of the viral DNA from the capsids of the input pores and are uncoated at these pores. Although it is likely that HSV DNA could be released by

Transcription

1 JOURNAL OF VIROLOGY, Dec. 1981, p X/81/ $02.00/0 Vol. 40, No. 3 Molecular Genetics of Herpes Simplex Virus V. Characterization of a Mutant Defective in Ability to Form Plaques at Low Temperatures and in a Viral Function Which Prevents Accumulation of Coreless Capsids at Nuclear Pores Late in Infection MAURO TOGNON,t DEIRDRE FURLONG, ANTHONY J. CONLEY,: AND BERNARD ROIZMAN* The Marjorie B. Kovler Viral Oncology Laboratories, The University of Chicago, Chicago, Illinois Received 5 March 1981/Accepted 17 July 1981 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: , 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 310C, accumulated coreless capsids at the nuclear pores late in infection in contrast to wild-type viruses. Recombinants selected for ability to produce plaques at 310C by marker rescue with digests of herpes simplex virus 2 DNA and selected cloned 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. In this paper we report that herpes simplex virus 1 (human herpesvirus 1, HSV-1) may specify a function which prevents the release of the viral DNA cores from progeny virus within the infected cell, possibly by preventing it from entering the infected cell after release. The evidence for such a function is suggested by the observation that an HSV-1 nonlethal mutant produced by recombination of wild-type DNA and in vitro-mutagenized fragments accumulated capsids totally devoid of cores at nuclear pores late in infection and produced plaques at 310C only after a considerable delay. Cells infected with recombinants obtained by rescue with wild-type DNA and selected for ability to form plaques at the expected time at 310C do not accumulate empty capsids at nuclear pores. Pertinent to this report are the following. (i) Accumulated evidence indicates that the HSV capsids accumulate in the cytoplasm (17) and that the input, parental DNA reaches the nucleus of the cell, but there is little or no information on the site or mechanism of release t Present address: Institute of Microbiology, University of Ferrara, Ferrara, Italy. f Present address: Institute for Molecular Virology, St. Louis University School of Medicine, St. Louis, MO of the viral DNA from the capsids of the input virus (23). Evidence presented by Dales and Chardonnet (2) indicates that adenoviruses capsids are transported vectorially along pathways furnished in part by microtubules to the nuclear pores and are uncoated at these pores. Although it is likely that HSV DNA could be released by a similar mechanism, the fate of the input capsids in HSV-infected cells has not been described. (ii) In HSV-infected cells the mature enveloped capsids (virions) accumulate in the space between the inner and outer lamellae of the nuclear membranes, in the cysternae of the endoplasmic membranes, and in structures enclosed by membranes, but are only infrequently found free in the cytoplasm (3, 26). Capsids containing cores are present free in the cytoplasm and in juxtaposition to cellular membranes, but their infectivity and ultimate contribution to the total yield of infectious progeny is not known (23). Coreless capsids are rarely seen in the cytoplasm late in infection and almost never in juxtaposition to the nuclear pores. Electron microscopic studies have suggested that in contrast to the unenveloped DNA viruses, the herpesvirus virions are actively transported from the site of maturation to the cell surface in 870

2 VOL. 40, 1981 structures that are totally enclosed in cellular membranes (3, 23, 26). (iii) A question that has not been resolved is what prevents the adsorption of progeny virus to the cells from which they had been released. One explanation is based on observations that viral glycoprotein constituents of the virion envelope are also inserted into the plasma membrane of the infected cells (8, 9, 24). It has been suggested that one or more viral glycoproteins on the surface of the cell may prevent the released virus from reentering the cell from which it was released (16). The characteristics of the mutant described in this paper constitute the first suggestive evidence that the virus may encode function(s) that specifically prevent entry of progeny virus into the infected cell. MATERIALS AND METHODS Viruses and cells. The isolation and relevant properties of HSV-l(mP) and HSV-2(G) strains were published elsewhere (4, 11). HSV-1(CVG2) is an HSV- 1 isolate containing an additional HsuI (HindIII