The Biology of HIV Infection

Clinical group

The Biology of HIV Infection

Duangrat Inthorn

Mahidol University

Duangrat Inthorn

The Biology of HIV Infection

This report includes general information on HIV history, the classes of retroviruses, and HIV composition and structure. It will also provide basic information on the HIV life cycle and identify possible targets for drug development. The HIV viral genome and the cause of the disease are also described. If you want more detailed information you can find it in the subsequent reports from the clinical group.

Introduction

History

We know now that AIDS is caused by the Human Immunodeficiency Virus (HIV), but it was originally observed by its effects on the immune system. An important clue was that AIDS patients often developed a lung infection (or pneumonia) caused by fungus called Pneumocystis carinii. This infection is very rare in healthy individuals, but patients with cancers of the immune system itself (lymphomas) were known to be susceptible to this disease. In 1981, a cluster of cases of Kaposi's sarcoma were observed in patients in San Francisco and New York. Kaposi's sarcoma normally occurs in elderly Jewish men but these patients were all young male homosexuals. Other diseases associated with immuno-compromisation also occurred in this same population; particularly of note was the occurrence of Pneumocystis pneumonia (which is an opportunistic infection) and lymphadenopathy. Later, a similar immunodeficiency was found in intravenous drug users who shared needles, persons who received blood transfusions and hemophiliaces. Moreover, the sex partners of these patients also got the disease. The disease was originally termed Gay-Related Immune Deficiency (GRID) but we now know it as Acquired Immuno-Deficiency Syndrome (AIDS).

HIV causes disease insidiously. The early stages of infection are often not apparent, without visible symptoms. The infected person may feel healthy and appears to be completely normal during that time (the incubation period) but such a person is able to transmit the infection. The HIV incubation period is of variable duration and can be quite long (on average 8 to 10 years). In contrast, for most common virus infections, such as colds or influenza, an incubation period of a few days or weeks will be followed by apparent disease. This adds greatly to the difficulty of studying and controlling AIDS, because many people infected with the virus have not yet developed the disease.

Retrovirus Family

HIV-1 is the predominant AIDS virus and is found worldwide, primarily in Central Africa , Europe and North and South America. A second virus HIV-2, closely related to the simian immunodeficiency virus (SIV), is shown to be endemic in parts of West Africa with limited spread in Western Europe. HIV-2 is only now beginning to appear in the Americas, mainly in the United States, Canada, and Brazil. Infection with either HIV-1 or HIV-2 results in a number of biologic and pathologic changes

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leading to a spectrum of immune dysfunctions, neurologic disorders, enteropathy and AIDS.

The human immunodeficiency viruses are members of the retrovirus family of viruses. The retroviruses are so called because at the beginning of their life cycle they reverse the usual flow of genetic information in a cell. In all living organism and in many other viruses, genetic information is stored as DNA and later transcribed into RNA. By contrast, retroviruses store their genetic information as RNA and contain a unique enzyme, reverse transcriptase (RT), which catalyzes the reverse transcription of the RNA genome (its entire complement of genes) into a DNA copy.

The retrovirus family is composed of three subfamilies : oncoviruses, spumaviruses and lentiviruses (Table 1). The oncoviruses cause cancer and are called cancer-causing viruses. The lentiviruses and spumaviruses do not cause cancer and do not integrate into the host's germ cell line. Both the lentiviruses and the spumaviruses produce a persistent lifelong infection of the host cells. Of the two, only the lentiviruses have been identified as causes of human and animal diseases. Classification of HIV as a lentivirus is based on its fine structure, biologic properties, protein and nucleic acid sequence homology (Table 2).

Table 1 Subfamilies of retroviruses (3) .

