TY - JOUR
T1 - HIV-1 pseudotype virus containing a Cocal virus genome and an HIV envelope
T2 - construction, assay and use
AU - Gregory, Susan
AU - Collman, Ronald
AU - James, William
AU - Gordon, Siamon
AU - Gonzalez-Scarano, Francisco
AU - Nathansona, Neal
N1 - Funding Information:
This study was supportedi n part by grantsN S 27405,H L 02358,a nd HL 07586f rom the United StatesP ublic Health ServiceU, SA, and by grantsf rom theA IDS DirectedP rogram,M edical ResearchC ouncil, UK. We would like to thank LaurenceT urley and VedapuriS hanmugamfo r expertt echnicasl upport, Robert Shopef or vesiculovirusesP,a ul Claphama nd Robin Weissf or advice, assistancea,n d reagentsa, nd BruceC hesebrof or CDCexpressing( 1022)c ells.
PY - 1993/10
Y1 - 1993/10
N2 - A method is described for the production and assay of pseudotype viruses between human immunodeficiency virus type 1 (HIV-1) and Cocal virus (COV), containing an HIV-1 envelope and a COV genome (COV(HIV)). COV(HIV) pseudotype virus is a useful tool for the investigation of a variety of questions regarding HIV entry into susceptible cells, including steps in virus binding, fusion, and internalization, and the role of molecules which inhibit entry. COV, a rhabdovirus closely related to vesicular stomatitis virus (VSV), replicated and caused cytopathic effect in primary cultures of human peripheral blood lymphocytes (PBLs) and monocyte-derived macrophages (MDM), and in human cell lines of lymphocytoid or monocytoid origin, making it an ideal candidate for pseudotype production. 174XCEM cells, which were permissive for selected macrophage-tropic strains as well as most lymphocyte-tropic strains of HIV-1, were used to produce stocks of putative pseudotype virus. To neutralize parental COV in these stocks, a rabbit antiserum was produced which had a neutralization index of > 107 at a dilution of 1:100. Using these methods, pseudotype viruses were produced with a titer of about 104 PFU per ml; these same stocks contained HIV-1 at a titer of about 105 TCD50 per ml and COV at a titer of about 108 PFU per ml. CD4-expressing HeLa cells were used to assay pseudotype stocks made with lymphocyte-tropic strains of HIV-1. The authenticity of the pseudotype stocks was validated by several controls, including their failure to register on congenic CD4-negative HeLa cells and their inhibition by monoclonal anti-CD4 antibodies such as Leu 3a.
AB - A method is described for the production and assay of pseudotype viruses between human immunodeficiency virus type 1 (HIV-1) and Cocal virus (COV), containing an HIV-1 envelope and a COV genome (COV(HIV)). COV(HIV) pseudotype virus is a useful tool for the investigation of a variety of questions regarding HIV entry into susceptible cells, including steps in virus binding, fusion, and internalization, and the role of molecules which inhibit entry. COV, a rhabdovirus closely related to vesicular stomatitis virus (VSV), replicated and caused cytopathic effect in primary cultures of human peripheral blood lymphocytes (PBLs) and monocyte-derived macrophages (MDM), and in human cell lines of lymphocytoid or monocytoid origin, making it an ideal candidate for pseudotype production. 174XCEM cells, which were permissive for selected macrophage-tropic strains as well as most lymphocyte-tropic strains of HIV-1, were used to produce stocks of putative pseudotype virus. To neutralize parental COV in these stocks, a rabbit antiserum was produced which had a neutralization index of > 107 at a dilution of 1:100. Using these methods, pseudotype viruses were produced with a titer of about 104 PFU per ml; these same stocks contained HIV-1 at a titer of about 105 TCD50 per ml and COV at a titer of about 108 PFU per ml. CD4-expressing HeLa cells were used to assay pseudotype stocks made with lymphocyte-tropic strains of HIV-1. The authenticity of the pseudotype stocks was validated by several controls, including their failure to register on congenic CD4-negative HeLa cells and their inhibition by monoclonal anti-CD4 antibodies such as Leu 3a.
KW - Cocal virus
KW - Human immunodeficiency virus
KW - Pseudotype virus
KW - Rhabdovirus
KW - Vesiculovirus
KW - Virus entry
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U2 - 10.1016/0166-0934(93)90064-X
DO - 10.1016/0166-0934(93)90064-X
M3 - Article
C2 - 8263122
AN - SCOPUS:0027359163
VL - 44
SP - 287
EP - 304
JO - Journal of Virological Methods
JF - Journal of Virological Methods
SN - 0166-0934
IS - 2-3
ER -