Analysis of a charge cluster mutation of herpes simplex virus type 1 UL34 and its extragenic suppressor suggests a novel interaction between pUL34 and pUL31 that is necessary for membrane curvature around capsids

Abstract

Interaction between pUL34 and pUL31 is essential for targeting both proteins to the inner nuclear membrane (INM). Sequences mediating the targeting interaction have been mapped by others with both proteins. We have previously reported identification of charge cluster mutants of herpes simplex virus type 1 UL34 that localize properly to the inner nuclear membrane, indicating interaction with UL31, but fail to complement a UL34 deletion. We have characterized one mutation (CL04) that alters a charge cluster near the N terminus of pUL34 and observed the following. (i) The CL04 mutant has a dominant-negative effect on pUL34 function, indicating disruption of some critical interaction. (ii) In infections with CL04 pUL34, capsids accumulate in close association with the INM, but no perinuclear enveloped viruses, cytoplasmic capsids, or virions or cell surface virions were observed, suggesting that CL04 UL34 does not support INM curvature around the capsid. (iii) Passage of UL34-null virus on a stable cell line that expresses CL04 resulted in selection of extragenic suppressor mutants that grew efficiently using the mutant pUL34. (iv) All extragenic suppressors contained an R229-->L mutation in pUL31 that was sufficient to suppress the CL04 phenotype. (v) Immunolocalization and coimmunoprecipitation experiments with truncated forms of pUL34 and pUL31 confirm that N-terminal sequences of pUL34 and a C-terminal domain of pUL31 mediate interaction but not nuclear membrane targeting. pUL34 and pUL31 may make two essential interactions-one for the targeting of the complex to the nuclear envelope and another for nuclear membrane curvature around capsids.

FIG. 1.

Wild-type and mutant UL31 and UL34 pcDNA3 expression constructs. Schematic drawings of expression constructs are shown. All constructs were expressed from the human cytomegalovirus immediate-early promoter in pcDNA3 and were constructed as described in Materials and Methods. The plasmids encoding each gene are the following: line 1, ppRRR1238; line 2, pRR1328; line 3, pRR1330; line 4, pRR1331; line 5, pRR1344; line 6, pRR1345; line 7, pRR1346; line 8, pRR1347; line 9, pRR1334; line 10; pRR1335; line 11, pRR1327; line 12, pRR1328.

FIG. 2.

CL04 is a dominant-negative mutant of UL34. Graphs of complementation indices for combinations of cotransfected plasmids are shown. The complementation index for each condition was calculated by dividing the infectivity produced in each culture by the infectivity produced when the wt UL34-expressing plasmid is transfected in the absence of mutant UL34. The numbers below each bar indicate the number of ng × 100 of the corresponding plasmid transfected into the culture. In addition to the transfected plasmids indicated below each graph, all cultures were transfected with pCMVβ and assayed for β-galactosidase activity to control for differences in transfection efficiency. Transfection efficiencies were similar (within 30%) in all samples. One representative of three independent experiments is shown.

FIG. 3.

Expression of wild-type and CL04 mutant UL34 by stable cell lines. Digital images of Western blots are shown. Vero cells (lanes 1 and 2) or cells stably expressing wt UL34 (lane 3) or CL04 UL34 (lane 4) were infected with wt HSV-1(F) (lane 1) or UL34-null vRRR1072(TK+) virus (lanes 2 to 4). Blotted infected cell proteins were probed for either scaffolding protein (top) or UL34 (bottom).

FIG. 4.

Formation of plaques on wt UL34 or CL04 mutant UL34-expressing cell lines. Digital micrographs of infected cell monolayers stained with amido black are shown. The cell line infected is indicated above each column of panels, and the infecting virus is indicated to the left of each row. Black arrowheads indicate minute plaques comprised of a few cells. The white arrowhead (F) indicates a robust plaque formed by an extragenic suppressor virus.

FIG. 5.

Single-step growth of wild-type and mutant viruses on complementing and noncomplementing cells. Replicate cultures of Vero (A), RepAC (B), or CL04AI cells (C) were infected at an MOI of 5 with HSV-1(F) (open circles), vRR1072(TK+) (closed circles), or CL04Rev (open squares). Residual virus was removed or inactivated with a low-pH wash, and at the indicated times, total culture virus was titrated on RepAC cells. Virus yields are expressed as the number of PFU per milliliter. Each data point represents the mean of three independent experiments. Error bars indicate the range of values.