SubfamilyExamples

Oncoviruses: associated with the activation of certain cell genes leading to tumor develop

Type AMouse intracisternal type A

Type BMouse mammary tumor virus

Type CMurine leukemia virus

Human T cell leukemia virus type I and II

Feline leukemia virus

Bovine leukemia virus

Type DMason-Pfizer virus

SAIDS virus

Spumavirus: readily isolated from humans and other primates, but have not been associated with any specific disease

Simian foamy virus

Human foamy virus

Lentiviruses: produce acute cytocidal infection followed by a slowly developing multisystem disease

Visna maedi virus

Caprine arthritis encephalitis virus

Equine infectious anemia virus

Feline immunodeficiency virus

Bovine immunodeficiency virus

Simian immunodeficiency virus

Human immunodeficiency virus type 1 (HIV-1) and type (HIV-2)

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Table 2 HIV characteristics resembling those of lentiviruses (3).

Biologic characteristics

Persistent or latent infection

Cytopathic effects (syncytia formation) on selected cells

Capable of infecting macrophages

Associated with immune suppression

Long incubation period

Central nervous system involvement

Affects hematopoietic system

Molecular biologic characteristics

Similar genomic organization

Morphology of virus (cone nucleoid)

Accumulation of unintegrated proviral DNA

Polymorphism, particularly in the envelope gene

Primer binding site (tRNAlys)

Origins of HIV-1 and HIV-2

The genetic similarity between HIV-1 and HIV-2 is significantly less (40-50% nucleotide identity) than is found among different HIV-1 isolates (>85% nucleotide identity). HIV-1 and HIV-2 and other lentiviruses have been discovered in a wide variety of nonhuman primates (Table 3). The nonhuman primate lentiviruses are collectively know as simian immunodeficiency viruses (SIVs). To date, natural SIV infections have not been shown to cause disease in the infected animal. HIV-1 and HIV-2 appear to be closely related to the primate lentiviruses isolated from chimpanzees and sooty mangabey monkeys.

Table 3 Primate lentiviruses (3)

VirusHost

HIV-1Human

HIV-2Human

SIVCPZChimpanzee

SIVSMSooty mangabey

SIVMACRhesus macaque

SIVMNEPig-tailed macaque

SIVSTMverStump-tailed macaque

SIVAGMgriVervet monkey

SIVAGMsabGrivet monkey

SIVAGMtanSabaeus monkey

SIVAGMTantalus monkey

SIVMNDMandrill

SIVSYKSykes' monkey

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Studies of the African green monkey and

their related simian virus (SIVagm) yield

important information in our understanding

of HIV-1 and HIV-2. As see in Fig.1 there are

three major species of African green monkeys

indigenous to Africa.

Fig. 1 African green monkeys (2)

Fig. 2 Geographic domain of African

green monkeys (2)

Fig. 2 shows a map of Africa and indicates

the native locations of different species

of the African green monkey. Within

each species, a unique family of SIVagm

has been show to exist

Structure of HIV

The structure of HIV resembles that

of all retroviruses but particularly that

of the lentiviruses (Fig.3). HIV

has a cycindrical eccentric nucleoid

or core. The nucleoid contains the

HIV genome, which is diploid.

Outer lipid bilayer coat studded with

Surface (SU, gp120), transmembrane

(TM, gp41) glycoprotein complexes.

Beneath the lipid bilayer are matrix

proteins (MA), the virion consist of

Internal capsular (CA) and nuclear

Capsular (NC) proteins which

surround the single stranded RNA

genome.Fig. 3 Structure of HIV (2)

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HIV virion

Fig. 4 shows the components and their

genetic source, the matrix (MA) internal

capsular (CA), and nuclear capsid (NC)

proteins produced by the gag region

contain gene products of the pol

region-RT, integrase and protease.

