Herpes simplex virus type 1 strain HSV1716 grown in baby hamster kidney cells has altered tropism for nonpermissive Chinese hamster ovary cells compared to HSV1716 grown in vero cells

Abstract

Chinese hamster ovary (CHO) cells are traditionally regarded as nonpermissive cells for herpes simplex virus type 1 (HSV-1) infection as they lack the specific entry receptors, and modified CHO cells have been instrumental in the identification of HSV-1 receptors in numerous studies. In this report we demonstrate that the HSV-1 strain 17+ variant HSV1716 is able to infect unmodified CHO cells but only if the virus is propagated in baby hamster kidney (BHK) cells. Infection of CHO cells by BHK-propagated HSV1716 results in expression of immediate-early, early, and late viral genes, and infectious progeny virions are produced. In normally cultured CHO cells, up to a maximum of 50% of cells were permissive for BHK-propagated HSV1716 infection, with 24 h of serum starvation increasing this to 100% of CHO cells, suggesting that the mechanism used by BHK-propagated virus to infect CHO cells was cell cycle dependent. The altered tropism of HSV1716 was also evident in another nonpermissive mouse melanoma cell line and is an exclusive property resulting from propagation of the virus using BHK cells, as viruses propagated on Vero, C8161 (a human melanoma cell line), or indeed, CHO cells were completely unable to infect either CHO or mouse melanoma cells.

FIG. 1.

Unprocessed images (magnification, ×184) from fluorescent microscopy of various cell lines after 8 h of infection with HSV1716gfp propagated either in BHK (a to h, o to r) or Vero (i to n) cells. Permissive BHK (a, i), C8161 (b, j), HeLa (c, k), or SK-N-SH (d, l) cells were infected with 5 × 10⁶ PFU HSV1716gfp. Nonpermissive CHO cells were infected with 1 × 10⁶ PFU, 5 × 10⁶ PFU, or 1 × 10⁷ PFU HSV1716gfp/BHK (e, f, g, respectively) or with 1 × 10⁷ PFU HSV1716gfp/Vero (m). Nonpermissive mouse melanoma cells were infected with 1 × 10⁷ PFU HSV1716gfp/BHK (h) or HSV1716gfp/Vero (n). Infection of BHK (o, p) or CHO (q, r) cells was completely neutralized using a sheep anti-HSV-1 antiserum (o and q, respectively) but was unaffected by normal sheep serum (p and r, respectively).

FIG. 2.

Unprocessed images (magnification, ×184) from fluorescent microscopy of CHO cells after 8 h of infection with either 5 PFU/cell HSV1716gfp (a to h) or HSV17+gfp (i, j) propagated alternatively in either BHK (b, d, e, g, i) or Vero (a, c, f, h, j) cells.

FIG. 3.

(a) Western blots developed with anti HSV-1 R1 antiserum 106. In duplicate, whole-cell extracts were prepared from Vero (lanes 1, 2, 7, 8), CHO (lanes 3, 4, 9, 10) or mouse melanoma (lanes 5, 6, 11, 12) cells infected with 5 PFU/cell HSV1716gfp/Vero (lanes 1 to 6) or HSV1716gfp/BHK (lanes 7 to 12). For panels b to f, Western blots developed with monoclonal antibodies against ICP4 (b), ICP0 (c), VP16 (d), gB (e), and gC (f) are shown. Whole-cell extracts were prepared from CHO (lanes 1 and 2) or BHK (lane 3) cells infected with 10 or 5 PFU/cell HSV1716gfp/Vero (lane 1 and 3, respectively) or 10 PFU/cell HSV1716gfp/BHK (lanes 2).

FIG. 4.

(a) Electron micrograph of CHO cells 24 h after infection with 5 PFU/cell HSV1716gfp/BHK. Examples of capsids in the nucleus (Nuc) are indicated by a black arrow, enveloped virions in the cytoplasm (Cyt) are indicated by a white arrow, and unenveloped capsids in the cytoplasm are indicated by a white arrowhead. Bar, 1 μm. For panels b to f, unprocessed images (magnification, ×200) from fluorescent microscopy are shown. (b) BHK cells after 8 h of infection with 5 PFU/cell HSV1716gfp propagated in CHO cells; (c) BHK cells after 8 h of infection with 5 PFU/cell HSV1716gfp propagated in Vero cells; (d) C8161 cells after 8 h of infection with 5 PFU/cell HSV1716gfp propagated in CHO cells; (e) BHK cells after 8 h of infection with 5 PFU/cell HSV1716gfp propagated in C8161 cells; (f) CHO cells after 8 h of infection with 5 PFU/cell HSV1716gfp propagated in CHO cells. For panels g, h, and i, unprocessed images (magnification, ×100) from fluorescent microscopy are shown for CHO (g), BHK (h), and Vero (i) cell lines 5 days after infection with 1 × 10³ PFU HSV1716gfp/BHK (g, h) or HSV1716gfp/CHO (i).

FIG. 4.

(a) Electron micrograph of CHO cells 24 h after infection with 5 PFU/cell HSV1716gfp/BHK. Examples of capsids in the nucleus (Nuc) are indicated by a black arrow, enveloped virions in the cytoplasm (Cyt) are indicated by a white arrow, and unenveloped capsids in the cytoplasm are indicated by a white arrowhead. Bar, 1 μm. For panels b to f, unprocessed images (magnification, ×200) from fluorescent microscopy are shown. (b) BHK cells after 8 h of infection with 5 PFU/cell HSV1716gfp propagated in CHO cells; (c) BHK cells after 8 h of infection with 5 PFU/cell HSV1716gfp propagated in Vero cells; (d) C8161 cells after 8 h of infection with 5 PFU/cell HSV1716gfp propagated in CHO cells; (e) BHK cells after 8 h of infection with 5 PFU/cell HSV1716gfp propagated in C8161 cells; (f) CHO cells after 8 h of infection with 5 PFU/cell HSV1716gfp propagated in CHO cells. For panels g, h, and i, unprocessed images (magnification, ×100) from fluorescent microscopy are shown for CHO (g), BHK (h), and Vero (i) cell lines 5 days after infection with 1 × 10³ PFU HSV1716gfp/BHK (g, h) or HSV1716gfp/CHO (i).

FIG. 5.

Unprocessed images (magnification, ×196) from fluorescent microscopy of various cell lines after 8 h of infection with HSV1716gfp or HSV17+gfp propagated either in BHK or Vero cells. For panels a to d, serum-starved or non-serum-starved BHK (a, b) or C8161 (c, d) cells were infected with 5 PFU/cell HSV1716gfp/BHK (a, b) or 5 PFU/cell HSV1716gfp/Vero (c, d) added either to non-serum-starved cells (b, d) or to cells simultaneously with the reintroduction of serum (a, c). For panels e to k, serum-starved or non-serum-starved CHO cells were infected with 5 PFU/cell HSV1716gfp/BHK added either to non-serum-starved cells (e) or to serum-starved cells simultaneously with the reintroduction of serum (f, j, k) or 4 (g), 8 (h), or 16 (i) hours after serum addition. For panels l and m, 5 PFU/cell HSV17+gfp/BHK was added to serum-starved CHO cells either simultaneously with serum addition (l) or 16 h after the reintroduction of serum (m). For panels n and o, 5 PFU/cell HSV1716gfp/Vero was added to either non-serum-starved CHO cells (n) or to serum-starved CHO cells simultaneously with the reintroduction of serum (o).