AZT "Resistance"! 
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 AZT "Resistance"!

When in vivo effectiveness of the antivirals is lost, based on surrogate
markers (which are questionable), resistant mutations in RT (nukes) or
PT (protease inhibitors) are blamed for this loss of effectiveness.

Resistance to AZT is based on changes in the reverse transcriptase (RT)
enzyme thereby losing susceptibility to the chain terminating effects of

Katz et al. state "Unexpectedly, the RTs purified from these mutant
viruses or bacterially expressed counterparts are not resistant to AZTTP
in vitro, and similar results have been reported from AZT-resistant
feline immunodeficiency virus."

To make this all more confusing, Lacey et al. (from the Wellcome
Research Laboratories) state "The contrast between the small differences
found in this study and the high level of viral resistance in tissue
culture presumably reflects an incomplete understanding of AZT
inhibition of HIV in the cell."

Interestingly, Williams and Loab state "In addition, recent evidence
indicates that specific mutations in the coding region for RT are
responsible for AIDS patients' reduced sensitivity after long-term
therapy with zidovudine (3'-azido-3'-deoxythymidine, AZT), a
chain-terminating nucleotide analogue. This resistance to a synthetic
drug may indicate an additional genomic 'plasticity' of HIV-1 lacking in
other retrovirus."

HIV-1 is one smart virus that has the capability to become resistant to
synthetic {*filter*}.

There are certainly many belief systems and assumptions that are
inherent in the supposed effectiveness of antivirals. First, there
was/is the CD4 T cell count.

Yet, Dr. Donald I. Abrams states that "Nevertheless, our study, like
others in the recent literature, calls into question the value of CD4 as
a surrogate end point in efficacy studies of antiretroviral {*filter*}."
(JAIDS 1996, 11:161-169)

Now there is the plasma viral load.

Yet, Dr. John M. Coffin states "Although, as concluded Levy et al., the
amount of virus detected could be made by relatively few cells, it
cannot be concluded that it is made by a few cells. An infected patient
is not a well-stirred system like a cell culture: The important sites of
replication are the lymphoreticular tissues (in lymph nodes, spleen,
tonsils, gut, skin, and elsewhere), which are not in free equilibrium
with {*filter*}. Only a fraction of the virus made at these sites can get to
the {*filter*}, and we have no idea what this fraction is and therefore have
no way of using the virus load to directly calculate the number of
infected cells. As I point out in my article, an important assumption in
the use of virus load measurements for this purpose is that not that the
fraction of virus release into the {*filter*} be large or small, only that it
be constant over the period of observation. While I believe this
assumption to be correct, I also consider it of first importance that it
be tested." (Science 1996, 271:671)

I wonder if there is the remote possibility that the antivirals just
don't work in vivo by interfering with HIV and that mutations in the
enzymes are just an excuse.

Considering the rate of virion production per day (in the billions) and
large number of quasispecies especially in the later stages of disease
progression, it would be no surprise that all sorts of mutations would
turn up. It has been shown that protease inhibitor resisitant mutations
exist in untreated individuals.


Katz RA; Skalka AM. The retroviral enzymes. Annual Review of
Biochemistry, 1994, 63:133-73.  

Abstract: We have reviewed the current state of knowledge concerning the
three enzymes common to all retroviruses. It is informative to consider
them together, since their activities are interrelated. The enzymatic
activities of RT and IN depend on processing of polyprotein precursors
by PR. Furthermore, RT produces the viral DNA substrate to be acted upon
by IN. All three of these retroviral enzymes function as multimers, and
it is conceivable that specific polyprotein precursor interactions
facilitate the multimerization of all of them. The multimeric structures
of the enzymes are, however, quite different. PR is a symmetric
homodimer whose subunits contribute to formation of a single active
site. RT (of HIV, at least) is an asymmetric heterodimer in which one
subunit appears to contribute all of the catalytic activity and the
second is catalytically inactive, but structurally important. IN also
functions minimally as a dimer for processing and joining. The
retroviral enzymes represent important targets for antiviral therapy.
Considerable effort continues to be focused on developing PR and RT
inhibitors. As more is learned about IN, such efforts can be extended.
Since these enzymes are critical at different stages in the retroviral
life cycle, one optimistic hope is that a combination of {*filter*} that
target all of them may be maximally effective as therapy for AIDS.

