Site-directed mutagenesis of the virion host shutoff gene (UL41) of herpes simplex virus (HSV): analysis of functional differences between HSV type 1 (HSV-1) and HSV-2 alleles

Abstract

During lytic herpes simplex virus (HSV) infections, the HSV virion host shutoff protein (UL41) accelerates the turnover of host and viral mRNAs. Although the UL41 polypeptides from HSV type 1 (HSV-1) strain KOS and HSV-2 strain 333 are 87% identical, HSV-2 strains generally shut off the host more rapidly and completely than HSV-1 strains. In a previous study, we identified three regions of the HSV-2 UL41 polypeptide (amino acids 1 to 135, 208 to 243, and 365 to 492) that enhance the activity of KOS when substituted for the corresponding portions of the KOS protein (D. N. Everly, Jr., and G. S. Read, J. Virol. 71:7157-7166, 1997). These results have been extended through the analysis of more than 50 site-directed mutants of UL41 in which selected HSV-2 amino acids were introduced into an HSV-1 background and HSV-1 amino acids were introduced into the HSV-2 allele. The HSV-2 amino acids R22 and E25 were found to contribute dramatically to the greater activity of the HSV-2 allele, as did the HSV-2 amino acids A396 and S423. The substitution of six HSV-2 amino acids between residues 210 and 242 enhanced the HSV-1 activity to a lesser extent. In most cases, individual substitutions or the substitution of combinations of fewer than all six amino acids reduced the UL41 activity to less than that of KOS. The results pinpoint several type-specific amino acids that are largely responsible for the greater activity of the UL41 polypeptide of HSV-2. In addition, several spontaneous mutations that abolish detectable UL41 activity were identified.

FIG. 1

Structures and activities of chimeric UL41 alleles. (A) The UL41 polypeptide encoded by HSV-1 (strain KOS) is represented by the open rectangle, and that encoded by HSV-2 (strain 333) is represented by the hatched rectangle. The short vertical lines above the rectangles indicate the sites where the KOS and 333 polypeptides differ (9). The polypeptides encoded by several chimeric UL41 alleles are shown, with the portion contributed by KOS represented by an open rectangle and that contributed by strain 333 represented by a hatched rectangle. The junctions between KOS and 333 sequences are indicated by the coordinates of the KOS amino acids. (B) Replicate Vero cell cultures were transfected with 3 μg of the reporter plasmid pSV-β-Galactosidase and the indicated amounts of UL41-expressing effector plasmids. lacZ expression was determined 40 to 48 h after transfection and expressed as a fraction of that observed for transfections involving the pcDNA1.1amp vector and no UL41 effector plasmid. Error bars represent standard errors of the means. For datum points where no error bars are visible, the error bars were smaller than the datum points.

FIG. 2

UL41 alleles with mutations in amino acids 1 to 135. The UL41 polypeptide of HSV-1 (strain KOS) is depicted by the open rectangle, with the portion (amino acids 1 to 135) containing site-directed mutations indicated by hatching. The structures of the UL41 polypeptides encoded by HSV-1(KOS) and the 3/K(135) chimera are shown, with only those amino acids that differ indicated. The UL41 polypeptides encoded by a number of mutant alleles are indicated, with only those amino acids at which the mutants differ from KOS indicated. Amino acid coordinates are those for the KOS allele. The [DNA]_0.3 for each allele is shown at the right of the figure, expressed as a fraction of the [DNA]_0.3 for KOS. The error bars represent standard errors of the means.

FIG. 3

UL41 alleles with mutations in amino acids 1 to 135. The UL41 polypeptide encoded by HSV-1 (strain KOS) is depicted by the open rectangle, with the portion (amino acids 1 to 135) containing site-directed mutations indicated by hatching. The structure of the KOS polypeptide is shown, with only those amino acids at which it differs from the 3/K(135) chimera shown. The UL41 polypeptides encoded by a number of mutant alleles are shown, with only those amino acids that differ from KOS indicated. Amino acid coordinates are those for the KOS allele. The [DNA]_0.3 for each allele is shown at the right of the figure, expressed as a fraction of the [DNA]_0.3 for KOS. The error bars represent standard errors of the means.

FIG. 4

UL41 alleles with mutations in amino acids 365 to 489. The UL41 polypeptide of HSV-1 (strain KOS) is depicted by the open rectangle, with the portion (amino acids 365 to 489) containing site-directed mutations indicated by hatching. The structures of the UL41 polypeptides encoded by HSV-1(KOS) and the K/3(365) chimera are shown, with only those amino acids that differ indicated. The UL41 polypeptides encoded by a number of mutant alleles are shown, with only those amino acids that differ from KOS indicated. Amino acid coordinates are those for the KOS allele. The [DNA]_0.3 for each allele is shown at the right of the figure, expressed as a fraction of the [DNA]_0.3 for KOS. The error bars represent standard errors of the means.

