HIV-1 pseudotype virus containing a Cocal virus genome and an HIV envelope: construction, assay and use.
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 > 10(7) at a dilution of 1:100. Using these methods, pseudotype viruses were produced with a titer of about 10(4) PFU per ml; these same stocks contained HIV-1 at a titer of about 10(5) TCD50 per ml and COV at a titer of about 10(8) 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.
Gregory
S
S
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104-6076.
Collman
R
R
James
W
W
Gordon
S
S
Gonzalez-Scarano
F
F
Nathanson
N
N
D000818:Animals; D015704:CD4 Antigens; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D003588:Cytopathogenic Effect, Viral; D016679:Genome, Viral; D015497:HIV-1 / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D008214:Lymphocytes / Q000382:microbiology; D008264:Macrophages / Q000382:microbiology; D009500:Neutralization Tests; D011817:Rabbits; D018116:Vesiculovirus / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D010948:Viral Plaque Assay; D014771:Virion / Q000502:physiology; D014779:Virus Replication
D015704:CD4 Antigens