuclei; (b) increased Feulgen-deoxyribonucleic acid (F-DNA) amounts in infected cells, probably due to viral DNA; (c) higher F-DNA levels in HSV-2 infected cells; and (d) increased rates of F-DNA hydrolysis in viral-infected as compared to uninfected nuclei.     

Ecto-ganglioside-sialidase activity of herpes simplex virus-transformed hamster embryo fibroblasts

Cellular location of ganglioside-sialidase activity was determined in confluent hamster embryo fibroblasts transformed with herpes simplex virus type 2. Approximately equal specific activities of ganglioside-sialidase activity were found to be associated with the crude lysosomal and crude plasma membrane fractions isolated from whole cell homogenates. Whole transformed cells hydrolyzed exogenous ganglioside substrate, suggesting a partial location of the cellular sialidase on the outer surface of the plasma membrane of these cells. Intact cells were treated with the diazonium salt of sulfanilic acid, a nonpenetrating reagent inhibitory to ecto-enzymes (DePierre, J.W., and M. L. Karnovsky. 1974. J. Biol. Chem. 249:7111-7120). Cytoplasmic lactate dehydrogenase activity was not inhibited by this treatment, and mitochondrial succinate dehydrogenase activity was inhibited only 10%, indicating that intracellular enzymes were not affected. 5'-Nucleotidase activity was diminished 90%, and sialidase very rapidly lost 40% of its exogenously directed activity. These results show that, in herpes simplex virus-transformed fibroblasts, ganglioside-sialidase is both a lysosomal and a plasma membrane enzyme. The plasma membrane sialidase is capable of acting on endogenous plasma membrane sialolipids and also functions in the cultured transformed cell as an ecto-enzyme which can attack exogenous substrates.     

Molecular genetics of herpes simplex virus. V. Characterization of a mutant defective in ability to form plaques at low temperatures and in a viral fraction which prevents accumulation of coreless capsids at nuclear pores late in infection.

In herpes simplex virus-infected cells, coreless capsids accumulate at the nuclear pores soon after infection, but subsequently disappear, suggesting that, as in adenovirus-infected cells (S. Dales and Y. Chardonnet, Virology 56:465-483, 1973), the release of viral DNA from nucleocapsids takes place at the nuclear pores. A nonlethal mutant, HSV-1(50B), produced by mutagenesis of HSV DNA fragments and selected for delayed production of plaques at 31 degrees C, accumulated coreless capsids at the nuclear pores late in infection in contrast to wild-type viruses. Recombinants selected for ability to produce plaques at 31 degrees C by marker rescue with digests of herpes simplex virus 2 DNA and selected clone fragments of HSV-1 DNA no longer accumulated empty capsids at nuclear pores late in infection. These results suggest that herpes simplex viruses encode a function which prevents accumulation of coreless capsids at nuclear pores, presumably by preventing uptake, unenvelopment, and DNA release from progeny virus, and indicate that the cold sensitivity of plaque formation and accumulation of coreless capsids might be related or comap in the S component of the genome.     

Characterization of herpes simplex virus 2 temperature-sensitive mutants whose lesions map in or near the coding sequences for the major DNA-binding protein.

By marker rescue with cloned herpes simplex virus 2 DNA fragments, we have mapped the temperature-sensitive mutations of a series of herpes simplex virus 2 mutants to a region of the herpes simplex virus 2 genome that lies within or near the coding sequences for the major DNA-binding protein, ICP8. In cells infected with certain of these mutants at the nonpermissive temperature, the association of the major DNA-binding protein with the cell nucleus was defective. In these cells, the DNA-binding protein accumulated in the cytoplasmic and the crude nuclear detergent wash fractions. At the permissive temperature, the maturation of the mutant ICP8 was similar to that of the wild-type viral protein. With the remainder of the mutants, the nuclear maturation of ICP8 was similar to that encoded by the wild-type virus at the nonpermissive and permissive temperatures as assayed by cell fractionation.     

Neutralizing antibodies specific for glycoprotein H of herpes simplex virus permit viral attachment to cells but prevent penetration.

Monoclonal antibodies specific for gH of herpes simplex virus were shown previously to neutralize viral infectivity. Results presented here demonstrate that these antibodies (at least three of them) block viral penetration without inhibiting adsorption of virus to cells. Penetration of herpes simplex virus is by fusion of the virion envelope with the plasma membrane of a susceptible cell. Electron microscopy of thin sections of cells exposed to virus revealed that neutralized virus bound to the cell surface but did not fuse with the plasma membrane. Quantitation of virus adsorption by measuring the binding of purified radiolabeled virus to cells revealed that the anti-gH antibodies had little or no effect on adsorption. Monitoring cell and viral protein synthesis after exposure of cells to infectious and neutralized virus gave results consistent with the electron microscopic finding that the anti-gH antibodies blocked viral penetration. On the basis of the results presented here and other information published elsewhere, it is suggested that gH is one of three glycoproteins essential for penetration of herpes simplex virus into cells.     

Partial functional complementation of a pseudorabies virus UL25 deletion mutant by herpes simplex virus type 1 pUL25 indicates overlapping functions of alphaherpesvirus pUL25 proteins

Homologs of the UL25 gene product of herpes simplex virus 1 (HSV-1) are highly conserved among the Herpesviridae. However, their exact function during viral replication is unknown. Current evidence suggests that in the alphaherpesvirus pseudorabies virus (PrV) the capsid-associated pUL25 plays a role in primary envelopment of DNA-containing mature capsids at the inner nuclear membrane. In the absence of pUL25, capsids were found in close association with the inner nuclear membrane, but nuclear egress was not observed (B. G. Klupp, H. Granzow, G. M. Keil, and T. C. Mettenleiter, J. Virol. 80:6235-6246, 2006). In contrast, HSV-1 pUL25 has been assigned a role in stable packaging of viral genomes (N. Stow, J. Virol. 75:10755-10765, 2001). Despite these apparently divergent functions, we wanted to assess whether the high sequence homology translates into functional homology. Therefore, we first analyzed a newly constructed HSV-1 UL25 deletion mutant in our assay system and observed a similar phenotype as in PrV. In the nuclei of infected cells, numerous electron-dense C capsids were detected, whereas primary envelopment of these capsids did not ensue. In agreement with results from PrV, vesicles were observed in the perinuclear space. Since these data indicated functional homology, we analyzed the ability of pUL25 of HSV-1 to complement a PrV UL25 deletion mutant and vice versa. Whereas a HSV-1 pUL25-expressing cell line partially complemented the pUL25 defect in PrV, reciprocal complementation of a HSV-1 UL25 deletion mutant by PrV pUL25 was not observed. Thus, our data demonstrate overlapping, although not identical functions of these two conserved herpesvirus proteins, and point to a conserved functional role in herpes virion formation.