Herpes simplex virus type 1 U(L)34 gene product is required for viral envelopment

Abstract

The herpes simplex virus type 1 U(L)34 gene encodes a protein that is conserved in all human herpesviruses. The association of the U(L)34 protein with membranes in the infected cell and its expression as a gamma-1 gene suggest a role in maturation or egress of the virus particle from the cell. To determine the function of this gene product, we have constructed a recombinant virus that fails to express the U(L)34 protein. This recombinant virus, in which the U(L)34 protein coding sequence has been replaced by green fluorescent protein, forms minute plaques and replicates in single-step growth experiments to titers 3 to 5 log orders of magnitude lower than wild-type or repair viruses. On Vero cells, the deletion virus synthesizes proteins of all kinetic classes in normal amounts. Electron microscopic and biochemical analyses show that morphogenesis of the deletion virus proceeds normally to the point of formation of DNA-containing nuclear capsids, but electron micrographs show no enveloped virus particles in the cytoplasm or at the surface of infected cells, suggesting that the U(L)34 protein is essential for efficient envelopment of capsids.

FIG. 1

Sequence arrangement of plasmids and viruses used in this study. (A) Schematic diagram of the HSV-1 genome in prototype arrangement, showing the unique sequences (lines) flanked by inverted repeats (boxes). (B) Expansion of the region of the HSV genome cloned in pRR1060, showing the positions of viral open reading frames (arrows) and selected restriction enzyme cleavage sites. (C) Schematic diagram of the plasmid pRR1099, produced by ligating the NheI-EcoNI fragment of pRR1060 between the NruI and XbaI sites of pcDNA3. (D) Schematic diagrams of the genomes of HSV-1 recombinants used in this study. The names and properties of these viruses as they relate to TK and U_L34 expression are indicated beneath each diagram. Restriction enzyme abbreviations: B, BglII; E, EcoNI; Nh, NheI; S, SpeI.

FIG. 2

Structures of recombinant virus genomes and U_L34 expression in recombinant viruses. (A and B) Sequence arrangement of viral genomes of Δ305 (A) and vRR1072 (B) around the U_L34 locus, showing the positions of viral open reading frames (filled arrows), GFP insertion (shaded arrow), and probes used for Southern analysis of recombinant virus genome structure. Restriction enzyme abbreviations: B, BamHI; Ec, EcoRI; H, HpaI; Nc, NcoI; No, NotI; X, XbaI. (C) Autoradiographic image of a Southern blot of NotI-digested viral DNAs from cells infected with Δ305 (lane 1), the U_L34 deletion virus vRR1072 (lane 2), or homologous repair virus vRR1072Rep probed with Southern probe I. (D) Same blot as in panel C but stripped and reprobed with Southern probe II. The sizes of migration standards (in kilobase pairs) are indicated between the panels. All lanes were from the same blot, but lane 1 was not adjacent to lanes 2 and 3 in the original autoradiogram. (E) Photographic image of a Western blot of SDS-PAGE-separated proteins from cells either mock infected (lane 1) or infected with Δ305 (lane 2), vRR1072 (lane 3), or vRR1072Rep (lane 4). The migration positions of size standards are indicated at the left. The position of the U_L34 signal is indicated by the arrowhead.

FIG. 3

Expression of U_L33, U_L34, and U_L35 mRNAs. Shown are autoradiographic images of Northern blots of formaldehyde agarose gel-separated RNAs purified from infected Vero cells and probed with RNA probe antisense to the U_L34 gene. (A) RNAs purified at various times after infection in the presence or absence of PAA. (B) RNAs purified at various times after infection with Δ305 (lanes 2, 5, 8, 11, and 14), the U_L34 deletion virus vRR1072 (lanes 3, 6, 9, 12, and 15), or the homologous repair virus vRR1072Rep (lanes 4, 7, 10, 13, and 16), separated on a 1.3% agarose gel and probed with labeled RNA antisense to sequences between the BspEI and XbaI sites that flank the U_L34 gene. Arrowheads indicate the migration positions of wild-type U_L33 and U_L34 mRNAs. These mRNAs migrate more slowly in the deletion virus due to the insertion of GFP sequences. (C) Same as panel B but run on a 1.8% agarose gel and probed with labeled RNA antisense to sequences between the BstBI and BspEI restriction sites that flank the U_L35 gene. The region of the gel containing U_L35 mRNA is shown, and an arrowhead indicates the migration position of U_L35 mRNA.

FIG. 4

Plaque formation by the U_L34 deletion virus on complementing and noncomplementing cells. Photographic images show 143/1099E cells (A) and 143B cells (B) infected with vRR1072, viewed with UV illumination to excite GFP fluorescence. Plaques in panel B are indicated with arrowheads.

FIG. 5

Single-step growth of U_L34 deletion, wild-type, and repair viruses on different cell lines. Shown are plots of the logarithm of PFU of virus accumulated in total culture or in medium against time after infection. Cultures were infected, harvested, and titered on 143/1099E cells as described in Materials and Methods. (A) Growth and release of vRR1072 on noncomplementing 143B cells and complementing 143/1099E cells. Each point represents the mean of three independent experiments; error bars indicate the sample standard deviation. (B to D) Growth of Δ305, vRR1072, and vRR1072Rep on HEp-2 (B), HEL299 (C), and Vero (D) cells. Results of representative experiments are shown.