FIG. 5

UL41 alleles with mutations in amino acids 365 to 489. The UL41 polypeptide of HSV-1 (strain KOS) is depicted by the open rectangle, with the portion (amino acids 365 to 489) containing site-directed mutations indicated by hatching. The structures of the UL41 polypeptides encoded by HSV-1(KOS) and the K/3(365) chimera are shown, with only those amino acids that differ indicated. The UL41 polypeptides encoded by a number of mutant alleles are shown, with only those amino acids that differ from KOS indicated. Amino acid coordinates are those for the KOS allele. Dose-response curves for the inhibition of reporter gene expression by the KOS, K/3(365), and mutant alleles are shown at the bottom. Error bars represent standard errors of the means. For datum points where no error bars are visible, the error bars were smaller than the datum points.

FIG. 6

UL41 alleles with mutations in amino acids 208 to 243. The UL41 polypeptide of HSV-1 (strain KOS) is depicted by the open rectangle, with the portion (amino acids 208 to 243) containing site-directed mutations indicated by hatching. The structures of the UL41 polypeptides encoded by HSV-1(KOS) and the K/3/K(208;243) chimera are shown, with only those amino acids that differ indicated. The UL41 polypeptides encoded by a number of mutant alleles are shown, with only those amino acids at which the mutants differ from KOS indicated. Amino acid coordinates are those for the KOS allele. The [DNA]_0.3 for each allele is shown at the right of the figure, expressed as a fraction of the [DNA]_0.3 for KOS. The error bars represent standard errors of the means.

FIG. 7

UL41 alleles with mutations in amino acids 208 to 243. The UL41 polypeptide of HSV-1 (strain KOS) is depicted by the open rectangle, with the portion (amino acids 208 to 243) containing site-directed mutations indicated by hatching. The structures of the UL41 polypeptides encoded by HSV-1(KOS) and the K/3/K(208;243) chimera are shown, with only those amino acids that differ indicated. The UL41 polypeptides encoded by a number of mutant alleles are shown, with only those amino acids at which the mutants differ from KOS indicated. Amino acid coordinates are those for the KOS allele. Dose-response curves for the inhibition of reporter gene expression by the KOS, K/3/K(208;243), and mutant alleles are shown at the bottom. Error bars represent standard errors of the means. For datum points where no error bars are visible, the error bars were smaller than the datum points.

FIG. 8

Dose-response curves for the inhibition of reporter gene expression by spontaneous and site-directed mutants of UL41. Replicate Vero cell cultures were transfected with 3 μg of the reporter plasmid pSV-β-Galactosidase and the indicated amounts of UL41-expressing effector plasmids. lacZ expression was determined 40 to 48 h after transfection and expressed as a fraction of that observed for transfections involving the pcDNA1.1amp vector and no UL41 effector plasmid. Error bars represent standard errors of the means. For datum points where no error bars are visible, the error bars were smaller than the datum points.

FIG. 9

Summary of the residues important to type-specific differences in UL41 activity. The UL41 polypeptide of HSV-1 (strain KOS) is depicted by the open rectangle at the top of the figure. The short vertical lines above the rectangle indicate the sites at which the strain KOS and 333 polypeptides differ (9). Replacement of the shaded regions of the KOS polypeptide (amino acids 1 to 135, 208 to 243, and 365 to 489) with the corresponding regions of the HSV-2 (strain 333) polypeptide has been shown to significantly enhance UL41 activity (9). The regions of the KOS polypeptide that are conserved in the UL41 homologues of the alphaherpesviruses are summarized, with roman numerals I through IV referring to the conserved regions identified by Berthomme and coworkers (3), while the region labeled A was identified by Jones and colleagues (19). The sequences of UL41 homologues corresponding to the portions of the HSV-1 and HSV-2 polypeptides that are responsible for type-specific differences are shown in the middle and lower portions of the figure. The sequences of the various UL41 homologues are taken from references cited in the text. The amino acids identified in this study as being important for the difference in UL41 activity of HSV-1(KOS) and HSV-2(333) are highlighted by white type in black boxes. Amino acid numbers refer to the positions of the KOS residues. Sequences between amino acids 398 and 421 were omitted (indicated by ∼). Threonine 214 that is mutated to isoleucine in the mutant vhs 1 is indicated by an arrow. The triplet of amino acids (leucine 200, tyrosine 201, and histidine 202) that is altered in one of the spontaneous mutants is indicated by the light shading in the bottom portion of the figure. Abbreviations: EHV, equine herpesvirus; HV-2, herpesvirus 2; BHV, bovine herpesvirus; PRV, pseudorabies virus; VZV, varicella-zoster virus.

FIG. 10

Sequences of UL41 homologues surrounding arginine 435. Arginine 435, which is invariant in all of the sequenced UL41 homologues of alphaherpesviruses, is highlighted by white type in black boxes. Alteration of this residue to histidine completely abolished UL41 activity. Amino acids that are identical to residues in HSV-1 (strain KOS) are shown in lightly shaded boxes, while conservative changes are shown in boldface and underlined. Abbreviations: EHV, equine herpesvirus; HV-2, herpesvirus 2; BHV, bovine herpesvirus; PRV, pseudorabies virus; VZV, varicella-zoster virus.