isoschizomer) cleavage site at 0.24 map units in the prototype arrangement of HSV-1 DNA (6). HSV- 1(CVG2) Ara Tr is a mutant lacking thymidine kinase; it was isolated as described elsewhere (25). Virus stocks were prepared and virus titers were determined on Vero or HEp-2 cells as previously described (4, 25). Rabbit skin cells were originally obtained from J. McLaren. Cloned HSV-1(F) DNA. The preparation and properties of cloned HSV-1(F) BamHI fragments were described elsewhere (21). Enzymes and radioisotopes. The restriction endonucleases BamHI and KpnI were obtained from New England Biolabs, Boston, Mass. The purification of the restriction endonuclease EcoRI has been described (18). D-['4C]glucosamine hydrochloride, ['4C]leucine, ['4C]isoleucine, and ['4C]valine (250 Ci/ mmol), and L-[nS]methionine (500 Ci/mmol) were purchased from New England Nuclear Corp., Boston, Mass. Preparation of viral DNA and restriction endonuclease digestion. Viral DNA was purified by NaI density gradient centrifugation of DNA extracted from infected whole-cell lysates. Digestion of viral DNA, preparative agarose gel electrophoresis, and isolation of individual restriction endonuclease-derived fragments were described elsewhere (1, 18, 21). Analytical gel electrophoresis was done on a horizontal apparatus at 2 V/cm. Mutagenic treatment ofthe DNA fragment and isolation of the 50B mutant. The HsuI A" fragment, molecular weight 16.7 x 106, mapping in the prototype arrangement of HSV DNA between coordinates 0.24 and 0.40 and containing the genetic marker for Ara T', was prepared from digests of HSV-1(CVG2) Ara TF DNA. This fragment was mutagenized with hydroxylamine as described elsewhere (1) and cotransfected with intact HSV-l(mP) into rabbit skin cells. Virus MOLECULAR GENETICS OF HSV 871 progeny from the transfection stock were isolated and tested for growth at 33 and 39 C in Vero cells. An isolate [HSV-1(50B)] which produced plaques at 39 C but not at 33 or 31 C was further purified by three successive plaque isolation passages. Stocks were prepared at a multiplicity of 0.01 PFU/cell in HEp-2 cell roller-bottle cultures. Intertypic marker rescue. Intertypic marker rescue was done essentially as previously described (1, 25), but with the following modifications. Briefly, approximately 0.5 yg of HSV-1(50B) mutant DNA was mixed with 1.0,g of HSV-2(G) which was previously digested with either HpaI or XbaI restriction endonucleases. After transfection, the rabbit skin cell monolayer (50% confluent) was immediately overlaid with 1 ml of Dulbecco modified Eagle medium supplemented with 2% serum; after 4 h the monolayer was overlaid with 5 ml of the same medium but containing 6% serum. Rabbit skin cell flask cultures were then incubated at 39 C for approximately 1 week. Titers of the progeny were then determined at 39 and 31 C on Vero cells. Recombinants which formed plaques at 31 C at 2 days postinfection were selected from the viral progeny of transfected cells and were plaque purified two times at 31 C before preparation of virus stocks. Intratypic marker rescue. The intratypic marker rescues were done as described above with individual, cloned HSV-1 BamHI fragments spanning the coordinates 0.24 to 0.40 and 0.86 to 0.94 map units on the prototype arrangement of HSV-1 DNA (21). Protein labeling and gel electrophoresis. Labeling of HSV-infected cells with ['4C]leucine, [14C]isoleucine, [14C]valine, D-[14C]glucosamine, and L-[3S]methionine, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell lysates, and autoradiography of electrophoretically separated polypeptides were performed as previously described (19). RESULTS Selection of the 50B mutant and its phenotype. The experimental design was to mutagenize with hydroxylamine a 16.7 x 106-molecular-weight fragment of HSV-1 DNA carrying the Ara Tr marker as described above. The mutagenized fragment was then cotransfected with intact Ara Tr HSV-l(mP) DNA, and from the progeny of the transfection, Ara Tr progeny were tested for the ability to produce plaques at 33.5 and 39 C. During the course of this study, we isolated several plaque morphology mutants (21; M. Tognon, A. J. Conley, and B. Roizman, manuscript in preparation). This report, however, concerns a mutant whose properties are distinct from any reported to date. Briefly (Table 1), the mutant designated HSV-1(50B) formed 10-4-fold more plaques on monolayers of infected cells maintained at 39 C than at 31 or 33.5 C at 2 days postinfection, the normal time of scoring plaques. The plaques formed at 39 C were smaller than those produced by the parent virus.

