Cell-free synthesis of herpes simplex virus proteins.

Polyribosomes isolated from herpes simplex virus type I (HSV-1)-infected cells have been used to program a eucaryotic cell-free translation system. At least 10 HSV-specific polypeptides, with apparent molecular weights of 25,000 to 160,000, are synthesized by wild-type HSV-infected polyribosomes. Polyribosomes prepared from thymidine kinase-negative mutants of HSV direct the synthesis of three putative nonsense termination polypeptides. HSV-specific polypeptides synthesized in vitro are precipitated with antiserum to HSV-infected cell proteins.     

Involvement of the portal at an early step in herpes simplex virus capsid assembly

DNA enters the herpes simplex virus capsid by way of a ring-shaped structure called the portal. Each capsid contains a single portal, located at a unique capsid vertex, that is composed of 12 UL6 protein molecules. The position of the portal requires that capsid formation take place in such a way that a portal is incorporated into one of the 12 capsid vertices and excluded from all other locations, including the remaining 11 vertices. Since initiation or nucleation of capsid formation is a unique step in the overall assembly process, involvement of the portal in initiation has the potential to cause its incorporation into a unique vertex. In such a mode of assembly, the portal would need to be involved in initiation but not able to be inserted in subsequent assembly steps. We have used an in vitro capsid assembly system to test whether the portal is involved selectively in initiation. Portal incorporation was compared in capsids assembled from reactions in which (i) portals were present at the beginning of the assembly process and (ii) portals were added after assembly was under way. The results showed that portal-containing capsids were formed only if portals were present at the outset of assembly. A delay caused formation of capsids lacking portals. The findings indicate that if portals are present in reaction mixtures, a portal is incorporated during initiation or another early step in assembly. If no portals are present, assembly is initiated in another, possibly related, way that does not involve a portal.     

Handedness of the herpes simplex virus capsid and procapsid

The capsid of herpes simplex virus has an icosahedral surface lattice with a nonskew triangulation number, T=16. Nevertheless, the proteins arrayed on this lattice necessarily have an intrinsic handedness. We have determined the handedness of both the herpes simplex virus type 1 capsid and its precursor procapsid by a cryoelectron microscopic tilting method.     

Cell-free synthesis of herpes simplex virus DNA: the influence of polyamines.

The effect of polyamines on cell-free DNA synthesis of herpes simplex virus DNA in two different systems is investigated. Purified nuclei from infected cells are devoid of measurable amounts of putrescine, spermidine, and spermine, while an unfractionated lysate contains the polyamines at close to their respective cellular concentrations. Spermine, 0.3 mM, and 0.5 mM spermidine, when added to the nuclear system, decrease the extent of viral DNA synthesis to the level found in the lysate system, the size of the cell-free viral DNA product is increased, and a specific inhibition of repair-type DNA synthesis is observed. These effects of the polyamines occur only in the presence of ATP and not the other three ribonucleoside triphosphates.     

Physical mapping and nucleotide sequence of a herpes simplex virus type 1 gene required for capsid assembly.

In this report, we describe some phenotypic properties of a temperature-sensitive mutant of herpes simplex type 1 (HSV-1) and present data concerning the physical location and nucleotide sequence of the genomic region harboring the mutation. The effect of shifts from the permissive to the nonpermissive temperature on infectious virus production by the mutant A44ts2 indicated that the mutated function is necessary throughout, or late in, the growth cycle. At the nonpermissive temperature, no major differences were detected in viral DNA or protein synthesis with respect to the parent A44ts+. On the other hand, electron microscopy of mutant-infected cells revealed that neither viral capsids nor capsid-related structures were assembled at the nonpermissive temperature. Additional analyses employing the Hirt extraction procedure showed that A44ts2 is also unable to mature replicated viral DNA into unit-length molecules under nonpermissive conditions. The results of marker rescue experiments with intact A44ts2 DNA and cloned restriction fragments of A44ts+ placed the lesion in the coordinate interval 0.553 to 0.565 (1,837 base pairs in region UL) of the HSV-1 physical map. No function has previously been assigned to this region, although it is known to be transcribed into two 5' coterminal mRNAs which code in vitro for a 54,000-molecular-weight polypeptide (K. P. Anderson, R. J. Frink, G. B. Devi, B. H. Gaylord, R. H. Costa, and E. K. Wagner, J. Virol. 37:1011-1027, 1981). We sequenced the interval 0.551 to 0.565 and found an open reading frame (ORF) for a 50,175-molecular-weight polypeptide. The predicted product of this ORF exhibits strong homology with the product of varicella-zoster virus ORF20 and lower, but significant, homology with the product of Epstein-Barr virus BORF1. For the three viruses, the corresponding ORFs lie just upstream of the gene coding for the large subunit of viral ribonucleotide reductase. The ORF described here corresponds to the ORF designated UL38 in the recently published nucleotide sequence of the HSV-1 UL region (D. J. McGeoch, M. A. Dalrymple, A. J. Davison, A. Dolan, M. C. Frame, D. McNab, L. J. Perry, J. E. Scott, and P. Taylor, J. Gen. Virol. 69:1531-1574, 1988).     

