Smith Strain Research Articles

HPMPC therapy of MCMV-induced retinal disease in the SCID mouse measured by electroretinography, a non-invasive technique

The purpose of these studies was to investigate the use of non-invasive electroretinography for the evaluation of retinal disease and its treatment in an ocular murine cytomegalovirus (MCMV) disease model. While under anesthesia, 10 2.6 plaque forming units (pfu) of salivary gland passaged, Smith strain MCMV was injected in the anterior chamber of 6- to 8-week-old severe combined immunodeficiency (SCID) mice. At various times post-inoculation, bright-flash scotopic electroretinogram, viral titer, and histology were obtained from the injected eye. Antiviral therapy was tested using 0.1 and 5 mg/kg/day subcutaneous injections of HPMPC (Cidofovir) once daily for 5 consecutive days. In infected animals, the a- and b-waves of the electroretinographic (ERG) signal were significantly reduced as of 10 days post-inoculation when compared to control animals. Therapy with HPMPC 0.1 mg/kg/day subcutaneously (s.c.) once daily for 5 consecutive days was able to delay the decrease in ERG wave amplitude and inhibit viral replication, whereas 5 mg/kg/day s.c. significantly protected the ERG, completely inhibited viral replication, and maintained ocular viral titer below the limit of detection for up to 17 days post-infection. The reduction of ERG activity during progression of retinal disease correlated well with reduction of disease pathology. ERG recording represents a valuable non-invasive technique to measure the progression of the retinal disease induced by MCMV and the efficacy of antiviral treatment in the ocular MCMV disease model.

NKG2D-mediated natural killer cell protection against cytomegalovirus is impaired by viral gp40 modulation of retinoic acid early inducible 1 gene molecules.

Natural killer (NK) cells play a critical role in the innate immune response against cytomegalovirus (CMV) infections. Although CMV encodes several gene products committed to evasion of adaptive immunity, viral modulation of NK cell activity is only beginning to be appreciated. A previous study demonstrated that the mouse CMV m152-encoded gp40 glycoprotein diminished expression of ligands for the activating NK cell receptor NKG2D on the surface of virus-infected cells. Here we have defined the precise ligands that are affected and have directly implicated NKG2D in immune responses to CMV infection in vitro and in vivo. Murine CMV (MCMV) infection potently induced transcription of all five known retinoic acid early inducible 1 (RAE-1) genes (RAE-1α, RAE-1β, RAE-1δ, RAE-1ɛ, and RAE-1γ), but not H-60. gp40 specifically down-regulated the cell surface expression of all RAE-1 proteins, but not H-60, and diminished NK cell interferon γ production against CMV-infected cells. Consistent with previous findings, a m152 deletion mutant virus (Δm152) was less virulent in vivo than the wild-type Smith strain of MCMV. Treatment of BALB/c mice with a neutralizing anti-NKG2D antibody before infection increased titers of Δm152 virus in the spleen and liver to levels seen with wild-type virus. These experiments demonstrate that gp40 impairs NK cell recognition of virus-infected cells through disrupting the RAE-1–NKG2D interaction.

In vitro and in vivo characterization of a murine cytomegalovirus with a mutation at open reading frame m166.

We have recently generated a pool of murine cytomegalovirus (MCMV) mutants by using a Tn3-based transposon mutagenesis approach. In this study, one of the mutants, Rvm166, which contained the transposon sequence at open reading frame m166, was characterized both in tissue culture and in immunocompetent BALB/c mice and immunodeficient SCID mice. The viral mutant replicated as well as the wild-type Smith strain in vitro in NIH 3T3 cells, whereas the transposon insertion precluded the expression of >65% of the m166 open reading frame. Compared to the wild-type strain and a rescued virus that restored the m166 region, the viral mutant was significantly attenuated in growth in both BALB/c and SCID mice that were intraperitoneally infected with the viruses. At 21 days postinfection, the titers of the viral mutant in the salivary glands, lungs, spleens, livers, and kidneys of the infected SCID mice were lower than the titers of the Smith strain and the rescued virus by about 30000-, 10000-, 1000-, 300-, and 800-fold, respectively. Moreover, the virulence of the mutant virus appears to be severely attenuated because no death was found in SCID mice infected with the viral mutant up to 90 days postinfection, whereas all of the animals infected with the wild-type and rescued viruses died at 27 days postinfection. Our results suggest that m166 probably encodes a virulence factor and is required for MCMV virulence in killing SCID mice and for optimal viral growth in vivo.

