Processing of herpes simplex virus-1 glycans in cells defective in glycosyl transferases of the golgi system: Relationship to cell fusion and virion egress

ABSTRACT Glycoprotein D (gD) interacts with two alternative protein receptors, nectin1 and HveA, to mediate herpes simplex virus (HSV) entry into cells. Fusion of the envelope with the plasma membrane requires, in addition to gD, glycoproteins gB, gH, and gL. Coexpression of the four glycoproteins (gD, gB, gH, and gL) promotes cell-cell fusion. gD delivered in trans is also capable of blocking the apoptosis induced by gD deletion viruses grown either in noncomplementing cells (gD−/−) or in complementing cells (gD−/+). While ectopic expression of cation-independent mannose-6 phosphate receptor blocks apoptosis induced by both stocks, other requirements differ. Thus, apoptosis induced by gD−/− virus is blocked by full-length gD (or two gD fragments reconstituting a full-length molecule), whereas ectopic expression of the gD ectodomain is sufficient to block apoptosis induced by gD−/+ virus. In this report we took advantage of a set of gD insertion-deletion mutants to map the domains of gD required to block apoptosis by gD−/− and gD−/+ viruses and those involved in cell-cell fusion. The mutations that resulted in failure to block apoptosis were the same for gD−/− and gD−/+ viruses and were located in three sites, one within the immunoglobulin-type core region (residues 125, 126, and 151), one in the upstream connector region (residues 34 and 43), and one in the C-terminal portion of the ectodomain (residue 277). A mutant that carried amino acid substitutions at the three glycosylation sites failed to block apoptosis but behaved like wild-type gD in all other assays. The mutations that inhibited polykaryocyte formation were located in the upstream connector region (residues 34 and 43), at the α1 helix (residue 77), in the immunoglobulin core and downstream regions (residue 151 and 187), and at the α3 helix (residues 243 and 246). Binding of soluble nectin1-Fc to cells expressing the mutant gDs was generally affected by the same mutations that affected fusion, with one notable exception (Δ277-310), which affected fusion without hampering nectin1 binding. This deletion likely identifies a region of gD involved in fusion activity at a post-nectin1-binding step. We conclude that whereas mutations that affected all functions (e.g., upstream connector region and residue 151) may be detrimental to overall gD structure, the mutations that affect specific activities identify domains of gD involved in the interactions with entry receptors and fusogenic glycoproteins and with cellular proteins required to block apoptosis. The evidence that glycosylation of gD is required for blocking apoptosis supports the conclusion that the interacting protein is the mannose-6 phosphate receptor.

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Mapping of Key Functions of the Herpes Simplex Virus 1 US3 Protein Kinase: the US3 Protein Can Form Functional Heteromultimeric Structures Derived from Overlapping Truncated Polypeptides

Journal of Virology ◽

10.1128/jvi.02265-06 ◽

2006 ◽

Vol 81 (4) ◽

pp. 1980-1989 ◽

Cited By ~ 11

Author(s):

Alice P. W. Poon ◽

Bernard Roizman

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Herpes Simplex Virus 1 ◽

Histone Deacetylase 1 ◽

Amino Terminal ◽

Helper Function ◽

Antiapoptotic Activity ◽

Carboxyl Terminal ◽

Simplex Virus ◽

In Cells

ABSTRACT Earlier studies have shown that the herpes simplex virus (HSV) US3 encodes two transcriptional units directing the synthesis of the US3 (residues 1 to 481) and US3.5 (residues 77 to 481) protein kinases. Both kinases phosphorylate histone deacetylase 1 (HDAC1) and HDAC2 and enable the expression of genes cotransduced into U2OS cells by recombinant baculoviruses, an activity designated the “helper function.” The two kinases differ with respect to antiapoptotic activity. In the studies reported here, we made a series of FLAG-tagged amino- and carboxyl-terminal truncations of US3 and these were tested for antiapoptotic activity, phosphorylation of HDAC1, and the helper function. We report the following. (i) HDAC1 phosphorylation and the helper function were expressed in cells transduced by the truncation encoding residues 182 to 481 but not in cells transduced by the truncation encoding residues 189 to 481 or the amino-terminal polypeptides encompassing the first 188 amino acids. (ii) The self-posttranslational modification requires residues 164 to 481. (iii) The antiapoptotic activity requires both the amino-terminal and the carboxyl-terminal domains, of which the truncated protein containing residues 1 to 163 and that containing residues 164 to 481, respectively, were the smallest fragments tested to be effective. The two domains need not be on the same molecule, but they must overlap. The smallest overlapping pair tested was the fragment containing residues 1 to 181 and that containing residues 164 to 481. Consistent with the hypothesis that the effective overlapping truncations form a heteromultimeric structure, antibody to FLAG coprecipitated untagged US3 from lysates of cells cotransduced with FLAG-tagged, truncated US3 constructs. Although US3 has been reported to be a monomeric enzyme, the results indicate that it can form enzymatically active multimeric structures.