FIG. 6.

Marker transfer and complementation assays for UL31 R229L mutant function. Digital micrographs of CL04AI cell monolayers infected with the progeny of cotransfection of UL34-null vRR1072(TK+) viral genomes with either no plasmid (A), plasmid carrying wt UL31 (B), or plasmid carrying UL31R229L (C). (D) Graph of complementation indices for transfected plasmids is shown. Complementation index for each plasmid was calculated by dividing the infectivity produced in each culture by the infectivity produced when plasmid that expressed wt UL34 alone was transfected. Plasmids for each condition are the following: no UL34/no UL31, pRR1072; wt UL34/no UL31, pRR1072Rep; CL04/no UL31, pRR1162; wt UL34/wt UL31, pRR1348; wt UL34/UL31 R229L, pRR1349; CL04/wt UL31, pRR1350; CL04/UL31 R299L, pRR1351. Each bar represents the mean of five independent experiments. Error bars indicate the standard deviation.

FIG. 7.

Growth of BAC-derived UL34-null, UL31 R229L mutant virus on wt and mutant UL34-expressing cells. (A and B) Digital images show electrophoretically separated PCR products that are either digested with restriction enzyme (lanes 3 and 5) or undigested (lanes 2 and 4). The sizes of the undigested and digested products are indicated on the right of the gel. Lambda BstEII digest size standards are shown in lane 1, and the sizes of standard molecular weight bands are indicated on the left of the gel. (A) PCR products from the UL31 locus in virus rescued from wild-type HSV-1 BAC (lanes 2 and 3) or UL34-null/UL31 R229L mutant (A, lanes 4 and 5) are shown. (B) PCR products from the UL34 locus in the same viruses are shown. Digital micrographs of immunofluorescently stained plaques formed on Vero (C to E), wt UL34-expressing RepAC cells (F to H), or CL04-expressing CL04AI cells (I to K) by viruses rescued from wt HSV-1(F) BAC (C, F, and I), UL34-null/UL31 wt BAC (D, G, and J), or UL34-null/UL31 R229L mutant BAC (E, H, and K).

FIG. 8.

Localization of wt UL34 and CL04 mutant UL34 in transfected, infected cells. Digital confocal images of cells transfected with pRR1072Rep expressing wt (A and C) or pRR1162 expressing CL04 mutant UL34 (B and D), subsequently infected with either vRR1072(TK+) (A and B) or CL04RevB (C and D), and immunofluorescently stained for UL34 are shown.

FIG. 9.

TEM analysis of nuclear egress from cells that express CL04 UL34. Digital micrographs show Vero (A) or CL04AI (B) cells infected with the UL34-null virus vRR1072(TK+) for 20 h. Black arrowheads (A) point to examples of intranuclear capsids. Black arrowheads (B) point to examples of capsids docked at the INM. The white arrowhead (B) points to an instance of a capsid docked with slight curvature of the membrane. Scale bars are shown at the lower left of each panel.

FIG. 10.

Interaction between wild-type and mutant UL34 deletion constructs and the C-terminal domain of UL31. Digital confocal micrographs of transfected Vero cells are shown. Cells were transfected either with a single plasmid (A to F) or with combinations of two plasmids (G to O) The plasmids used are those depicted in Fig. 1 and are indicated within each panel. All UL31 constructs were FLAG tagged and were detected with anti-FLAG primary antibody and either green (E-F, G to I, and K to N) or red (J and O) secondary antibody. All UL34 constructs were detected with anti-UL34 primary antibody and red secondary antibody. EGFP emerin was detected by GFP fluorescence in green (J and O). For combination transfections, the color of the text in the panel corresponds to the color of the antibody used to detect that construct. Only merged images are shown.

FIG. 11.

Digital images of Western blots are shown. Unfractionated lysates (lanes 1 to 3) or proteins immunoprecipitated with anti-FLAG antibody (lanes 4 to 6) were separated by SDS-PAGE and probed with antibody directed against FLAG epitope to detect UL31 (top row) or with antibody directed against pUL34 (bottom row).