Fig. 4 HIV Virion (2)

Life cycle of the human immunodeficiency virus

Fig. 4 Life cycle of HIV (2)

HIV and related lentiviruses have growth cycles that are typical of all retroviruses. It is convenient to think of viral growth as four distinct stages:

• Infection: The HIV-1 virus binds to the CD4 receptor complex on the surface of CD4+ cells. The virion then enters the cell, and uncoats.
• reverse transcription and integration : The HIV-1 virus undergoes the process of reverse transcription in which viral RNA is transcribed into complementary DNA. This is the portion of the life cycle at which all currently available antiretroviral agents are designed to intercede.
• viral gene expression : After reverse transcription, the DNA becomes double-stranded and migrates to the cell nucleus, where it is integrated into the host genomic DNA as a provirus.

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• virus assembly and maturation: The virus can then be transcribed back into mRNA and genomic RNA, and the resultant proteins and genomic RNA are assembled near the surface of the cell and packaged into a new virion, which buds from the cell membrane (2).

Soon after infection of the cells, the viral RNA genome is reverse transcribed into a DNA copy, transported to the nucleus and integrated at random sites in the chromosome. Once integrated, the proviral genome is subject to transcriptional regulation by the host cell, as well as its own transcriptional control mechanisms. The later stages of the life cycle involve expression of the viral genes and eventual assembly and release of virus particles

How it might be stopped (Drug develpoment )

• RNA is converted into double strand DNA by RT

-Drugs called RT inhibitors can interrupt this process. ART inhibitor drugs, such as AZT and 3TC, can disrupt the early stage of viral reproduction.

• The integrase enzyme incorporates the virus genetic material into the T cell's DNA

-Drugs called integrase inhibitors, which are designed to halt this process, are in development

• Disrupting the assembly line

The protease enzyme cuts viral proteins into shorter pieces so that they can become functional proteins and allow the infection of other cells.

-Protease inhibitors block this stage of reproduction by neutralizing the enzyme. They are even more effective when combine with RT inhibitors.

In this electron micrograph

(see Fig. 5), the virus can be seen

budding forth from the surface of

a cell. Note how the outer membrane

of the virus is composed of the lipid

bilayer membrane of the host cell

studded with integrated protein

products (envelop proteins) of

the virus (2).

Fig. 5show viral budding (2)

The Genome of HIV

The HIV genome is 9749 nucleotides, about the same size as any other retrovirus, for example Rous sarcoma virus (RSV). But the genome of HIV is more complex than RSV since it has extra open reading frames that clearly code for small proteins. Some of these are protein synthesis controlling proteins.

The HIV genome has nine open reading frames but 15 proteins are made in all.

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The GAG/POL polyprotein and the ENV polyprotein are cleaved into several proteins:

GAG is cleaved to : MA (matrix), CA (capsid), NC (nucleocapsid), p6

POL is cleaved to: PR (protease), RT (reverse transcriptase), IN (integrase)

ENV is cleaved to: SU (gp120) and TM (gp41)

Of the other proteins, three are incorporated into the virus (Vif, Vpr and Nef) while three are not found in the mature virus: Tat and rev are regulatory proteins and Vpu indirestly assists in assembly.

TAT: Trans-Activator of Transcription

REV: Regulator of Virion protein expression

NEF: Negative Regulatory Factor

VIF: Virion Infective Factor

VPU: Viral Protein U

VPR: Viral Protein R

These genes encode small proteins. They overlap with the structural genes (especially ENV) but are different reading frames. Note some are encoded by two exons (unlike the structural genes) and therefore their mRNAs can be derived by alternative splicing of structure gene mRNAs. Mutants in the TAT and REV genes show that they are both vital to any production of virus.

TAT gene product binds to a sequence in all the genes and positively stimulates transcription. It is thus a positive regulator of protein synthesis, including its own synthesis.

REV bind to an element only in the mRNA of structural proteins (GAG/POL/ENV) and regulates the ratio of GAG/POL/ENV to non-structural, controlling protein (TAT/REV) synthesis.

NEF despite its small size NEF has several functions

a)Homeostasis leads to several problem for the parasitic: super-infection by other HIV particles.

b)By a different mechanism from its down regulation of CD4 antigen, NEF reduces surface expression of MHC class molecules. This alters antigen presentation by the infected cell and is proposed to protect the infected cell from attack by cytotoxic T cells.

c)NEF is important for HIV replication in vivo but there seems to be little effect of NEF in an in vitro cell culture situation.