Isolation and analysis of an aciclovir-resistant murine cytomegalovirus mutant

An aciclovir (ACV)-resistant murine cytomegalovirus (MCMV) was isolated from the Smith strain and the mutant was analysed. Attempts were also made to identify directly the mutated gene. The 50% inhibitory concentration (IC 50) of ACV for the mutant strain was ∼30 times higher than that for the wild-type strain. The mutant strain was equally sensitive to ganciclovir (GCV), but slightly resistant to cidofovir (CDV) and foscarnet (PFA) when compared with the wild-type. Molecular analysis of the mutant strain revealed that a single base mutation of cytosine (C) to guanine (G) occurred at the 2476th nucleotide position in the DNA polymerase gene region, resulting in an amino acid substitution of proline (Pro) with alanine (Ala) at codon 826. The marker transfer experiment confirmed that this mutation conferred ACV resistance to MCMV. This mutation at codon 826 was easily identified by means of Hae III digestion of the selected PCR product and electrophoresis.

Murine cytomegalovirus containing a mutation at open reading frame M37 is severely attenuated in growth and virulence in vivo.

A pool of murine cytomegalovirus (MCMV) mutants was generated by using a Tn3-based transposon mutagenesis procedure. One of the mutants, RvM37, which contained the transposon sequence at open reading frame M37, was characterized both in tissue culture and in immunocompetent BALB/c and immunodeficient SCID mice. Our results provide the first direct evidence to suggest that M37 is not essential for viral replication in vitro in NIH 3T3 cells. Compared to the wild-type strain and a rescued virus that restored the M37 region, the viral mutant was severely attenuated in growth in both BALB/c and SCID mice after intraperitoneal infection. Specifically, titers of the Smith strain and rescued virus in the salivary glands, lungs, spleens, livers, and kidneys of the SCID mice at 21 days postinfection were about 5 x 10(5), 2 x 10(5), 5 x 10(4), 5 x 10(3), and 1 x 10(4) PFU/ml of organ homogenate, respectively; in contrast, titers of RvM37 in these organs were less than 10(2) PFU/ml of organ homogenate. Moreover, the virulence of the mutant virus appeared to be significantly attenuated because none of the SCID mice infected with RvM37 had died by 120 days postinfection, while all animals infected with the wild-type and rescued viruses had died by 26 days postinfection. Our results suggest that M37 probably encodes a virulence factor and is required for MCMV virulence in SCID mice and for optimal viral growth in vivo.

Isolation of Rickettsia in vero cell culture

The diagnosis of spotted fever is based on characteristic signs and symptoms but requires laboratorial confirmation because of the possible differential diagnosis from other diseases like leptospirosis, enterovirus, meningococcemia and typhoid fever. Laboratorial confirmation may be done by detection of specific antibodies which is possible only 5-10 days after the onset of the symptoms or by the isolation of Rickettsiae from blood and/or skin biopsy and from ticks collected in the patient or in the animal reservoir. The isolation of Rickettsiae from blood or skin biopsy results in an early diagnosis of spotted fever since in the rickettsiemic phase of the disease there is no detectable level of antibodies in the serum. With the purpose of facilitating the diagnosis of Spotted Fever we have standardized the isolation of Rickettsiae in cell culture by a method that is less time consuming and that reduces the biological risks than isolation in guinea pigs. Vero cell cultures were inoculated with the Sheyla Smith strain of Rickettsia rickettsii provided by CDC (Atlanta-USA). The identification was performed by indirect immunofluorescence technique. The presence of green fluorescent organisms characterized the growth of the agent. Ulterior confirmation of the methodology was done by isolation of the spotted fever agent from skin biopsy of a patient from an endemic area and from Amblyomma ticks that are the reservoir and vector of the Brazilian spotted fever.

A model system of primary murine hepatocytes infected by murine cytomegalovirus.

In order to establish a model system of the murine hepatocyte infection by murine cytomegalovirus (MCMV), the primary cultured murine hepatocytes were obtained in a modified low-serum medium system by a non-perfusion method, and then infected by Smith strain MCMV. Infected hepatocytes showed characteristic cytopathic effect (CPE) at 30 h after infection, in which a large number of viral particles was found and ultrastructures were destroyed (as revealed by disappearance of bile canalicula and organelles) under the electron microscope and MCMV immediate-early genes were detected by in situ hybridization. Meanwhile, infected cells produced albumin significantly less than corresponding uninfected controls. On the contrary, uninfected controls simultaneously cultured under the same conditions showed normal function and ultrastructure (glycogen rosettes, bile canalicula, wheel-like mitochondria and well-developed rough and smooth endoplasmic reticula). These results demonstrated that a model system of primary cultured murine hepatocytes infected by MCMV was successfully set up.