3 872 TOGNON ET AL. J. VIROL. TABLE 1. Titration of HSV-1(50B) and HSV-1(mP) on Vero and HEp-2 cell monolayer cultures at different temperaturesa Titer at time postinfection: CelLs Temp (0C) 2 days 5 days 50B mp 50B mp Vero 31 <104 2 x x x <104 4 x105 4 x108 5 x x x x x108 HEp-2 31 <104 2 x 108 <103 2 x i <105 4 x 108 <106 4 x 10W 39 4 x x x x108 a These virus stocks were prepared in Vero cell cultures and titers were determined in Vero or HEp-2 cell cultures at temperatures shown. Several experiments, however, indicated that the replication of this virus was not cold sensitive. First, identical numbers of plaques were produced in Vero cells, but not in HEp-2 cells at all temperatures tested when the cultures were incubated for 5 days (Table 1). Second, the relative yields of mutant and parent virus from cells infected with 5 PFU/cell and incubated at various temperatures were similar if not identical (Table 2). Mutant 50B therefore appeared defective in ability to produce plaques at 310C but not in replication at that temperature. Electron microscopy of cells infected with mutant and parent viruses. Secondary scanning electron micrographs of Vero cells infected with 5 PFU/cell (Fig. 1) revealed that at 18 h postinfection, cells infected with the mutant and incubated at 31 C showed a typical flat, spread-out appearance which could not be differentiated from that of uninfected cells (not shown). Typical cell rounding and aggregation could be seen in cultures of cells infected with 50B and incubated at 39 C. Rounding and aggregation of infected cells were seen in cultures infected with HSV-1(mP) and incubated at both 31 and 390C. Thin sections of cells infected with HSV- 1(50B) at a multiplicity of 5 PFU/cell and maintained for 48 h at 390C showed a striking accumulation of coreless capsids in the cytoplasm at the nuclear pores (Fig. 2B). In some sections, a coreless capsid was juxtaposed to nearly every nuclear pore. In contrast, cells infected with HSV-1(mP) at the same multiplicity and maintained at the same temperature showed very few coreless capsids at nuclear pores (Table 3). Coreless capsids were present, but in fewer numbers, in cells infected with 50B and maintained for 48 h at 33 C. They were absent from cells infected with mp and maintained under the same conditions. In an attempt to define the significance of the coreless capsids at the nuclear pores, we exam- TABLE 2. Yield of HSV-1(50B) and mp in Vero cells infected and maintained at 31 and 390C' Viral yield Temp (00) 50B mp 31 8x x x x108 a The cells were infected at 5 PFU/cell and incubated at temperatures shown. The cells were harvested at 25 h postinfection, and the virus titers were determined in Vero cells incubated at 370C. ined cells infected with HSV-l(mP), HSV-1(F), HSV-1(50B), and HSV-1(HFEM) at different times after infection. These studies showed that coreless capsids were found at the nuclear pores in thin sections of cells fixed before 2 h postinfection (Fig. 2A). In cells infected at relatively low multiplicities ('5 PFU/cell) the number of such coreless capsids at the nuclear pores was relatively low (less than 1 per 100 cells), and this ratio appeared to decrease in cells fixed after 2 h postinfection. Analyses of the polypeptides specified by 50B and its parent, HSV-l(mP). Two series of experiments were done. In the first, replicate cultures of cells infected with either 50B or mp virus and incubated at 33 and 39 C were pulselabeled with [35S]methionine or with "C-amino acids from 5 to 7 or 16 to 18 h postinfection. The cells were then harvested, solubilized, and subjected to electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The autoradiographic images revealed no significant differences in the electrophoretic profiles of the polypeptides specified by 50B and mp at either temperature. In the second series of experiments, the cells were labeled at 31 and 390C from 5 to 20 h postinfection with ['4C]glucosamine. Again, the autoradiograms of the electrophoretically separated polypeptides revealed no significant differences in the accumulation and electrophoretic