Fig. 6 HIV genomic map (2)

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This genetic map of the HIV-1 viral genome depicts the structural and regulatory genes in their relative position as well as their products and functions. The 9-kb RNA virus is flanked by a long terminal repeat (LTR) section on both the 5' and 3' ends of the virus, which serves as a promoter and binding site for host and viral transactivating factors. The TAR element exists within the R region of the LTR and serves as a binding point for the tat gene product (a potent transcriptionalactivator). The gag region encodes the nucleocapsid core and matrix proteins. The pol gene codes the reverse transcriptase, protease, and integrase enzyme. The envelope region (env) is responsible for the viral-coat glycoproteins, gp120 and gp41, which mediate CD4 binding and membrane fusion. The remaining genes (vif, vpr, vpu, rev and nef) are regulatory genes whose products play critical roles in controlling viral expression, trafficking of viral gene products within the infected cell, and viral infectivity. The rev-responsive element (RRE) is the binding site for the rev gene product, which is important for the transport of unspliced and singly spliced RNA massage from the nucleus (2).

HIV subtype

The two types of HIV can be distinguished genetically and antigenically.

HIV-1 is the cause of the current pandemic while HIV-2 is found in west Africa and rarely elsewhere. The HIV-2 type is closely related to simian immunodeficiency virus (SIV) found in west Africa. There are also 10 different HIV-1 subtypes. The major one in the US and Europe is type B. In some countries, mosaics between different subtypes have been found.

HIV binding via CD4 receptor

The HIV virus binds to the cell surface of a CD4

Lymphocyte. The binding attachment occurs

through an interaction of the viral glycoprotein

gp120/gp41 and the CD4 receptor complex on

the cell surface (see in Fig. 7).

Fig. 7 HIV binding via CD4 receptor (2)

The gp41 fragment consists of cytoplasmic

tail and a hydrophobic membrane-spanning

domain and is joined with the larger gp120

component via a fusion domain. The gp120

glycoprotein has several glycosylation sites

and hypervariable loops (eg. V3), which lead

to antigenic variation between viral strains.

The cd4-binding region is located toward

the center of the complex and consists of

components from both the gp120 and gp41

fragment.

Fig.8 Envelope glycoprotein complex of HIV-1 (2)

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The couse of the disease (5)

Usually from the time of infection till the clinical manifestation of AIDS a period of 8-10 years goes by. However, in certain cases this period may be two year or less. Approximately 10% of patients succumb to AIDS within 2 to 3 years.

• Acute infection (acute retroviral syndrome)

HIV infection produces a mild disease that is self-limiting. This is not seen in all patients. In the period immediately after infection, virus titer rises (about 4 to 11 days after infection) and continues at a high level over a period of a few weeks. The patient experiences some mononucleosis-like symptom (fever, rash, swollen lymph glands but none of these are life-threatening. The result is an initial fall in the number of CD4+ cells and a rise in CD8+ cells but the numbers quickly return to near normal levels.

• A strong cell-mediated and humoral anti-HIV immune defense

Cytotoxic B and T lymphocytes mount a strong defense and the virus largely disappears from circulation. During this period, more than 10 billion new particles are produced each day but they are rapidly cleared by the immune system and have a half life of only 5-6 hours. The infected cells that are producing this virus are destroyed either by the immune system or by the virus (half life about 1 day). However, the rate of production of CD4+ cells can compensate for the loss of cells and a steadstate is set up in which a very small fraction of the resting memory CD4+ cells carry an integrated HIV genome. Most CD4 cells at this stage are uninfected.

The virus disseminates to other regions including lymphoid and nervous tissue. This is the most infectious phase of the disease. Seroconversion occurs between one and four weeks after infection.