Immunotherapy for Infectious Diseases

246 Dornburg and Pomerantz
Fig. 5. Retroviral packaging system derived from HIV-1. (A) A provirus of HIV-1. (B–D)
Plasmid constructs to express pseudotyped HIV-1 retroviral particles. (E) A plasmid construct to
encapsidate and transduce genes with a HIV-1 vector (the plasmid sequences to propagate such
constructs in bacteria are not shown). Besides the genes encoding for HIV-1 proteins, which form
the core of the virus (e.g., Gag: structural core proteins; P, protease; Pol, reverse transcriptase
and integrase) and the envelope (e.g., env [envelope protein]), the HIV-1 genome also codes for
several regulatory proteins (termed Vif, U [� Vpu], V [� Vpr], Tat, Rev, and Nef), which are
expressed from spliced mRNAs and which have important functions in the viral life cycle.
(B) Plasmid construct to express the core and regulatory proteins. To avoid encapsidation and
transduction of genes coding for such proteins, the following modifications have been made: the
5� long terminal repeat (LTR) promoter of the HIV-1 provirus has been replaced with the promoter
of cytomegalovirus (CMV) to enable constitutive gene expression; the 3� LTR has been
partially replaced with the polyadenylation signal sequence of simian virus 40 (polyA); the
encapsidation signal has been deleted (��); the reading frames for the envelope and vpu genes
have been blocked. (C), (D) Plasmid constructs used to express the envelope proteins of the
vesicular stomatitis virus (VSV-G) or the envelope protein of murine leukemia virus (MLV),
respectively. In the absence of HIV-1 envelope proteins, which are rather toxic to the cell, HIV-1
efficiently incorporates the envelope proteins of VSV or MLV into virions. The use of such
envelopes also further reduces the risk of the reconstitution of a replication-competent HIV-1 by
homologous recombination between the plasmid constructs. (E) A retroviral vector used to package
and transduce a gene of interest (T-gene) with HIV-1-derived vectors. Since the encapsidation
sequence extends into the Gag region, part of the gag gene (G) has been conserved in the
vector. However, the ATG start codon has been mutated. The gene of interest is expressed from
an internal promoter, since the HIV-1 LTR promoter is silent without Tat. sd, splice donor site.
ANIMAL MODEL SYSTEMS
One of the major problems with any therapeutic agent against HIV-1 infection is the
lack of an appropriate and inexpensive animal model system to test the efficiency of an
antiviral agent. Since HIV-1 only causes AIDS in humans, it is very difficult to test and
evaluate the therapeutic effect of novel antiviral agents in vivo. In addition, the evalua-