Mouse cytomegalovirus crosses the species barrier with help from a few human cytomegalovirus proteins

Abstract

Strong species specificity and similar tropisms suggest mouse cytomegalovirus (mCMV) as a potential vector for transgenes into human cells. We reexamined the dogma that mouse cytomegalovirus cannot productively replicate in human cells and found that mouse cytomegalovirus can produce infectious particles albeit at a level that does not sustain an infection. This finding demonstrates that mouse cytomegalovirus can undergo all processes of its life cycle in human cells but may not be well adapted to circumvent the human cell's intrinsic defenses. The suppression of mCMV production in human cells is affected at several levels, which additively or synergistically result in the appearance of species specificity. Hydrolysis of most newly replicated viral DNA and very low capsid protein transcription reduced the potential particle production to insignificant levels. These effects can be ameliorated by adding human cytomegalovirus tegument proteins and immediate-early protein 1. They function synergistically to produce significant amounts of mCMV in human cells. While the possibility that mouse cytomegalovirus might replicate in human cells raises caution in the use of this virus as a transgene vector, manipulation of the mouse cytomegalovirus genome to allow limited spread to other human cells might also provide an advantage for the distribution of certain transgenic products.

FIG. 1.

Expression of immediate-early and early proteins at different time (hours) of mCMV infection (MOI of 1). The membrane was stripped each time before probing with a specific antibody occurred. Tubulin was used as a loading control. m, mock infected.

FIG. 2.

mCMV DNA replication and IE1 distribution in human fibroblasts. (A) In situ hybridization of mCMV-infected 3T3 cells 24 h p.i., counterstained with antibodies to M112/113 gene products (red). Green dots represent replicated mCMV DNA. (B) Human fibroblasts probed as described for panel A. (C) Human fibroblasts infected with mCMV for 3 h and probed with antibodies against IE1 and PML. (D) Same as panel C, except at 24 h p.i., IE1 (arrows) is segregated beside PML-labeled aggregates. (E) Human fibroblasts infected with mCMV for 24 h and probed with antibodies to M112/113 and HDAC2. Note the accumulation of HDAC2 in the M112/113 domains. (F) Same as panel E but showing only the HDAC2 layer. (G) Human fibroblasts infected for 24 h with mCMV and probed with antibodies to IE1 and HDAC2. (H) Same as panel G but showing only the HDAC2 staining.

FIG. 3.

(A) Southern blot of mCMV-infected mouse and human cells. 3T3 and human fibroblasts were infected with 1 PFU per cell, and cells were harvested at different times after infection. Coexisting cellular DNA was used as a loading control. (B) Pulsed-field gel electrophoresis-separated DNA from infected and uninfected cells exposed or unexposed to UV. Smears indicate DNA degradation.

FIG. 4.

Plaque formation of GFP-producing mCMV infection in IE1-expressing human cells. Confluent cell lines (indicated at lower left) were infected with equal amounts of mCMV expressing GFP (RVG102), with titers that induced ∼1% GFP-producing 3T3 cells. Much higher numbers of U373 astrocytoma cells produce GFP (C), and more than 50% of the hCMV IE1-expressing U373 cells were labeled with GFP (D). (E to H) Size increase of a plaque over time in U373-IE1 cells; the different days (e.g., D5, day 5) are indicated at the upper right.

FIG. 5.

(A) Expression of immediate-early and early proteins in human U373 and U373-IE1 cells at different times (hours) post-mCMV infection. The membrane was stripped each time before probing with a different specific antibody occurred. Tubulin was used as a loading control. The gel was loaded with less cell lysate for the U373-IE1 sample. (B) mCMV DNA production in mCMV-infected human U373 and U373-IE1 cells, as determined by Southern blotting. Cells were harvested at different times (hours) after infection. m, mock infected.

FIG. 6.

Immunolocalization of pp71 (A) and pp65 (B) introduced by UV-treated hCMV 3 h p.i., relative to PML. pp71 localization in uninfected (uninf.) HF pp71 (C) relative to PML and (D) relative to Daxx.

FIG. 7.

RT-PCR analysis of late transcripts in mCMV-infected normal and hCMV IE1-producing human cells. The early transcript of M112/113 was used as a positive control, as it is transcribed in all mCMV-infected human cells. M, marker.