4 FIG. 1. Secondary scanning electron micrograph of Vero cells 18 h after infection with 5 PFU of 50B (A and B) or mp (C and D). The cells were grown on plastic cover slips. The infected cells were fixed with 2% glutaraldehyde for 1 h, dehydrated through graded alcohols, and critical-point dried out of CO2. The cells were coated with gold. At 310C (A) the cells infected with mutant 50B showed no obvious signs of infection in that they retained the flat, spread-out appearance of uninfected cells. Sparse microvilli were present as in the uninfected cells, and nuclei were prominent. At 39 C (B) the cells were rounded and clumped, leaving large, nearly empty areas. The majority of the cytoplasm in each cell constricted around the nucleus, which has become more spherical. Panels C (at 31 C) and D (at 39 C) show rounded clumps of mp-infected cells. The majority of cells are in clumps; the few remaining flat cells appeared to be partially torn off the supporting surface (lower left in C and upper center in D). The clumps of cells tended to be larger at 39 C (D) than at 31 C (C), and thin cytoplasmic bridges connecting cells within the small aggregates were common. Bar, 20 -n. 873

5 ~~~~~~~~~~~~ TOGNON ET AL. :,. 4 < e ru r~~~~r..44 t,; i ]W - i. A i4 -t t Adt 4 Vi? 74p:, 4qr~~~~~~~~4 I, ~ ~ ~ ~ 4 4"., l~~~~~~~~~~~~~~~~~j -+b qw Jg~~~~~~~~4 4 4 R-t ~ ~ ~ 4 ~ C. -J V., 1,.t 4,.. ': lw?,;-." Wi i..*.-, {-s 1. * r. V 0~~ -. 't.q..... K I.@ dml.. b0 ;4. s. J. VIROL. rṣ o~~a FIG. 2. Thin sections of infected Vero cells. The cells were fixed for 1 h with 2% glutaraldehyde, postfixed for 1 h with 1% osmium tetroxide, and dehydrated. After embedding in Epon thin sections were cut and stained with 2% uranyl acetate. (A) Empty capsid in the cytoplasm of a Vero cell 2 h after infection with 10 PFU of mp at 39 C. The capsid abuts a nuclear pore and has lost its core. (B) Part of a cell infected with 5 PFU of 50B for 48 h at 39 C. Four capsids without cores can be seen at nuclear pores in the left part of this panel Two nucleocapsids are seen to the lower right free in the cytoplasm. One large and several small vesicles are present in the cytoplasm. Bar, 200 nm. mobility of the major viral glycoproteins (Fig. were transfected with mixtures of HpaI or XbaI 3). digests of HSV-2(G) DNA and intact 50B DNA Mapping of the mutation of HSV-1(50B). and incubated at 39 C as described in Materials In this series of experiments, rabbit skin cells and Methods. The progeny of individual plaques

6 VOL. 40, 1981 TABLE 3. Distribution of empty capsids at nuclear pores in cells infected with HSV-1(mP), HSV-1(50B), and 50B x HSV-2(G) recombinants selected for plaque production at 31C Ca No. of celrs with No. of total Virus empty particles at nuclear pores/ empty cap- s 100 cells HSV-l(mP) 1 1 HSV-1(50B) R50BG6 0 0 R50BG a For each virus strain, thin sections of a total of 100 Vero cells from cultures harvested 48 h postinfection were examined for the presence of coreless viral capsids at the nuclear membranes. The cells were incubated at 39 C. Studies on Vero cells infected with 50B or mp and incubated at 33 C yielded similar results. were harvested, plaque purified five times by passage and selection in Vero cells at 31 C, and then analyzed for plaque formation at 31 and 39 C. Approximately 30 plaque-purified recombinants, obtained from independently transfected cells and capable of forming plaques at 31 and 39 C, were then analyzed with restriction endonucleases to determine the extent of replacement of HSV-1 DNA sequences with HSV- 2 DNA. The results of these studies (Fig. 4 and 5) showed that when the HSV-2(G) DNA sequences could be detected, they were uniformly present in the S component of HSV DNA, and that all of the recombinants fall into four classes based on restriction enzyme maps of the HSV-1 sequences substituted by HSV-2 DNA. The significant features of these four classes of recombinants were the following. (i) No HSV-2 DNA sequences were detected in the DNAs of four recombinants, suggesting that they are either revertants or recombinants containing HSV-2 sequences within a region of the genome not cleaved by any of the enzymes tested. Recombinant R50BG4, a member of this group, has been shown by Para et al. (M. F. Para, L. Goldstein, and P. G. Spear, manuscript in preparation; P. G. Spear, personal communication) to specify the HSV-2 glycoprotein ge mapping in the S component and therefore contains HSV-2 sequences. (ii) In recombinant R50BG6, the HSV-2 DNA sequences substituted for the HSV-1 DNA sequences containing BamHI fragments N, J, X, and Z. All recombinants representative of this group retained the HSV-1 EcoRI cleavage sites KI-H and H-K2. The KpnI digests of these recombinants contained a fragment representing the fusion of HSV-2 KpnI A with the termini of HSV-1 KpnI fragments I and K. MOLECULAR GENETICS OF HSV 875 (iii) In recombinant R50BG8, the substitution of HSV-1 sequences by HSV-2 DNA was more extensive and included a portion of the HSV-1 50 B mp mock _ ' -- _ t DEl.4p,.. FIG. 3. Autoradiographic images ofelectrophoretically separated polypeptides from cells infected with mp or 50B and labeled with ['4C]glucosamine. The major glycoproteins were designated according to Spear (28).