Murine cytomegalovirus induces a Sjögren's syndrome-like disease in C57Bl/6-lpr/lpr mice.

To analyze Fas and tumor necrosis factor receptor I (TNFRI) apoptosis pathways in salivary gland inflammatory disease induced by murine cytomegalovirus (MCMV) infection. Four different strains of mice (C57BI/6 [B6]-+/+, Fas-deficient B6-lpr/lpr, TNFRI-deficient B6-tnfr1(0/0), and B6-tnfr1(0/0)-lpr/lpr mice) were infected intraperitoneally with the Smith strain of MCMV (1 x 10(5) plaque-forming units). Viral load was determined by a plaque assay, inflammation and apoptosis by immunohistochemistry and staining with terminal dUTP nickend labeling, and autoantibodies by enzyme-linked immunosorbent assay. Infectious MCMV was not detectable by day 100. Although all MCMV-infected mice developed acute sialadenitis by day 28, a chronic (>100 days), severe salivary gland inflammation and anti-Ro and anti-La antibodies developed only in the B6-lpr/lpr mice. Apoptotic cells were detected during the acute, but not the chronic, phase of inflammation. Both Fas- and TNFRI-mediated apoptosis contribute to the clearance of MCMV-infected cells in the salivary glands. However, because Fas-mediated apoptosis is necessary for the down-modulation of the immune response, a defect in this process can lead to a postinfection, chronic inflammatory response that resembles Sjögren's syndrome.

Murine cytomegalovirus induces a Sjögren's syndrome‐like disease in C57Bl/6‐lprlpr mice

Objective. To analyze Fas and tumor necrosis factor receptor I (TNFRI) apoptosis pathways in salivary gland inflammatory disease induced by murine cytomegalovirus (MCMV) infection. Methods. Four different strains of mice (C57Bl/6 [B6]-+/+, Fas-deficient B6-lpr/lpr, TNFRI-deficient B6-tnfr1 0/0 , and B6-tnfr1 0/0 -lpr/lpr mice) were infected intraperitoneally with the Smith strain of MCMV (1 x 10 5 plaque-forming units). Viral load was determined by a plaque assay, inflammation and apoptosis by immunohistochemistry and staining with terminal dUTP nick-end labeling, and autoantibodies by enzyme-linked immunosorbent assay. Results. Infectious MCMV was not detectable by day 100. Although all MCMV-infected mice developed acute sialadenitis by day 28, a chronic (>100 days), severe salivary gland inflammation and anti-Ro and anti-La antibodies developed only in the B6-lpr/lpr mice. Apoptotic cells were detected during the acute, but not the chronic, phase of inflammation. Conclusion. Both Fas- and TNFRI-mediated apoptosis contribute to the clearance of MCMV-infected cells in the salivary glands. However, because Fas-mediated apoptosis is necessary for the down-modulation of the immune response, a defect in this process can lead to a postinfection, chronic inflammatory response that resembles Sjogren's syndrome.

NK Cell Modulation of Murine Cytomegalovirus Retinitis

CMV retinitis, the most common ophthalmic infection of AIDS patients, causes blindness if left untreated. To study the role of NK cells in the modulation of CMV ocular infection, 9.0 x 10(2) plaque-forming units of the Smith strain of murine CMV (MCMV) was injected into the supraciliary space of the left eyes of BALB/c mice. Lysis of NK-sensitive target cells (YAC-1) by effectors from the draining lymph nodes peaked at day 5 postinfection, while the splenic cytolytic response was biphasic, with peaks at days 2 and 7 postinfection. Flow cytometry showed that NK cells (DX-5+) increased in spleens and eyes 5 days after supraciliary infection with MCMV compared with uninfected or mock-infected controls. Eight days after supraciliary injection with 9.0 x 10(2) plaque-forming units of MCMV, 7 of 10 NK-depleted mice developed retinitis compared with only 2 of 10 non-NK-depleted control mice. Poly(I-C) activation of NK cells in T cell-depleted animals protected mice from MCMV retinitis; only 2 of 10 mice in the poly(I-C)-treated group developed retinitis compared with 8 of 10 T cell-depleted, non-poly(I-C)-treated control mice. These results show the importance of NK cells in preventing MCMV retinitis and suggest that NK cells may also be involved in modulation of cytomegalovirus retinitis in human patients.

