Replication-competent herpes simplex virus type 1 mutant expressing an autofluorescent glycoprotein H fusion protein.
The heterocomplex of glycoproteins H (gH) and L (gL) of herpes simplex virus type 1 (HSV-1) is essential for viral infectivity and is involved in viral penetration, cell-to-cell spread, and syncytium formation. We constructed an HSV-1 mutant expressing a gH-EGFP (enhanced green fluorescent protein) fusion protein under the control of the gH true late promoter. The EGFP coding sequence was cloned after the gH signal peptide into the HSV-1 genome. Superinfection of transfected, gH-nontranscomplementing cells with gH-negative HSV-1 resulted in a replication-competent recombinant virus. Cells infected with the recombinant virus exhibited strong and stable EGFP-specific fluorescence late in infection, and autofluorescence was detected in purified virions. The recombinant genotype of the mutant was confirmed by PCR. The 140-kD gH-EGFP fusion protein showed an N-linked glycosylation pattern similar to gH-1, was recognized by the conformation-dependent gH-specific monoclonal antibodies 52S and LP11 and formed a heterocomplex with gL which was transported to the cell surface and integrated into the viral envelope. Infectivity of the gH-EGFP mutant was neutralized by antibodies 52S and LP11. To our knowledge, this is the first replication-competent HSV-1 mutant expressing an autofluorescent essential glycoprotein which will be a versatile tool for studies of penetration, late gene expression, transport and tissue spread.
Lorentzen
E U
EU
Abteilung Klinische Virologie, Institut für Medizinische Mikrobiologie, Universität Münster, Von-Stauffenberg-Strasse 36, D-48151 Münster, Germany.
Eing
B R
BR
Hafezi
W
W
Manservigi
R
R
Kühn
J E
JE
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D005453:Fluorescence; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006561:Herpes Simplex / Q000821:virology*; D008164:Luminescent Proteins / Q000235:genetics; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D018139:Simplexvirus / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics*; D014779:Virus Replication
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C044200:glycoprotein H, herpes simplex virus type 1; D049452:Green Fluorescent Proteins