7 876 TOGNON ET AL. D-H A-A R P U w B co 7 X a co La Lf La a L Ln 0 c N ~ jmm no 0 0LI, cr _~~~~~~~~~~~ J. VIROL. co z _ X T (D000 0o0 000 CIO Do In In J L In 0 In CY m CY ac a C 0 r 0 c: VI a nl I L La 0 (x 0 A EK < ~~~~~~~~E K A KM J M F *W4 K -_. 8 af -4,*O L P h v */w X. Q.. N R 3 Ban HI Kon Eco Rl FIG. 4. Photographs of electrophoretically separated BamHI, KpnI, and EcoRI digests of HSV-1(50B), HSV-2(G), and recombinants produced by marker rescue of 50B. Note that in 50B, as in the parent, HSV- 1(mP), the KpnI M fragment is cleaved into M' and M". The BamHI, KpnI, and EcoRI fragments containing the L-S junction are labeled with double letters according to previously established nomenclature (12). In HSV-1 mp, the BamHI fragment D is fused to H (D-H), whereas A is fused to A' (A-A'). The HSV-2 fragments are underlined. BamHI P fragment. However, the substitution did not extend to the end of the S component because the HSV-2 KpnI cleavage sites R1-A and A-R2 were not present. (iv) In recombinant R50BG13, the extent of the substitution of HSV-1 DNA sequences was still more extensive; the HSV-2 KpnI cleavage sites R1-A and A-R2 were present, generating an intact HSV-2 KpnI A fragment (Fig. 4). (v) Recombinant R50BG14 is unique among the recombinants tested in that it was found to contain HSV-2 DNA sequences in both L and S components. The substitution in S component was similar if not identical to that observed in R50BG8. In the L component, the substitution was reflected in the loss of the BamHI G-V cleavage sites. Biological properties of the HSV-1 x HSV-2 recombinants produced by rescue of the defect in mutant 50B. Analyses of the recombinants showed that they do not differ from the parent, HSV-l(mP), with respect to plaque fonnation or ability to replicate at 31 C (data not shown). Of particular interest is the. I *a T J M M N O finding that in cells infected with the recombinants and maintained at either 31 or 39 C, there was no accumulation of coreless capsids at the nuclear pores late in infection. These experiments indicate that the defect resulting in accumulation of coreless capsids at the nuclear pores late in infection was "rescued" by the substitution for the HSV-1 sequences by HSV-2 sequences in these recombinants. Rescue of 50B with cloned HSV-1 DNA fragments. The rescue of 50B with cloned HSV-1 DNA fragments was done to resolve the contradiction between the apparent provenance of the mutagenized DNA and the site of substitution of the HSV-1 DNA sequences by HSV-2 DNA in the course of the marker rescue tests with the latter DNA. Cloned HSV-1 DNA BamHI fragments spanning the regions from 0.24 to 0.40 and 0.86 to 0.94 map units in the prototype arrangement (21) were used individuaily in marker rescue tests as described in Materials and Methods. In repeated tests, the HSV-1 fragments from the 0.24 to 0.40 map unit region and of the 0.86 to 0.90 map unit region of

8 VOL. VOL. 40, 40, ~~~MOLECULAR GENETICS OF HSV 877 o L b caa c s c a 3 9 J I I. 0'0 01 0: ' '8 ; 0'9- I0 R 50BG O '4 H SV -I Kpn I M S N P V~X, i i i I~1 H SV-2 Eco RIN4 F Bom HI U GE' GC V RW Kpn Eco RI G M G A 0 U. Q0 F K II ~~~iii E *K1 H K2 B s1,py N J XZ-YP I;. I;;... c RI AR I; H K N O M FIG. 5. Summary of the results of marker rescue of tshal with restriction endonuclease digests of HSV-2 DNA. The top two lines represent sequence arrangement and map coordinates of HSV DNA in the prototype arrangement. All the lines below represent expansions of two regions of HSV DNA in L and S components. The top and bottom lines to the right of the individual designations of the recombinants represent HSV-1 and HSV-2 sequences, respectively. The heavy line identifies the sequences (HSV-1 or HSV-2) present in the recombinant DNAs. Although only one recombinant is listed for each line, the ones listed are representative of other recombinants exhibiting similar map positions of HSV-1 sequences replaced by HSV-2 DNA. The bottom portion of the figure shows the relevant restriction endonuclease maps of the expanded regions of the HSV-1 and HSV-2 DNAs. The BamHI and KpnI maps of HSV-1 mp were determined from the maps published by Locker and Frenkel (15) and Post et al. (21). All other maps were published elsewhere (12, 18). the genome failed to convert 5OB to wild-type phenotype. The cloned BamHl J fragment (0.89 to 0.94 map units) rescued the SOB mutant with high efficiency. Thus, the titers of the progeny of transfection tests were 17.1 x 107 and 4.7 x 107 PFU at 39 and 31'C, respectively, indicating that 28% of the progeny had the phenotype of wild-type virus. In contrast, titers of