Practical human being immunodeficiency virus type -1 clones have been widely used for vaccine design, neutralization assays, and pathogenesis studies. (86%) of the gene cassettes were functional. Pseudoviruses generated with pPCR products or related plasmid DNA showed similar level of sensitivity to six HIV-1 positive sera and three monoclonal antibodies, suggesting neutralization properties are not modified in pPCR pseudovirions. Furthermore, adequate amounts of pseudovirions can be obtained for a large number of neutralization assays. The new pPCR method eliminates cloning, transformation, and plasmid DNA preparation methods in the generation of HIV-1 pseudovirions, this allows for quick analysis of multiple PRI-724 kinase inhibitor genes from HIV-1 infected individuals. manifestation plasmid (Helseth et al., 1990). This allows for the study of genes from a large number of HIV-1 isolates (Derdeyn et al., 2004; Gao et al., 1996; Wei et al., 2003; Li et al., 2005; Li et al., 2006). Traditionally, genes are amplified from either proviral DNA or PRI-724 kinase inhibitor viral RNA through RT-PCR and the products are then cloned into manifestation vectors for generation of pseudovirions. However, the gene products amplified through bulk PCR may be affected by recombination or resampling during bulk PCR amplification (Fang et al., 1998; Liu et al., 1996). Since small sequence changes can affect the biological functions of envelope glycoproteins (Cordonnier et al., 1989; Kalia et al., 2005; LaBranche et al., 1995; Morris et al., 1994; Shimizu et al., 1999; Shioda et al., 1994), it is important to avoid studying genes comprising artificial elements generated during PCR in vitro. Non recombinant genes can be obtained using limiting dilution or solitary genome amplification (SGA) methods (Liu et al., 1996; Palmer et al., 2005; Simmonds et al., 1990). Since only one amplifiable viral genome is definitely amplified in each PCR reaction in either method, the products are not affected by recombination or resampling. PRI-724 kinase inhibitor However, because the PCR products are acquired through multiple reactions, several purifications, ligations, transformations, and plasmid DNA preparations are required to test the functionality of the PCR products. As a result, this cloning step is time consuming, labor intensive, and costly. The use of Env pseudotype viruses in a single round infection system has greatly improved the accuracy and simplicity of the evaluation of neutralization activity in vaccinated humans and experimental animals (Derdeyn et al., 2004; Gao et al., 1996; Helseth et al., 1990; Li et al., 2005; Wei et al., 2003). Given the extent of viral diversity that is seen among patients and even within a single individual, a large number of clones are needed to understand neutralization profiles. For this reason, two panels of subtype B and C viruses have been proposed for use as standards for the FGF5 evaluation of anti-HIV-1 neutralization activity in anti-HIV-1 sera (Li et al., 2005; Li et al., 2006). Analysis of a large number of Env pseudovirions from many different individuals using autologous and heterologous sera may lead to identification of signature sequences of critical neutralization epitopes and assist vaccine design. Therefore, a more efficient system is needed to generate a large number of Env pseudovirions. This study describes a promoter PCR (pPCR) method that can significantly decrease the labor, time, and cost needed to obtain a large numbers of Env pseudovirions by eliminating the cloning step. 2. Methods and Materials 2.1 Amplification of HIV-1 env genes Seven plasma samples were collected from HIV-1 positive individuals enrolled in a study of current HIV-1 strains in Ndola, Zambia. The study was approved by the ethics committee of the Tropical Disease Research Centre, the Duke University Institutional Review Board, and the National Institutes of Health. Viral RNA was extracted from the plasma and eluted into 55 l of elution buffer using QiaAmp Viral RNA Mini kit (Qiagen; Valencia, CA). Reverse transcription was performed with 25 l of vRNA and 25 pmol primer OFM19 5-GCACTCAAGGCAAGCTTTATTGAGGCTTA-3 in 100 l using Superscript III (Invitrogen; Carlsbad, CA). Single genome amplification (SGA) of the cDNA was performed to obtain the.