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Background:  The recently developed HIV/HCV pseudovirus system has opened areas of study previously unavailable due to the lack of a cell culture based system for HCV virions. We hypothesized that sequence polymorphisms in E1and E2 glycoproteins affect infectivity in a pseudotype system. To address this, we analyzed sequence differences between E1E2 clones that produce infectious and noninfectious pseudovirus. 

Methods:  RNA was extracted from serum samples of acutely infected injection drug users and used to amplify and clone the 5.2-kb region from the 5’UTR to the NS3/4A junction by large-amplicon RT-PCR. E1E2 expression clones were generated, fully sequenced, and co-transfected into 293T cells with the pNL4-3. Luc.R-E- plasmid, which contains an env-deficient HIV proviral genome as well as the luciferase gene. Supernatants containing HCV/HIV pseudoparticles were harvested and used to transduce Hep3B target cells. Infectivity was determined by measuring luciferase activity.

Results:  We produced E1E2 expression clones representing multiple variants from subjects VHC11, VHC13, and VHC44, at 2 time-points during acute infection. Of 39 in-frame E1E2 clones, 12 were infectious and 27 non-infectious in the pseudotype system. Eight of 20 clones representing VHC11, 2/18 from VHC13, and 2 of 11 from VHC44 were infectious, showing a strong bias towards sequences from VHC11. For VHC11 time-point 1, and VHC13 time-point 2, we had 10 or more clones to analyze that represented both infectious and noninfectious outcomes. At these time-points, 4 clones from VHC11 produced infectious pseudovirus while 6 did not, whereas 2 clones from VHC13 produced infectious pseudovirus while 9 did not. Comparison within study subjects showed noninfectious clones differing from infectious clones by no more than 4 and as few as 1 nonsynonymous (amino acid altering) nucleotide changes.

Conclusions:  Progress using HCV/HIV pseudovirus is slowed by the inefficiency of HCV E1E2 expression clones to produce infectious pseudoparticles. At the sequence level, it is apparent that very few amino acid changes can determine the difference between an infectious or non-infectious pseudovirus. Future studies may determine the underlying mechanisms, and identify key residues required for infectivity, which could streamline the production of infectious pseudovirus and increase understanding of HCV pathogenesis.

Keywords: HCV; pseudovirus; glycoprotein