Induced extrusion of DNA from the capsid of herpes simplex virus type 1.

DNA-filled capsids (C capsids) of herpes simplex virus type 1 were treated in vitro with guanidine-HCl (GuHCl) and analyzed for DNA loss by sucrose density gradient ultracentrifugation and electron microscopy. DNA was found to be lost quantitatively from virtually all capsids treated with GuHCl at concentrations of 0.5 M or higher, while 0.1 M GuHCl had little or no effect. DNA removal from 0.5 M GuHCl-treated capsids was effected without significant change in the capsid protein composition, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, or in its structure, as judged by electron microscopy. Electron microscopic examination of capsids in the process of emptying showed that DNA was extruded from multiple, discrete sites which appeared to coincide with capsid vertices. DNA exited the capsid in the form of thick strands or fibers that varied in diameter from approximately 4 to 13 nm with preferred diameters of 7 and 11 nm. The fibers most probably correspond to multiple, laterally aligned DNA segments, as their diameters are nearly all greater than that of a single DNA double helix. The results suggest that GuHCl treatment promotes an alteration in the capsid pentons which allows DNA to escape locally. Hexons must be more resistant to this change, since DNA loss appears to be restricted to the pentons. The ability of GuHCl to cause loss of DNA from C capsids with no accompanying change in capsid morphology or protein composition suggests that penton sites may open transiently to permit DNA exist and then return to their original state.     

Function of dynein and dynactin in herpes simplex virus capsid transport

After fusion of the viral envelope with the plasma membrane, herpes simplex virus type 1 (HSV1) capsids are transported along microtubules (MTs) from the cell periphery to the nucleus. The motor ATPase cytoplasmic dynein and its multisubunit cofactor dynactin mediate most transport processes directed toward the minus-ends of MTs. Immunofluorescence microscopy experiments demonstrated that HSV1 capsids colocalized with cytoplasmic dynein and dynactin. We blocked the function of dynein by overexpressing the dynactin subunit dynamitin, which leads to the disruption of the dynactin complex. We then infected such cells with HSV1 and measured the efficiency of particle binding, virus entry, capsid transport to the nucleus, and the expression of immediate-early viral genes. High concentrations of dynamitin and dynamitin-GFP reduced the number of viral capsids transported to the nucleus. Moreover, viral protein synthesis was inhibited, whereas virus binding to the plasma membrane, its internalization, and the organization of the MT network were not affected. We concluded that incoming HSV1 capsids are propelled along MTs by dynein and that dynein and dynactin are required for efficient viral capsid transport to the nucleus.     

Architecture of the herpes simplex virus major capsid protein derived from structural bioinformatics

The dispositions of 39 alpha helices of greater than 2.5 turns and four beta sheets in the major capsid protein (VP5, 149 kDa) of herpes simplex virus type 1 were identified by computational and visualization analysis from the 8.5A electron cryomicroscopy structure of the whole capsid. The assignment of helices in the VP5 upper domain was validated by comparison with the recently determined crystal structure of this region. Analysis of the spatial arrangement of helices in the middle domain of VP5 revealed that the organization of a tightly associated bundle of ten helices closely resembled that of a domain fold found in the annexin family of proteins. Structure-based sequence searches suggested that sequences in both the N and C-terminal portions of the VP5 sequence contribute to this domain. The long helices seen in the floor domain of VP5 form an interconnected network within and across capsomeres. The combined structural and sequence-based informatics has led to an architectural model of VP5. This model placed in the context of the capsid provides insights into the strategies used to achieve viral capsid stability.     

Dynamics of herpes simplex virus capsid maturation visualized by time-lapse cryo-electron microscopy

The capsid of the herpes simplex virus initially assembles as a procapsid that matures through a massive conformational change of its 182 MDa surface shell. This transition, which stabilizes the fragile procapsid, is facilitated by the viral protease that releases the interaction between the shell and the underlying scaffold; however, protease-deficient procapsids mature slowly in vitro. To study procapsid maturation as a time-resolved process, we monitored this reaction by cryo-electron microscopy (cryo-EM). The resulting images were sorted into 17 distinct classes, and three-dimensional density maps were calculated for each. When arranged in a chronological series, these maps yielded molecular movies of procapsid maturation. A single major switching event takes place at stages 8-9, preceded by relatively subtle adjustments in the pattern of interactions and followed by similarly small 'aftershocks'. The primary mechanism underlying maturation is relative rotations of domains of VP5, the major capsid protein.     