Effects of moxibustion on the enhancement of serum antibody in rabbit against Staphylococcus aureus.

The titer and activity of antibody in rabbits immunized with heat-killed vaccine were assessed with and without moxibustion treatment. Enzyme-linked immunosorbent assay (ELISA) was applied for the detection of immunoglobulins to Smith strain of Staphylococcus aureus. Positive IgM titer of more than 0.4 were observed with this assay against the moxibustion group (P < 0.05). The titer of IgG antibody also increased; however, there was no significant difference between the moxibustion group and the control group. The ELISA inhibition test showed significantly higher protective activity of the sera in the moxibustion group at the 9th week after the first immunization (P < 0.05).

Analysis of the complete DNA sequence of murine cytomegalovirus.

The complete DNA sequence of the Smith strain of murine cytomegalovirus (MCMV) was determined from virion DNA by using a whole-genome shotgun approach. The genome has an overall G+C content of 58.7%, consists of 230,278 bp, and is arranged as a single unique sequence with short (31-bp) terminal direct repeats and several short internal repeats. Significant similarity to the genome of the sequenced human cytomegalovirus (HCMV) strain AD169 is evident, particularly for 78 open reading frames encoded by the central part of the genome. There is a very similar distribution of G+C content across the two genomes. Sequences toward the ends of the MCMV genome encode tandem arrays of homologous glycoproteins (gps) arranged as two gene families. The left end encodes 15 gps that represent one family, and the right end encodes a different family of 11 gps. A homolog (m144) of cellular major histocompatibility complex (MHC) class I genes is located at the end of the genome opposite the HCMV MHC class I homolog (UL18). G protein-coupled receptor (GCR) homologs (M33 and M78) occur in positions congruent with two (UL33 and UL78) of the four putative HCMV GCR homologs. Counterparts of all of the known enzyme homologs in HCMV are present in the MCMV genome, including the phosphotransferase gene (M97), whose product phosphorylates ganciclovir in HCMV-infected cells, and the assembly protein (M80).

Effect of natural sequence variation at the H-2Ld-restricted CD8+ T cell epitope of the murine cytomegalovirus ie1-encoded pp89 on T cell recognition.

The amino acid sequence YPHFMPTNL of pp89, the ie1-encoded product of murine cytomegalovirus (MCMV; Smith strain), constitutes an immunodominant T cell epitope recognized in association with H-2Ld. Nucleotide sequencing of MCMV isolates derived from wild mice identified variation between amino acids 147-192 of pp89 in 19 of 27 isolates, including the region encompassing the CTL epitope (amino acid residues 168-176). Four groups of isolates with naturally occurring variant sequences for the CTL epitope were defined: (1) YPHFMPPNL; (2) YPHFMPPSL; (3) YPHFIPPSL; and (4) YLDFMPPNL. The remaining isolates, and the laboratory strains K181 and Vancouver, showed complete identity with the Smith strain. Polyclonal pp89 (Smith strain)-specific CTL only weakly recognized target cells infected with MCMV from most variant groups. No lysis of cells infected with isolate N1 from group 4 was detected. Analyses of cross-reactive recognition of YPHFMPTNL peptide-coated targets by CTL primed with variant MCMV isolates showed that the group 2 and 3 isolates, G4 and K6, respectively, but not the group 4 isolate N1, elicited CTL that exhibited a cross-reactive response. Furthermore, while the group 2 and 3 isolates G4 and K6 were able to prime CTL responses that displayed reactivity to homologous pp89 variant nonapeptides, the group 4 isolate N1 failed to do so. Finally, while immunization of mice with the nonapeptide YPHFMPTNL conferred significant protection against the laboratory strain K181 [correction of Kl81], no evidence of protection was observed for the group 2 and 4 variants G4 and N1, respectively. These observations raise the possibility that clinical isolates of HCMV may also differ in sequence from potential vaccine strains at immunodominant epitopes for CD8+ T cells thus reducing the efficacy of vaccination.

An absolute and restricted requirement for IL-12 in natural killer cell IFN-gamma production and antiviral defense. Studies of natural killer and T cell responses in contrasting viral infections.