the progeny of transfection of 50B alone were 8.7 x 107 and x 107 PFU at 39 and 31'C, respectively, indicating that only 0.17% had the phenotype of wild-type virus. The results of the marker rescue tests with the cloned HSV-1 fragments are therefore consistent with the results of the marker rescue tests with HSV-2 DNA and indicate that the mutation maps within map units 0.90 to 0.94 on the physical map of the prototype arrangement of the genome. DISCUSSION Mapping of the defect in 50B. The salient features of the mapping studies described in this paper were as follows. (i The marker rescue studies with both HSV- 2 DNA and cloned HSV-1 DNA fragments indicate that the defect in mutant SOB maps in the S component even though the mutagenized DNA purified from electrophoretically separated digests of HSV-1 DNA contained the fragment spanning the coordinates from 0.24 to 0.40 map units and all of the selected mutants, including SOB, expressed the Ara TFr genetic marker carried by that fragment. One likely explanation is that DNA fragments purified from electrophoretically separated fragments generated by digestion of viral DNA with restriction endonuclease were probably contaminated with DNA arising from other regions of the genome, and that mutant SOB arose by recoinbination of the parental genome with two fragments, i.e., the fragment carrying Ara T resistance (thymnidine kinase minus) and the mutagenized fragment mapping in the S component of the genome. The conclusion that the mutagenesis of the fragment carrying the Ara T r marker did not contribute to the phenotype of this mutant is reflected in the observation that the mutant was converted to wild-type pheno-

9 878 TOGNON ET AL. type by marker rescue with a cloned HSV-1 fragment from the S component but not with any of the cloned DNA fragments from 0.20 to 0.40 map units. (ii) It is of interest that in all of the HSV-1 x HSV-2 recombinants produced by marker rescue of 50B, the HSV-2 sequence extended symmetrically into the reiterated regions of the S component. The results of analysis of these recombinants are consistent with those of other HSV-1 x HSV-2 recombinants which showed that the terminal regions of the S component were identical (14). Phenotype of mutant 50B. The initial interest in mutant 50B stemmed from the observation that its efficiency of plating at 33.5 or 310C was considerably lower than that at 390C. Subsequent studies showed that the relative yields of 50B at the various temperatures of incubation were not significantly different from those of the parent virus, but that the transmission of the virus from cell to cell and the destruction of cells manifest in the form of plaques were grossly delayed at the lower temperatures. However, the significant aspects of this report emerged from the electron microscopic studies and center on the finding, late in infection, of numerous coreless capsids at the nuclear pores in cells infected with the mutant 50B. Our results therefore indicate that mutant 50B is defective in a function manifested in the accumulation of coreless capsids in the cytoplasm at nuclear pores late in infection. Although coreless herpesvirus nucleocapsids juxtaposed to nuclear pores have not been reported previously, Dales and Chardonnet (2) showed that after infection, adenovirus 5 virions are transported to nuclear pores. They also reported evidence that the uncoating of adenovirus 5 takes place in juxtaposition to or upon binding to the nuclear pore structures. Consistent with their results, we have found that coreless HSV capsids accumulated at the nuclear pores early in infection. In cells infected with wild-type virus, these capsids were rarely seen late in infection. The conclusion that coreless capsids at the nuclear pores represent parental viral capsids which had released viral DNA is reinforced by recent studies on cells infected with mutant HSV-1(HFEM) tsb7. At the nonpermissive temperature (390C), this mutant expresses no detectable viral functions (13). In cells infected and maintained at the nonpermissive temperature, capsids containing cores accumulate at the nuclear pores, but the cores largely disappear within 30 min after shift down to permissive (330C) temperatures (W. Batterson, D. Furlong, and B. Roizman, manuscript in preparation). J. VIROL. The significance of our results stems from the following considerations. (i) In cells infected with 50B, the time course of accumulation and disappearance of coreless capsids at the nuclear membranes early in infection was similar to that observed in cells infected with wild-type virus. Furthermore, the