Reconstitution of herpes simplex virus type 1 nuclear capsid egress in vitro

Newly assembled herpesvirus capsids travel from the nucleus to the plasma membrane by a mechanism that is poorly understood. Furthermore, the contribution of cellular proteins to this egress has yet to be clarified. To address these issues, an in vitro nuclear egress assay that reproduces the exit of herpes simplex virus type 1 (HSV-1) capsids from nuclei isolated from infected cells was established. As expected, the assay has all the hallmarks of intracellular transport assays, namely, a dependence on time, energy, and temperature. Surprisingly, it is also dependent on cytosol and was slightly enhanced by infected cytosol, suggesting an implication of both host and viral proteins in the process. The capsids escaped these nuclei by budding through the inner nuclear membrane, accumulated as enveloped capsids between the two nuclear membranes, and were released in cytosol exclusively as naked capsids, exactly as in intact cells. This is most consistent with the view that the virus escapes by crossing the two nuclear membranes rather than through nuclear pores. Unexpectedly, nuclei isolated at the nonpermissive temperature from cells infected with a U(L)26 thermosensitive protease mutant (V701) supported capsid egress. Although electron microscopy, biochemical, and PCR analyses hinted at a likely reconstitution of capsid maturation, DNA encapsidation could not be confirmed by a traditional SQ test. This assay should prove very useful for identification of the molecular players involved in HSV-1 nuclear egress.     

Study of herpes simplex virus maturation during a synchronous wave of assembly.

Production of an infectious herpes simplex virus (HSV) particle requires sequential progression of maturing virions through a series of complex assembly events. Capsids must be constructed in the nucleus, packaged with the viral genome, and transported to the nuclear periphery. They then bud into the nuclear membrane to acquire an envelope, traffic through the cytoplasm, and are released from the cell. Most of these phenomena are very poorly defined, and no suitable model system has previously been available to facilitate molecular analyses of genomic DNA packaging, capsid envelopment, and intracellular virion trafficking. We report the development of such an assay system for HSV type 1 (HSV-1). Using a reversible temperature-sensitive mutation in capsid assembly, we have developed conditions in which an accumulated population of immature capsids can be rapidly, efficiently, and synchronously chased to maturity. By assaying synchronized scaffold cleavage, DNA packaging, and acquisition of infectivity, we have demonstrated the kinetics with which these events occur. Kinetic and morphological features of intranuclear and extranuclear virion trafficking have similarly been examined by indirect immunofluorescence microscopy and electron microscopy. This system should prove a generally useful tool for the molecular dissection of many late events in HSV-1 biogenesis.     

Cell-free synthesis of herpes simplex virus-coded pyrimidine deoxyribonucleoside kinase enzyme.

The incubation of a cell-free protein-synthesizing system prepared from rabbit reticulocytes with cytoplasmic RNA from herpes simplex virus (HSV)-infected cells resulted in increased thymidine kinase activity. This enzyme activity was specifically inhibited by anti-HSV antiserum and was relatively unaffected by TTP, an inhibitor of cellular thymidine kinases. Induction of the new activity was prevented by addition of inhibitors of eucaryotic protein synthesis, and no new activity was detected when RNA from cells infected with pyrimidine deoxyribonucleoside kinase-deficient mutants, instead of wild-type HSV, was added. An increased deoxycytidine kinase activity with similar properties to the HSV-specified enzyme activity was also present in cell-free systems incubated with RNA from HSV-infected cells. Phosphorylation of thymidine and deoxycytidine at 30 degrees C continued for longer than 11 h. The findings are consistent with the accurate synthesis in vitro of enzymically active HSV-specified pyrimidine deoxyribonucleoside kinase.     