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The patterns of accumulation of cellular RNAs in cells infected with a wild-type and a mutant herpes simplex virus 1 lacking the virion host shutoff gene

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.252588599 ◽

2002 ◽

Vol 99 (26) ◽

pp. 17031-17036 ◽

Cited By ~ 73

Author(s):

B. Taddeo ◽

A. Esclatine ◽

B. Roizman

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Wild Type ◽

Herpes Simplex Virus 1 ◽

Virion Host Shutoff ◽

Host Shutoff ◽

Simplex Virus ◽

In Cells

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Posttranslational Processing of Infected Cell Proteins 0 and 4 of Herpes Simplex Virus 1 Is Sequential and Reflects the Subcellular Compartment in Which the Proteins Localize

Journal of Virology ◽

10.1128/jvi.75.17.7904-7912.2001 ◽

2001 ◽

Vol 75 (17) ◽

pp. 7904-7912 ◽

Cited By ~ 27

Author(s):

Sunil J. Advani ◽

Ryan Hagglund ◽

Ralph R. Weichselbaum ◽

Bernard Roizman

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Infected Cell ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Herpes Simplex Virus 1 ◽

Simplex Virus ◽

Made In ◽

In Cells

ABSTRACT The herpes simplex virus 1 (HSV-1) infected cell proteins 0 and 4 (ICP0 and ICP4) are multifunctional proteins extensively posttranscriptionally processed by both cellular and viral enzymes. We examined by two-dimensional separations the posttranslational forms of ICP0 and ICP4 in HEp-2 cells and in human embryonic lung (HEL) fibroblasts infected with wild-type virus, mutant R325, lacking the sequences encoding the US1.5 protein and the overlapping carboxyl-terminal domain of ICP22, or R7914, in which the aspartic acid 199 of ICP0 was replaced by alanine. We report the following (i) Both ICP0 and ICP4 were sequentially posttranslationally modified at least until 12 h after infection. In HEL fibroblasts, the processing of ICP0 shifted from A+B forms at 4 h to D+G forms at 8 h and finally to G, E, and F forms at 12 h. The ICP4 progression was from the A′ form noted at 2 h to B′ and C′ forms noted at 4 h to the additional D′ and E′ forms noted at 12 h. The progression tended to be toward more highly charged forms of the proteins. (ii) Although the overall patterns were similar, the mobility of proteins made in HEp-2 cells differed from those made in HEL fibroblasts. (iii) The processing of ICP0 forms E and F was blocked in HEL fibroblasts infected with R325 or with wild-type virus and treated with roscovitine, a specific inhibitor of cell cycle-dependent kinases cdc2, cdk2, and cdk5. R325-infected HEp-2 cells lacked the D′ form of ICP4, and roscovitine blocked the appearance of the most highly charged E′ form of ICP4. (iv) A characteristic of ICP0 is that it is translocated into the cytoplasm of HEL fibroblasts between 5 and 9 h after infection. Addition of MG132 to the cultures late in infection resulted in rapid relocation of cytoplasmic ICP0 back into the nucleus. Exposure of HEL fibroblasts to MG132 late in infection resulted in the disappearance of the highly charged ICP0 G isoform. The G form of ICP0 was also absent in cells infected with R7914 mutant. In cells infected with this mutant, ICP0 is not translocated to the cytoplasm. (v) Last, cdc2 was active in infected cells, and this activity was inhibited by roscovitine. In contrast, the activity of cdk2 exhibited by immunoprecipitated protein was reduced and resistant to roscovitine and may represent a contaminating kinase activity. We conclude from these results that the ICP0 G isoform is the cytoplasmic form, that it may be phosphorylated by cdc2, consistent with evidence published earlier (S. J., Advani, R. R. Weichselbaum, and B. Roizman, Proc. Natl. Acad. Sci. USA 96:10996–11001, 2000), and that the processing is reversed upon relocation of the G isoform from the cytoplasm into the nucleus. The processing of ICP4 is also affected by R325 and roscovitine. The latter result suggests that ICP4 may also be a substrate of cdc2 late in infection. Last, additional modifications are superimposed by cell-type-specific enzymes.

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Glycoprotein D or J Delivered in transBlocks Apoptosis in SK-N-SH Cells Induced by a Herpes Simplex Virus 1 Mutant Lacking Intact Genes Expressing Both Glycoproteins

Journal of Virology ◽

10.1128/jvi.74.24.11782-11791.2000 ◽

2000 ◽

Vol 74 (24) ◽

pp. 11782-11791 ◽

Cited By ~ 107

Author(s):

Guoying Zhou ◽

Veronica Galvan ◽

Gabriella Campadelli-Fiume ◽

Bernard Roizman

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Dna Sequences ◽

De Novo ◽

Cell Protein ◽

Herpes Simplex Virus 1 ◽

Simplex Virus ◽

Apoptotic Cascade ◽

Made In ◽

In Cells

ABSTRACT We have made two stocks of a herpes simplex virus 1 mutant lacking intact US5 and US6 open reading frames encoding glycoproteins J (gJ) and D (gD), respectively. The stock designated gD−/+, made in cells carrying US6 and expressing gD, was capable of productively infecting cells, whereas the stock designated gD−/−, made in cells lacking viral DNA sequences, was known to attach but not initiate infection. We report the following. (i) Both stocks of virus induced apoptosis in SK-N-SH cells. Thus, annexin V binding to cell surfaces was detected as early as 8 h after infection. (ii) US5 or US6 cloned into the baculovirus under the human cytomegalovirus immediate-early promoter was expressed in SK-N-SH cells and blocked apoptosis in cells infected with either gD−/+ or gD−/− virus, whereas glycoprotein B, infected cell protein 22, or the wild-type baculovirus did not block apoptosis. (iii) In SK-N-SH cells, internalized, partially degraded virus particles were detected at 30 min after exposure to gD−/− virus but not at later intervals. (iv) Concurrent infection of cells with baculoviruses did not alter the failure of gD−/− virus from expressing its genes or, conversely, the expression of viral genes by gD−/+ virus. These results underscore the capacity of herpes simplex virus to initiate the apoptotic cascade in the absence of de novo protein synthesis and indicate that both gD and gJ independently, and most likely at different stages in the reproductive cycle, play a key role in blocking the apoptotic cascade leading to cell death.

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