Biology Reference
In-Depth Information
viruses. The authors suggest that GFP gene insertion may interfere with the
appropriate splicing or expression of other viral products. Recombinant virus
with GFP inserted into the nef locus replicated with wild-type kinetics and
produced brightly ¯uorescent cells, similar to HIV-1. SIV Dnef EGFP plasmid
produced infectious SIV. A related plasmid with the HIV-1 nef sequence ex-
pressed by downstream placement of an internal ribosomal entry site produced
similar results. The strains were infectious in rhesus monkey, and infected cells
could be detected in the tissue of infected animals by FACS analysis and ¯uo-
rescence microscopy. The GFP gene was found to be expressed through several
rounds of viral replication in cultured cells. However, GFP expression in vivo
turned out to be much less stable. Seven days after inoculation, all SIV yielded
from PBMCs were ¯uorescent, whereas <10% of the virus recovered at day 21
had detectable ¯uorescence. By day 35, none of the recovered SIV caused cells
to ¯uoresce. Analysis of polymerase chain reaction ( PCR)-ampli®ed regions of
SIV genome from genomic DNA isolated from PBMCs of infected animals re-
vealed deletions within the GFP sequence. Thus, SIV recombinants expressing
GFP were suggested as a tool in studying early events in SIV infection. The
instability of the genomes in vivo hampers application of GFP-tagged SIV for
long-term studies of virus-host interaction.
LABELING OF HIV-1 VIRIONS IN TRANS
Fluorescent viral particles allow for the microscopic studies of virus tra½ck-
ing, which is important for understanding the mechanisms of viral entry. The
attachment of the virus to the cells and the subsequent entry steps leading to
infection have become popular targets in the development of novel antiviral
agents. The potential of the development of potent antiviral agents stimulated
the studies of mechanisms of viral attachments and the pathways of the virions'
tra½cking. Electron microscopy still remains the major method to visualize the
interactions between a virus and the cell, but it does not allow for observation
of viral tra½cking in real time in live cells. It is also very laborious, especially
when numerous cells have to be examined; and the harsh ®xation procedures
can give rise to artifacts. In the past, chemical modi®cations of assembled virus
particles and intercalation of ¯uorescent lipids into the particle envelopes were
used for labeling of virions. Greber and colleagues successfully studied the
nuclear entry of adenovirus labeled with ¯uorescein isothiocyanate ( FITC)
(Greber et al., 1997). Nichols and co-workers have used FITC-labeled in¯uenza
virus and ¯ow cytometry to assess binding and internalization of virions by
monocytes-macrophages and lymphocytes ( Nichols et al., 1993). The vesicular
stomatitis virus labeled with FITC enabled observation of endocytosis and
intracellular tra½cking of the virus to the nucleus of infected cells ( Da Poian et
al., 1996). Kinetics of intracellular disassembly and processing of the vesicular
stomatitis virus were probed by Bodipy ¯uorescence dequenching ( Da Poian et
al., 1998). The virus was covalently labeled with the ¯uorescent probe Bodipy-