IL-12 is a potent induce of IFN-gamma and is associated with a wide variety of immunoregulatory activities. Using murine CMV (MCMV) and lymphocytic choriomeningitis virus (LCMV) models of acute viral infection, we show here that IL-12 is integrally involved in certain aspects of antiviral immunity. Experiments evaluating the production and function of IL-12 demonstrated the induction of biologically active factor early during Smith strain MCMV, but not Armstrong strain LCMV, infection. The MCMV-induced IL-12 was responsible for early NK cell IFN-gamma production and viral control, as in vivo IL-12 neutralization by Ab treatment blocked both of these. In contrast, NK cell IFN-gamma production could not be detected during LCMV infection, and IL-12 neutralization had no effect on early LCMV replication. In both infections, treatments neutralizing IL-12 failed to alter either early NK cell cytotoxicity or later T cell responses, including IFN-gamma production and viral clearance on days 7 and 9 after infection. These data demonstrate the induction of IL-12 production during certain viral infections and the function of IL-12 for eliciting NK cell IFN-gamma production and antiviral defense. Furthermore, they suggest the existence of IL-12-independent mechanisms for IFN-gamma induction and viral control by T cells.

Analysis of the role of CD4 + T-cells during murine cytomegalovirus infection in different strains of mice

We examined the role of CD4 + T-cells in peritoneal exudate cells (PECs) during the course of acute murine cytomegalovirus (MCMV) infection in two strains of mice. Cell counts of PECs and cytofluorometric analysis showed that C57BL/6, a resistant strain, had more CD4 + T-cells than BALB/c, a susceptible strain, after intraperitoneal infection of 3 × 10 3 PFU of the Smith strain of MCMV, though both strains had an equivalent number of CD8 + T-cells. CD4 + T-cells of both strains expressed mRNA of IFN-γ, IL-2, and IL-4 on days 5 and 7 after infection, with much higher expression of these cytokines in C57BL/6 than in BALB/c. At the same time point after infection, macrophages were shown to express mRNA of IL-1α and TNF-α with higher expression of IL-1α in C57BL/6 than in BALB/c. Production of nitric oxide, recently shown to be one of the antiviral effector mechanisms of macrophages, by macrophages of both strains was examined showing more production of nitric oxide on day 7 after infection in C57BL/6 than in BALB/c. From these findings, we suggest the possibility that CD4 + T-cells contribute to the protection against MCMV infection via the secretion of cytokines and the resultant activation of macrophages to produce nitric oxide.

Identification, Sequencing and Expression of the Glycoprotein L Gene of Murine Cytomegalovirus

DNA sequence analysis of the genome of the Smith strain of murine cytomegalovirus (MCMV) revealed an open reading frame (ORF) with amino acid sequence identity to glycoprotein L (gL) of other herpesviruses. The ORF is 822 bp in size and has the capacity to encode a protein of 274 amino acids. It has significant identity with the gL genes of human CMV and human herpesvirus 6. The coding sequence of the gL gene of MCMV strain K181 was also determined, and expressed in Escherichia coli as a fusion protein with glutathione S-transferase using the pGEX expression system. Two antibody-binding regions were identified on the basis of the reactivity of a series of truncated gL constructs with anti-MCMV antibodies. One was mapped to residues 1 to 38 and the other between residues 230 and 274. Polyclonal antibodies specific to gL were raised against the full-length gL fusion protein. The antisera were shown to react with a 46K protein present in purified virions by Western blotting. Treatment of purified virions with endoglycosidase-H or -F resulted in reductions in M(r) of the 46K species to 42K and 31K, respectively. The antisera did not exhibit any neutralizing activity in a plaque reduction assay.

Pathogenicity of murine cytomegalovirus for newborn mice: analysis with the attenuated mutants.

Mutants (ts21, rev21, and rec21w) of murine cytomegalovirus (MCMV) with reduced ability to kill newborn mice have been isolated from the Smith strain (wild type; wt). The postulated mutations for these mutants are as follows; a mutation(s) responsible for temperature sensitivity (ts); a mutation(s) responsible for attenuation (att); a mutation(s) which modifies temperature sensitivity (mts); and a mutation(s) responsible for modified growth characteristics (mgc). Genotypes of the mutants have been proposed from their phenotypes; ts21 (ts, att, mts+, mgc+); rev21 (ts, att, mts, mgc+) or (ts+, att, mts+, mgc); and rec21w (ts+, att, mts+, mgc+). Inoculation of 2.0 x 10(4) plaque forming units (PFU) of wt intraperitoneally into mice resulted in lethal infection accompanying intraperitoneal hemorrhage and pathognomonic changes in the target organs. Such changes were less in rec21w-infected mice than in wt-infected mice, and almost absent in ts21- and rev21-infected mice, on the sixth day after inoculation. The lesser damage to the organs correlated with poor growth of these viruses in the corresponding organs. On the other hand, in the case of rec21w-infected mice, virus titer in the target organs was almost equivalent to that of wt-infected mice. The growth characteristic of rec21w in the liver was also similar to that of wt. Nevertheless, damage to the organs by rec21w was less than that by wt. Comparison of the phenotypic characteristics between rec21w and wt demonstrated that att affects the ability of MCMV to induce the organ damage without affecting viral growth. Similar comparison between ts21 and rec21w revealed that ts affects the growth of MCMV in most organs.

