From: Billi Goldberg <bigoldberg@igc.apc.org>
Subject: HIV, AZT and PCR
Note: Copyright 1992, Dan R. Greening. Non-commercial reproduction allowed.
Archive-Number: 18
Organization: unspecified
Date: Sat, 23 Jan 93 08:01:42 PST

        PCR FINDINGS ON HIV CELLULAR INFECTION AND AZT RESISTANCE
                         by Billi Goldberg

Both of these articles are very important and well-worth reading in 
their entirety. Dr. Haase of the University of Michigan Medical School 
and David Ho of the Aaron Diamond AIDS Research Center are experts in 
their field.

The Haase et al. article states that their procedures allowed them to 
pick up on cells that "probably only have a single copy of HIV DNA that 
would not be identified as infected cells by in situ hybridization." It 
must be remembered that CD4 lymphocytes proliferate when activated; 
antibody production in HIV is excessive. Latently infected cells, when 
activated, could easily result in millions of HIV virions from the 
single provirus per cell. Nuclear Factor Kappa B and Special protein-1 
are known promoters/enhancers of HIV when mitosis of cells (M1 cycle) 
takes place.

The Ho et al. article undercuts the foundation of treatment with AZT. He 
states that "Of the four patients who had highly resistant virus, three 
patients contracted HIV-1 in the pre-AZT era. Given that HIV-1 exists in 
vivo as a population of diverse yet related viruses, we believe that 
resistance develops de novo, perhaps due to the error-prone reverse 
transcription step. Initially, the fraction of drug-resistant HIV-1 is 
probably small. As viral replication increase with disease progression, 
the chance of having AZT-resistant virus also increases." Ho concludes 
the article by stating "It is possible that the reported short-term 
benefits may eventually be offset by the earlier development of drug-
resistant virus and that with widespread use of AZT, the modes 
beneficial effect of the drug may diminish in time. This concern 
emphasizes the urgent need to quickly develop; other anti-HIV drugs for 
clinical use."

Isn't it interesting how PCR tests are changing and increasing the 
knowledge about HIV/AIDS.
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Embretson J, Zupancic M, Beneke J, Till M, Wolinsky S, Ribas JL, Burek 
A, and Haase AT. Analysis of human immunodeficiency virus-infect tissues 
by amplification and in situ hybridization reveals latent and permissive 
infections at single-cell resolution. Proc Natl Acad Sci USA, January 
1993;90:357-361.

Abstract: Latent and productive viral infections are at the extremes of 
the spectrum of virus-cell interactions that are thought to play a major 
role in the ability of such important pathogens as human 
immunodeficiency virus (HIV) to elude host defenses and cause disease. 
The recent development of PCR-based methods to amplify target sequences 
in individual cells in routinely fixed tissues affords opportunities to 
directly examine the subtle and covert virus-cell relationships at the 
latent end of the spectrum that are inaccessible to analysis by 
conventional in situ hybridization techniques. We have now used PCR in 
situ with in situ hybridization to document latent and permissive HIV in 
routinely fixed and paraffin embedded tissue. In one of the first 
specimens we examined, a tumor biopsy from an HIV-infected individual, 
we found many of the lymphocytes and lymphocytes infiltrating the tumor 
had HIV DNA that was detectable only by PCR in situ. The fraction of 
positive cells varied regionally, but there were foci where most of the 
cells contained HIV DNA. Most of these lymphocytes and macrophages are 
latently infected, as we could detect HIV RNA in fewer than one in a 
thousand of these cells. We also detected HIV RNA, surprisingly, in 6% 
of the tumor cells, where the number of copies of viral RNA per cells 
was equivalent to productively infected cell lines. The alternative 
states of HIV-gene expression and high local concentration of latently 
infected lymphocytes and monocytes revealed by these studies 
conceptually supports models of lentiviral pathogenesis that attribute 
persistence to the reservoir of latently infected cells and disease to 
the consequences of viral-gene expression in this population. The 
magnitude of infection of lymphocytes documented in this report is also 
consistent with the emerging view that HIV infection per se could 
contribute substantially to depletion of immune cells in AIDS.
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Mohri H, Singh Mandaleshwar K, Ching WTW, Ho DD. Quantitation of 
zidovudine-resistant human immunodeficiency virus type 1 in the blood of 
treated and untreated patients. Proc Natl Acad Sci USA, January 
1993;90:25-29.

Abstract: A nonselective ex vivo assay was used to directly detect and 
quantify zidovudine (AZT)-resistant human immunodeficiency virus type 1 
(HIV-1) in the blood of treated and untreated patients. In contrast to 
previous reports, drug-resistant virus was detected in peripheral blood 
mononuclear cells of a few of the patients who had never received AZT. 
The AZT resistance of HIV-1 isolated from one untreated individual was 
confirmed by further susceptibility studies in vitro and by the finding 
of a characteristic mutation (Lys -> Arg at codon 70) in the reverse 
transcriptase. In patients who were clinically stable while on AZT, 
HIV-1 titers in plasma and mononuclear cells were generally low but 
resistant viruses already predominated. In these individuals who were 
deteriorating despite AZT administration, high levels of viremia were 
observed, and the resistance phenotype was nearly universal. These 
findings serve to emphasize the magnitude of the AZT-resistance problem 
in patients on drug treatment.

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