Page 155: AZT-resistance viruses appear during the course of treatment
of AIDS patients and in tissue culture. Nucleotide sequencing of highly
resistant viruses from patients revealed several amino-acid changes in
RT (e.g. D67N, K70R, T215F/Y, K219Q, and M41L) that appear in an ordered
fashion with a concomitant increase in resistance. Unexpectedly, the RTs
purified from these mutant viruses or bacterially expressed counterparts
are not resistant to AZTTP in vitro, and similar results have been
reported from AZT-resistant feline immunodeficiency virus. Thus, the
biochemical basis for AZT resistance is obscure. One possible
explanation for these observations is that AZT is further metabolized in
vivo to its active from and the genetic changes in RT do not produce
resistance to the pro-drug. Residues associated with AZT, ddI, and ddC
resistance lie in the fingers and palm and probably do not play a direct
role in recognition of these nucleoside inhibitors, but may affect
template interactions.


Lacey SF; Reardon JE; Furfine ES; Kunkel TA; Bebenek K; Eckert KA; Kemp
SD; Larder BA. Biochemical studies on the reverse transcriptase and
RNase H activities from human immunodeficiency virus strains resistant
to 3'-azido-3'-deoxythymidine. Journal of Biological Chemistry, 1992 Aug
5, 267(22):15789-94.

Abstract: A series of biochemical investigations to compare the DNA
polymerase and RNase H functions of the reverse transcriptases (RTs)
corresponding to azidothymidine (AZT)-sensitive and -resistant human
immunodeficiency virus (HIV) strains are described. Steady-state kinetic
studies with purified recombinant enzymes utilizing several templates
and three inhibitors, 3' azido-3' deoxythymidine triphosphate (AZTTP),
3-amino-thymidine 5'-triphosphate, and 2',3'-didehydro-2',3'-
dideoxythymidine 5'-triphosphate, found consistent 2-4-fold differences
between the enzymes from the two strains over a wide pH range. A strong
pH dependence for all three inhibitors was found at pH values below 7.4
and suggested an ionizable group on the enzyme with a pK of about 7. The
sensitivities of the RNase H activities of the two enzymes to AZTTP
and AZTMP were also compared and found to be similar. The nucleotide
incorporation fidelities of recombinant RTs corresponding to
AZT-sensitive and -resistant clinical isolates were compared and the
error specificities determined. No significant differences were found.
Both enzymes were equally able to incorporate AZTTP into an elongating
M13 DNA strand with concomitant chain termination. Purified wild-type
and mutant virions from cell-culture supernatants were compared in
"endogenous" DNA synthesis reactions, and the sensitivities of this
activity to AZTTP were found to be similar. The contrast between the
small differences found in this study and the high level of viral
resistance in tissue culture presumably reflects an incomplete
understanding of AZT inhibition of HIV in the cell.  


Remington KM; Chesebro B; Wehrly K; Pedersen NC; North TW. Mutants of
feline immunodeficiency virus resistant to 3'-azido-3'-deoxythymidine.
Journal of Virology, 1991 Jan, 65(1):308-12.  

Abstract: We selected 3'-azido-3'-deoxythymidine (AZT)-resistant mutants
of feline immunodeficiency virus (FIV) in a cat cell culture system. The
characterization of one of these mutants was facilitated by the
development of a focal immunoassay which could accurately measure FIV
infectivity. This assay was used to quantitate the susceptibility of FIV
to various inhibitors. The AZT-resistant mutant was found to be cross-
resistant to 3'-azido-2',3'-dideoxyuridine and 3'-azido-2',3'-
dideoxyguanosine but remained sensitive to several other inhibitors
(2',3'-dideoxyinosine, 2',3'-dideoxy-2',3'-didehydrothymidine, and
phosphonoformate). These patterns of cross-resistance and sensitivity
were similar to those of the AZT-resistant human immunodeficiency virus
(HIV) that has recently been isolated from patients with AIDS (B. A.
Larder and S. D. Kemp, Science 246:1155-1158, 1989). Like the AZT-
resistant HIV, purified reverse transcriptase from mutant FIV failed to
show resistance to the 5'-triphosphate of AZT. This mutant can be used
in the FIV model system to study the mechanisms of drug resistance and
to determine the pathogenicity of AZT-resistant mutants.


Williams KJ; Loeb LA. Retroviral reverse transcriptases: error
mutagenesis. Current Topics in Microbiology and Immunology, 1992,

Sun, 10 Dec 2000 03:00:00 GMT
 [ 1 post ] 

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