Characterization of a strain of murine cytomegalovirus which fails to grow in the salivary glands of mice.

Characterization of a tissue culture-adapted strain of murine cytomegalovirus (MCMV), the Vancouver strain, which demonstrated altered tissue tropism in mice was undertaken to help understand the mechanism of pathogenesis of cytomegaloviruses. The Vancouver strain grew to a limited extent in the spleen but failed to grow in the salivary glands of inoculated mice. This mutation probably arose during multiple in vitro passaging of the parental Smith strain. The Vancouver strain replicated more quickly and produced a greater yield of virus per cycle than the Smith strain in vitro, resulting in a larger plaque size. In addition to these phenotypic differences, the Vancouver strain was found to have a 9.4 kb deletion spanning the XbaI I/L junction of the parental Smith strain (0.960 to 0.995 map units), and a 0.9 kb insertion which mapped to the EcoRI K fragment (0.37 to 0.47 map units). Analysis of virus-induced proteins at various times post-infection identified only one major change in Vancouver strain-infected cells, the absence of a 42K protein found in Smith-infected cells at early and late times.

Effect of subclinical hyperammonaemia on phagocytic activity of polymorphonuclear leukocytes isolated from peripheral blood of sheep

In in vitro experiments, the one-hour incubation of neutrophils from peripheral blood of 10 sheep (Polish Merino, aged 1 year) with ammonium acetate to final ammonia concentrations of 7.1, 71.0, 355.0, and 710.0 μmol/l caused no changes in cell viability. Incubation prolonged to 2 h significantly reduced cell viability at all ammonia concentrations. In contrast, incubation of neutrophils for 45 min with ammonium acetate significantly decreased their phagocytic activity against non-opsonized bacteria of Staphylococcus aureus 305 (all test concentrations) and against opsonized bacteria of S. aureus Smith strain (355.0 and 710.0 μmol/l only). In in vivo experiments, subclinical hyperammonaemia was produced in 10 sheep by continuous, three-day infusion of ammonium acetate solution into the right jugular vein. Ten control animals received an infusion of 0.9% sodium chloride. Blood was sampled from the left jugular vein before infusion, and 24, 48 and 72 h after beginning, and 48 h after the end of infusion. A significant rise in blood ammonia concentration occurred 24 and 48 h after beginning infusion. This increase in the experimental group correlated with a significant fall in phagocytic activitty of neutrophils against non-opsonized S. aureus 305 bacteria as compared to values before the infusion and to the controls. In contrast to the results of the in vitro experiment, no statistically significant influence of hyperammonaemia on neutrophil phagocytic activity against opsonized bacteria of S. aureus Smith was found in vivo.

Characterization of a membrane-associated phosphoprotein of murine cytomegalovirus (pp50) and its immunological cross-reactivity with a human cytomegalovirus protein

We have identified an abundant 50K phosphoprotein (pp50) in MCMV-infected 3T3-L1 cells and shown by immunofluorescence microscopy and surface-iodination experiments that pp50 is localized to the plasma membrane of the infected cell. Furthermore, the kinetics of its synthesis suggests that it belongs to the early-late class of herpesvirus proteins. Using monoclonal antibodies specific for pp50 to screen a λ ZAP II expression library constructed from poly(A)+ mRNA of MCMV-infected cells, we have isolated a cDNA clone that synthesizes a truncated form of pp50 as a β-galactosidase fusion protein. This allowed us to localize the partial pp50 transcript to a region between map coordinates 0.228 and 0.260 of the MCMV genome (Smith strain, Vancouver). Finally, we demonstrated that the MAb 5H10.21 A recognizes an antigenic determinant that is conserved between pp50 and a 50K human cytomegalovirus (HCMV) nonstructural protein.