Simulation of HIV – 1 Detection

12/03/2012

Purpose:

The objective of the experiment is for students to understand the molecular biology of the human immunodeficiency virus and the pathogenesis of acquired immune deficiency syndrome. The experimental concepts and methodology involved with enzyme-linked immune sorbent (ELISA) assays will be introduced in the context of the clinical screening of serum samples for antibodies to the HIV virus.
Background:

Acquired immune deficiency syndrome (AIDS) is a disease characterized by the progressive deterioration of an individual's immune system. The immunological impairment allows infectious agents such as viruses, bacteria, fungi and parasites to invade the body and propagate unchecked. In addition, the incidence of certain cancers dramatically increases in these patients because of their compromised immune system. AIDS is a serious threat to human health and is a global problem. Intensive research is being done to advance methods of detection, clinical treatment and prevention.
The AIDS etiologic agent (HIV-1) is the human immunodeficiency virus type 1, which is a retrovirus. HIV-1 contains an RNA genome and the RNA dependent DNA polymerase also termed reverse transcriptase. Members of the retrovirus family are involved in the pathogenesis of certain types of leukemia and other sarcomas in humans and animals. The structure and replication mechanism of HIV is very similar to other retroviruses. HIV is unique in some of its properties since it specifically targets the immune system, is very immune-evasive, forms significant amounts of progeny virus in vivo during the later stages of the disease and can be transmitted during sexual activity.
The HIV viral particle is surrounded by a lipid bilayer derived from the host cell membrane during budding. The viral proteins are identified by the prefix gp (glycoprotein) or p (protein) followed by a number indicating the approximate molecular weight in kilo-daltons. The lipid bilayer contains gp 160, gp 120 and gp 41. The gp 41 anchors gp 120 in the bilayer. Beneath the bilayer is a capsid consisting of p17 and p18. Within this shell is the viral core. The walls of the core consist of p24 and p25. Within the core are two identical RNA molecules 9000 nucleotides in length. Hydrogen bonded to each viral RNA, is a cellular tRNA molecule. The core also contains approximately 50 molecules of reverse transcriptase.
Large quantities of virus can be grown in tissue culture for diagnostic and research purposes. Several of the viral proteins have been cloned and expressed in relatively large quantities.

Figure 1: Shows Mechanism of HIV infection, taken from EDVOTEK handout.

MECHANISM OF HIV INFECTION:
An individual can be infected with HIV through an abrasion in a mucosal surface (e.g. genital and rectal walls), a blood transfusion or by intravenous injection with a contaminated needle. Virus or virally infected cells are found in bodily fluids such as semen and blood. During the early stages of infection in an immune competent person the HIV virus elicits Humoral and Cellular immunity responses that result in a variety of circulating IgG molecules directed at several viral epitopes. However, since the virus has a high mutation rate the variants survive and produce progeny having a similar capacity to escape immune surveillance.
Unlike other cellular DNA polymerases, HIV DNA polymerase (reverse transcriptase) has a high error rate. These frequent mutations continually change the viral protein epitopes. This is believed to be the main mechanism of HIV immune evasion. The most important target for the virus is hemato-poietic cells such as bone marrow derived monocytes, myelocytes and immune system lymphocytes. Infection of immune system effector cells such as T cells and macrophages ultimately produce the most profound clinical consequences. Gp 120 binds to the CD4 receptors on the surface of T helper (TH) cells. These receptors are