FIG. 6

Protein synthesis in Vero cells infected with wild-type, U_L34-negative, and repair viruses. Shown is an autoradiographic image of SDS-PAGE-separated proteins from Vero cells either mock infected (lane 1) or infected with the indicated virus for 4 h (lanes 2 to 5), 8 h (lanes 6 to 8), 12 h (lanes 9 to 11), 18 h (lanes 12 to 14), or 24 h (lanes 14 to 16) and labeled with [³⁵S]methionine for 2 h prior to harvest. The migration positions of size standards (in kilodaltons) are indicated at the left. The migration positions of U_L34 and GFP are indicated by arrowheads at the right. The overall lower level of label seen in lane 6 is due to a corresponding overall lower level of protein loaded in that lane. p.i., postinfection.

FIG. 7

Accumulation of late gene products in wild-type, U_L34-negative, and repair viruses. Photographic images show Western blots of proteins from Vero cells either mock infected (lane 1) or infected with the indicated virus for 16 h. (A) Blot probed with anti-gD monoclonal antibody; (B) blot probed with anti-gE monoclonal antibody; (C) blot probed with anti-U_S11 monoclonal antibody.

FIG. 8

Nuclear capsids and encapsidated DNA in wild-type and U_L34-negative virus. Photographic images show electrophoretically separated and ethidium bromide-stained DNA (A and C) and Western-blotted proteins (B and D) from sucrose gradient fractionation of DNase I-treated nuclear lysates from cells infected with either vRR1072 (A and B) or Δ305 (C and D).

FIG. 9

Transmission EM analysis of cells infected with wild-type and U_L34-negative virus. Micrographs show Vero cells infected with either HSV-1(F) (panels A and B) or vRR1072(TK⁺) (panels C to F) for 20 h. (A) Wild-type-infected Vero cells showing envelopment of capsid at the inner nuclear membrane, enveloped virus particles in vesicles in the cytoplasm, and enveloped virus at the surface of, and between cells. Examples of each are indicated by arrowheads. Final magnification is ×13,500. (B) Wild-type-infected Vero cell showing capsids in the nucleus and enveloped virus particles between the inner and outer nuclear membranes (one example indicated by arrow). Final magnification is ×40,500. (C) Deletion mutant-infected Vero cell showing capsids in the nucleus (a few examples indicated with arrowheads) but no cytoplasmic or cell-surface-associated enveloped virus particles. Final magnification is ×13,500. (D) Cell fragment from deletion mutant-infected Vero cells containing numerous viral capsids. Final magnification is ×40,500.

FIG. 9

Transmission EM analysis of cells infected with wild-type and U_L34-negative virus. Micrographs show Vero cells infected with either HSV-1(F) (panels A and B) or vRR1072(TK⁺) (panels C to F) for 20 h. (A) Wild-type-infected Vero cells showing envelopment of capsid at the inner nuclear membrane, enveloped virus particles in vesicles in the cytoplasm, and enveloped virus at the surface of, and between cells. Examples of each are indicated by arrowheads. Final magnification is ×13,500. (B) Wild-type-infected Vero cell showing capsids in the nucleus and enveloped virus particles between the inner and outer nuclear membranes (one example indicated by arrow). Final magnification is ×40,500. (C) Deletion mutant-infected Vero cell showing capsids in the nucleus (a few examples indicated with arrowheads) but no cytoplasmic or cell-surface-associated enveloped virus particles. Final magnification is ×13,500. (D) Cell fragment from deletion mutant-infected Vero cells containing numerous viral capsids. Final magnification is ×40,500.

FIG. 9

Transmission EM analysis of cells infected with wild-type and U_L34-negative virus. Micrographs show Vero cells infected with either HSV-1(F) (panels A and B) or vRR1072(TK⁺) (panels C to F) for 20 h. (A) Wild-type-infected Vero cells showing envelopment of capsid at the inner nuclear membrane, enveloped virus particles in vesicles in the cytoplasm, and enveloped virus at the surface of, and between cells. Examples of each are indicated by arrowheads. Final magnification is ×13,500. (B) Wild-type-infected Vero cell showing capsids in the nucleus and enveloped virus particles between the inner and outer nuclear membranes (one example indicated by arrow). Final magnification is ×40,500. (C) Deletion mutant-infected Vero cell showing capsids in the nucleus (a few examples indicated with arrowheads) but no cytoplasmic or cell-surface-associated enveloped virus particles. Final magnification is ×13,500. (D) Cell fragment from deletion mutant-infected Vero cells containing numerous viral capsids. Final magnification is ×40,500.

FIG. 9

Transmission EM analysis of cells infected with wild-type and U_L34-negative virus. Micrographs show Vero cells infected with either HSV-1(F) (panels A and B) or vRR1072(TK⁺) (panels C to F) for 20 h. (A) Wild-type-infected Vero cells showing envelopment of capsid at the inner nuclear membrane, enveloped virus particles in vesicles in the cytoplasm, and enveloped virus at the surface of, and between cells. Examples of each are indicated by arrowheads. Final magnification is ×13,500. (B) Wild-type-infected Vero cell showing capsids in the nucleus and enveloped virus particles between the inner and outer nuclear membranes (one example indicated by arrow). Final magnification is ×40,500. (C) Deletion mutant-infected Vero cell showing capsids in the nucleus (a few examples indicated with arrowheads) but no cytoplasmic or cell-surface-associated enveloped virus particles. Final magnification is ×13,500. (D) Cell fragment from deletion mutant-infected Vero cells containing numerous viral capsids. Final magnification is ×40,500.