number of coreless capsids at the nuclear pores late in infection was a significant fraction of the total cytoplasmic particles and by a large factor more than could be seen between 0.5 and 2 h postinfection. We conclude therefore that the coreless capsids represent progeny virus rather than failure of disaggregation of input, parental viral capsids. (ii) It has been generally observed that coreless capsids are rarely enveloped (23). In thin sections of infected cells 48 h postinfection with wild-type virus, they represent fewer than 5% of total capsids accumulating in the cytoplasm. Furthermore, few if any are juxtaposed to the nuclear pore (D. Furlong and B. Roizman, unpublished data). This conclusion was recently reinforced by the studies of D. Vlazny and N. Frenkel (personal communication), which showed that although capsids containing less than full-length DNA molecules could be detected in nuclear extracts, they were not present among the enveloped progeny accumulating in the cytoplasm. These observations suggest that the coreless capsids represent particles from which the DNA was released possibly in a fashion analogous to the release of DNA from nucleocapsids of input parental virus early in infection. (iii) Finally, the marker rescue data suggest that the failure to produce plaques at 310C and the defect responsible for the accumulation of empty capsids at the nuclear pores at least comap and could be related. One hypothesis that could explain these data is that the function of one or more polypeptides specified by the virus is to prevent thr readsorption of virus particles to the cells from which they were released and that this function has been mutagenized and is defective in mutant 50B. The observation that production of plaques by mutant 50B is considerably delayed at 330C than at 390C and yet empty capsids accumulate at nuclear pores in cells maintained at both temperatures is not inconsistent with this hypothesis inasmuch as the release of virus from cells infected with wildtype virus at the lower temperatures is considerably less efficient than at the higher temperatures (10). Consequently, adsorption of released virus from cells incubated at 31 C would have a more drastic effect on cell-to-cell transmission of virus than that in cultures maintained at 390C. The hypothesis that HSV encodes a function

10 VOL. 40, 1981 that precludes the adsorption of released progeny virus may explain a phenomenon known for many years. Early studies have shown that the plasma membranes of HSV-infected cells acquire an immunological specificity indistinguishable from that of the virion envelope (5, 7, 20, 22, 24, 27), and subsequent studies have shown that these membranes incorporate viral glycoproteins in the course of viral replication (8, 9, 24). One explanation for the presence of viral glycoprotein in the plasma membrane of cells infected with HSV, apart from the formal explanation that they represent an excess which flows into the plasma membrane, is that one or more viral membrane proteins prevent readsorption of released virus (16). The defect in 50B could therefore be an impaired flow of glycoproteins into the plasma membrane or the loss of this function by viral gene products accumulating on the surface of infected cells. It is noteworthy that the DNA sequences which rescue 50B contain the genes for glycoprotein D (25) and glycoprotein E (Para, Goldstein, and Spear, personal communication). It is conceivable that these or other genes mapping that region may be responsible for this function. ACKNOWLEDGMENTS These studies were aided by Public Health Service grants CA and CA from the National Cancer Institute and by American Cancer Society Grant MV-2Q. M. T. was a James E. Davis Fellow. A.J.C. was a Public Health Service postdoctoral trainee (grant GM ). D.F. is a Public Health Service postdoctoral trainee (grant AI-07182). LITERATURE CITED 1. Conley, A. F., D. M. Knipe, P. C. Jones, and B. Roizman Molecular genetics of herpes simplex virus. VII. Characterization of a temperature sensitive mutant produced by in vitro mutagenesis and defective in DNA synthesis and accumulation of a polypeptides. J. Virol. 37: Dales, S., and Y. Chardonnet Early events in the interaction of adenoviruses with HeLa cells. IV. Association with microtubules and the nuclear pore complex during vectorial movement of the inoculum. Virology 56: Darlington, R. W., and L. H. Moss III Herpesvirus envelopment. J. Virol. 2: Ejercito, P. M., E. D. Kieff, and B. Roizman Characterization of herpes simplex virus strains differing in their effect on social behavior of infected cells. J. Gen. Virol. 