Structural analysis of the capsid polypeptides of herpes simplex virus types 1 and 2

Capsids of herpes simplex virus (HSV) types 1 and 2 contain seven polypeptides ranging in molecular weight from 154,000 to 12,000 (termed NC-1 through NC-7 in order of descending molecular weight). Antibodies prepared to HSV-1 capsid polypeptides isolated from sodium dodecyl sulfate-polyacrylamide gels reacted in an immunofluorescence assay against HSV-1-infected KB cells. Three of the antibodies (anti-NC-1, anti-NC-2, and anti-NC-3,4) also reacted with HSV-2-infected cells. Tryptic peptide analysis showed that each of the HSV-1 capsid polypeptides had a unique methionine peptide profile, and none appeared to be derived from the major capsid polypeptide. Comparative peptide analysis of HSV-1 and HSV-2 showed that one polypeptide (NC-7, 12,000 molecular weight) had an identical methionine peptide profile and a very similar arginine peptide profile in both virus types. The arginine peptide profile of NC-7 of HSV-1 was very different from the arginine profile of KB histone H4. Although there were certain intertypic similarities in the methionine peptide profiles of the other capsid components especially in NC-1 (the major capsid protein), there was no case where the tryptic peptides were identical in the two virus types.     

The pseudorabies virus homology of the herpes simplex virus UL21 gene product is a capsid protein which is involved in capsid maturation.

We mutagenized, mapped, and sequenced the pseudorabies virus (PRV) homology of gene UL21 of herpes simplex virus type 1. A polyclonal mouse antiserum against the protein encoded by the UL21 homolog was generated and used to monitor the expression and subcellular localization of the UL21-encoded protein. We found that the protein is identical to a previously detected PRV capsid protein. We analyzed viable PRV strains encoding mutant UL21 homologys, truncated by insertion of an oligonucleotide that contains stop codons in all reading frames. In two PRV mutants carrying the oligonucleotide at two sites within the gene, processing of newly replicated viral DNA was impaired. In addition, we show that one of the UL21 mutants has strongly reduced virulence for mice.     

Dynamic interactions of the UL16 tegument protein with the capsid of herpes simplex virus

The UL16 tegument protein of herpes simplex virus is conserved throughout the herpesvirus family. It has been reported to be capsid associated and may be involved in budding by providing an interaction with the membrane-bound UL11 protein. UL16 has been shown to be present in all the major locations that capsids are found (i.e., the nucleus, cytoplasm, and virions), but whether it is actually capsid associated in each of these has not been reported. Therefore, capsids were purified from each compartment, and it was found that UL16 was present on cytoplasmic but not nuclear capsids. In extracellular virions, the majority of UL16 (87%) was once again not capsid associated, which suggests that the interaction is transient during egress. Because herpes simplex virus (HSV) buds into the acidic compartment of the trans-Golgi network (TGN), the effect of pH on the interaction was examined. The amount of capsid-associated UL16 dramatically increased when extracellular virions were exposed to mildly acidic medium (pH 5.0 to 5.5), and this association was fully reversible. After budding into the TGN, capsid and tegument proteins also encounter an oxidizing environment, which is conducive to disulfide bond formation. UL16 contains 20 cysteines, including five that are conserved within a putative zinc finger. Any free cysteines that are involved in the capsid interaction or release mechanism of UL16 would be expected to be modified by N-ethylmaleimide, and, consistent with this, the amount of capsid-associated UL16 dramatically increased when virions were incubated with this compound. Taken together, these data suggest a transient interaction between UL16 and capsids, possibly modified in the acidic compartment of secretory vesicles and requiring a release mechanism that involves cysteines.     

Pseudorabies virus protein homologous to herpes simplex virus type 1 ICP18.5 is necessary for capsid maturation.

In pseudorabies virus (PrV), an open reading frame that partially overlaps the gene for the essential glycoprotein gII has been shown to encode a protein homologous to the ICP18.5 polypeptide of herpes simplex virus type 1 (N. Pederson and L. Enquist, Nucleic Acids Res. 17:3597, 1989). To study the function of this protein during the viral replicative cycle, a PrV mutant which carries a beta-galactosidase expression cassette interrupting the ICP18.5(PrV) gene was constructed. This mutant could be propagated only on cell lines that were able to provide ICP18.5(PrV) in trans after transformation with a corresponding genomic PrV DNA fragment. Detailed analysis showed that inactivation of the ICP18.5(PrV) gene did not impair infection of noncomplementing cells, nor did it impair early or late gene expression, as shown by immunoprecipitation of glycoproteins gII, gIII, and gp50. Surface localization of glycoproteins as demonstrated by fluorescence-activated cell sorting analyses was also not affected. Southern blot hybridizations, however, showed that cleavage of replicative concatemeric viral DNA did not occur in noncomplementing cells infected by the ICP18.5 mutant PrV. In addition, electron microscopic analysis revealed an accumulation of empty capsids in the nucleus of mutant-infected noncomplementing cells. We conclude that the ICP18.5(PrV) protein is necessary for viral replication and plays an essential role in the process of mature capsid formation.