3: Glorioso, J. C., L. A. Wilson, T. W. Fenger, and J. W. Smith Complement-mediated cytolysis of HSV- 1 and HSV-2 infected cells: plasma membrane antigens reactive with type-specific and cross-reactive antibody. J. Gen. Virol. 40: Hammer, S. M., T. G. Buchman, L. J. D'Angelo, A. W. Karchmer, B. Roizman, and M. S. Hirsch Temporal cluster of herpes simplex encephalitis: investigation by restriction endonuclease cleavage of viral DNA. J. Infect. Dis. 141: Hayashi, D., A. Niwa, J. Rosenthal, and A. L. Notkins Detection of virus-induced membrane and cytoplasmic antigen: comparison of the '25M-labeled MOLECULAR GENETICS OF HSV 879 antiviral antibody binding technique with immunofluorescence. Intervirology 2: Heine, J. W., and B. Roizman Proteins specified by herpes simplex virus. IX. Contiguity of host and viral proteins in the plasma membrane of infected cells. J. Virol. 11: Heine, J. W., P. G. Spear, and B. Roizman The proteins specified by herpes simplex virus. VI. Viral protein in the plasma membrane. J. Virol. 9: Hoggan, M. D., and B. Roizman The effect of the temperature of incubation on the formation and release of herpes simplex virus in infected FL cells. Virology 8: Hoggan, M. D., and B. Roizman The isolation and properties of a variant of herpes simplex producing multinucleated giant cells in monolayer cultures in the presence of antibody. Am. J. Hyg. 70: Jones, P. C., G. S. Hayward, and B. Roizman Anatomy of herpes simplex virus DNA. VI. arna is homologous to noncontiguous sites in both L and S components of viral DNA. J. Virol. 21: Knipe, D. M., W. Batterson, C. Nosal, B. Roizman, and A. Buchan Molecular genetics of herpes simplex virus. VI. Characterization of a temperaturesensitive mutant defective in the expression of all early viral gene products. J. Virol. 38: Knipe, D. M., W. T. Ruyechan, B. Roizman, and L. W. Halliburton Molecular genetics of herpes simplex virus. Demonstration of regions of obligatory and non-obligatory identity in diploid regions of the genome by sequence replacement and insertion. Proc. Natl. Acad. Sci. U.S.A. 75: Locker, H., and N. Frenkel BamI, KpnI, and Sail restriction enzyme maps of the DNAs of herpes simplex virus strains Justin and F: occurrence of heterogeneities in defined regions of the viral DNA. J. Virol. 32: Manservigi, R., P. G. Spear, and A. Buchan Cell fusion induced by herpes simplex virus is promoted and suppressed by different viral glycoproteins. Proc. Natl. Acad. Sci. U.S.A. 74: Morgan, C., H. M. Rose, and B. Mednis Electron microscopy of herpes simplex virus. I. Entry. J. Virol. 2: Morse, L. S., T. G. Buchman, B. Roizman, and P. A. Schaffer Anatomy of herpes simplex virus DNA. IX. Apparent exclusion of some parental DNA arrangements in the generation of intertypic (HSV-1 x HSV-2) recombinants. J. Virol. 24: Morse, L. S., L. Pereira, B. Roizman, and P. A. Schaffer Anatomy of herpes simplex virus (HSV) DNA. X. Mapping of vital genes by analysis of polypeptides and functions specified by HSV-1 x SV-2 recombinants. J. Virol. 26: Norrild, B., 0. J. Bjerrum, H. Ludwig, and B. F. Vestergard Analysis of herpes simplex virus type 1 antigens exposed on the surface of infected tissue culture cells. Virology 87: Post, L. E., A. J. Conley, E. S. Mocarski, and B. Roizman Cloning of reiterated and non-reitereated herpes simplex virus 1 sequences as Bam HI fragments. Proc. Natl. Acad. Sci. U.S.A. 77: Roane, P. R., Jr., and B. Roizman Studies of the determinant antigens of viable cells. II. Demonstration of altered antigenic reactivity of HEp-2 cells infected with herpes simplex virus. Virology 22: Roizman, B., and D. Furlong The replication of herpesviruses. Compr. Virol. 3: Roizman, B., and P. G. Spear Herpesvirus antigens on cell membranes detected by centrifugation of membrane-antibody complexes. Science 171: Ruyechan, W. T., L S. Morse, D. M. Knipe, and B.

11 880 TOGNON ET AL. Roizman Molecular genetics of herpes simplex virus. II. Mapping of the major viral glycoproteins and of the genetic loci specifying the social behavior of infected cells. J. Virol. 29: Schwartz, J., and B. Roizman Concerning the egress of herpes simplex virus from infected cells: electron microscope observations. Virology 38: Shore, S. L., C. M. Black, F. M. Melewixz, P. A. Wood, J. VIROL. and A. J. Nahmias Antibody dependent cellmediated cytotoxicity to target cells infected with type 1 and type 2 herpes simplex virus. J. Immunol. 116: Spear, P. G Membrane proteins specified by herpes simplex viruses. I. Identification of four glycoprotein precursors and their products in type 1-infected cells. J. Virol. 17: