9.0 Virology Lecture synopses BS2005 Back
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Brief synopses and Notes
The course begins with a video to introduce viruses and to illustrate the versatility of viruses; a fact we ignore them at our peril. To aid your understanding, you will be given a set of questions to answer from the video as it runs. However, this is an introductory session only, and you will not be examined on it.
Virus Structure and Replication. You should understand the self assembly mode of replication to overcome small gnomic size; the geometrical constraints governing virus structure and the packaging of nucleic acid and he replication cycle of viruses with particular reference to Semliki Forest Virus and to Influenza Virus.
Influenza virus and its infections. a) Entry into host cells; b) The importance of the segmented RNA genome and its relationship to reassortment of antigens; c) The functions of the neuraminidase and the haemagglutin; d) The relationship between antigenic drift and antigenic shift and flu virus epidemics and pandemics; e) The role of respiratory tract immunoglobulins and of interferon in protection; g) Persons at high risk from influenza and how they may be protected by vaccination or by chemotherapy.
Immunity to virus infections. The role of a) Neutralising antibodies, ADCC, antibody and complement; b) NK cells and interferons; c) T Cells. You will be expected to be able to use influenza virus infections as named examples of the importance of these systems.
Chlamydial infections. a) How chlamydia differ from other bacteria and from viruses; b) the advantages and disadvantages of their adaptation to intracellular life; c) outline understanding of the chlamydial replication cycle and the role of elementary bodies and reticulate bodies; d) classification and possible evolution of the genus Chlamydia; e) important human infections caused by Chlamydia; f) mechanisms of protection and immunopathology in chlamydial infections.
Human Immunodeficiency Virus (HIV) and AIDS:
Epidemiology. At the end of 1999 a total of 34.3 million people were living with HIV/AIDS and the total number of deaths since the beginning of the epidemic had risen to 3.8 million. In 1999 there were 5.4 million people newly infected with HIV and 2.8 million AIDS deaths occurred worldwide. In sub-Saharan Africa 55% of HIV-1 positive adults are women with the main mode of transmission being heterosexual. These figures supplied by the WHO suggest that the HIV/AIDS pandemic is still a growing health problem and is now threatening the heterosexual population as well as those individuals in the so-called "high risk behavior groups". With the current lack of an effective vaccine and the poor availability of expensive anti-HIV drugs for the vast majority of infected people, the future outlook for the pandemic remains bleak.
Virology. HIV, the primary etiological agent for AIDS, is a member of the subfamily of retroviruses known as the lentiviruses. These viruses typically display long periods of latent infection prior to causing immunological and neurological diseases. HIV and it closely related simian (SIV), feline (FIV), caprine (CIV), equine (EIAV) and bovine (BIV) immunodeficiency viruses cause a depletion of CD4+ helper T cells that over several years leads to a profound loss of immune function resulting in increased susceptibility to opportunistic infections and neoplasms. HIV exists in two major forms, HIV-1 and HIV-2, which have a common ancestor in SIV. HIV-1 is responsible for the vast majority of infections throughout the world and is a highly pathogenic virus, by contrast HIV-2 infections are limited to regions of West Africa and are associated with a very mild form of disease.
Figure 1. Structure of HIV
Biochemical structure of HIV-1. HIV is a roughly spherical particle with a diameter of 110nm and is covered in a lipid bilayer from which 72 knob-like structures are projected. These surface projections are composed of multimers of two viral glycoproteins, gp120 and gp41, that enable the virus to recognise and bind to chemokine and CD4 molecules found at the surface of helper T lymphocytes and macrophages. The main shape and structural features of HIV are provided by the gag polyprotein gene products, Matrix, Capsid and Nucleocapsid. The core of the virus is formed by the Capsid and contains the genetic and biochemical information required for HIV replication in human cells. This information includes two identical copies of the HIV RNA genome and three viral enzymes, reverse transcriptase (RT), integrase (IN) and protease (PR). The HIV RNA genome is approximately 10,000 base pairs in length and is arranged as a series of overlapping open reading frames that encode the viral proteins.
Figure 2. Proteins of HIV.
Protein Gene Function
gp120 env Attachment of HIV to cell surface
gp41 env Fusion of viral and cell lipid bilayers
Matrix gag Viral structure + nuclear import of viral
Capsid gag Formation of viral core
Nucleocapsids gag Packaging of genomic RNA and reverse
RT pol Reverse transcription of viral RNA to DNA
PR pol Maturation of Gag and Gag-Pol polyproteins
IN pol Integration of viral genome with host DNA
Tat tat Regulation of viral gene transcription
Rev rev Regulation of viral mRNA transport
Nef nef Viral infectivity and immune evasion
Vpu vpu Immune evasion
Vif vif Enhancement of viral infectivity
Vpr vpr Nuclear import of viral genome
HIV infection. The life cycle of HIV begins with recognition and attachment of HIV via surface gp120 to its primary receptor CD4 found at the surface of helper T cells and macrophages. Subsequent interaction of gp120 with cell surface chemokine receptors (CCR5, CCR3, CXCR4 etc) leads to a conformational change in the glycoprotein that promotes fusion of the viral and cellular lipid bilayer. Naturally occurring mutations in the CCR5 chemokine receptor afford a degree of protection to individuals against infection, as does expression of high levels of circulating chemokines.
Figure 3. HIV attachment and fusion with human cell.
Following fusion the viral core is injected into the cell cytoplasm and undergoes uncoating to enable the process of reverse transcription. The viral reverse transcriptase then converts the viral RNA genome into a double stranded DNA that can be integrated into the host cell genome. Reverse transcriptase is an error-prone DNA polymerase that introduces roughly 1bp mutation into the viral genome, this poor fidelity serves as the driving force for the genetic variation of HIV which is critical for spread of infection and emergence of drug-resistant viruses. Reverse transcriptase is a target for many highly effective anti-HIV drugs including AZT. During reverse transcription the viral genome is imported into the host cell nucleus guided by associated viral Matrix and Vpr proteins. Completion of reverse transcription and nuclear import is followed by integration of the viral DNA into the host cell genome, this even is catalysed by the viral Integrase. Infection is now complete and irreversible, moreover if the infected cell is capable of division all daughter cells will also carry the HIV genome.
HIV Replication. The integrated HIV provirus uses a combination of viral and cellular transcription factors to replicate new copies of its RNA genome. Some of these viral RNAs are exported to the cytoplasm to serve as new viral genomes, however a percentage of the viral RNA is spliced into smaller mRNA species that are translated by the host cell ribosomal machinery. The viral regulatory proteins Tat and Rev tightly control transcription and transport of viral mRNA to the cell cytoplasm and both proteins are essential for HIV replication. After translation, the three major viral polyprotein precursors, gp160, Gag and Gag-Pol are transported to the plasma membrane region of the cell. Gag and Gag-Pol precursors package viral genomes. gp120 and gp41 envelope glycoproteins, which are processed from gp160 by cellular proteases, are inserted into the plasma membrane of the cell. New immature viral particles bud off the plasma membrane carrying an outer lipid bilayer captured from the host cell surface together with the correct number of gp120 and gp41 surface receptors. The final event is known as maturation and involves cleavage of the Gag and Gag-Pol polyproteins by the viral Protease. Cleavage of the Gag and Gag-Pol polyproteins results in assembly of the matrix and capsid structures and release of active reverse transcriptase and integrase into the viral particle. This final event of replication is essential for production of infectious HIV particles and is the target for protease inhibitors, which are one of the most effective classes of anti-HIV drugs.
Current HIV therapies and the hopes for a vaccine. The most effective anti-HIV drugs target the activity of viral reverse transcriptase and protease proteins. Reverse transcriptase function can be inhibited by either nucleoside or non-nucleoside inhibitors. The nucleoside class of inhibitor that resemble the natural substrates of reverse transcriptase and act as competitors for the active site of the polymerase to block further DNA synthesis, the best example of this type of drug is AZT. Protease inhibitors are peptide analogs that capture the enzyme in an irreversible form that resembles the transition state for cleavage of the Gag and Gag-Pol polyproteins. Three major disadvantages are associated with the use of reverse transcriptase and protease inhibitors:
1. The rapid emergence of drug resistant strains of HIV. Reverse transcription creates a large pool of variant viruses (known as a quasi-species) some of which are resistant to reverse transcriptase and protease inhibitors. These resistant viruses are selected during therapy and eventually emerge as the dominant viral species. This problem has been overcome in recent year by the use of combination therapy in which patients are treated with a combination of reverse transcriptase and protease inhibitors. Emergence of resistant viruses is less likely since they would have to be modified to replicate in the presence of several drugs targeted at two different viral enzymes.
2. Side effects and management. Nucleoside inhibitors of reverse transcriptase and drugs targeting the viral protease have a range of unpleasant side effects that are often not tolerated by patients. Combination therapy requires the daily use of several different drugs which must be taken for the rest of the patient's life, this routine is often difficult to maintain when the patient is suffering side effects such as vomiting or drowsiness.
3. Cost. Therapy for an HIV-1 positive patient is costly (roughly £10,000 per annum) and is therefore not available for the vast majority of infected people living in poor and under-developed countries.
The best worldwide solutions to the HIV/AIDS pandemic are therefore education and production of a vaccine. Education is aimed at dispelling local taboos and encouraging the practice of safe sex through the use of condoms. To date there is no effective vaccine, however a group of AIDS researchers in Oxford have recently developed a promising new DNA-based vaccine that is in clinical trials.
Useful web sites:
The site at Leicester University gives a good overview of HIV virology and molecular biology - http://www-micro.msb.le.ac.uk/335/AIDS.html
The WHO site is an excellent resource for reviewing the latest epidemiological data on the AIDS pandemic - http://www.unaids.org/epidemic_update/report/index.html.
Fields Virology 3rd Edition provides good chapters on HIV/AIDS biology and therapies (available in the General Hospital Library).
What you should know from this document and the accompanying session:
The current epidemiology of HIV/AIDS
What type of virus HIV is
The basic structure and biochemical features of HIV
The function of the major HIV proteins
The mechanism by which HIV attaches and fuses with human cells
The types of cells susceptible to HIV infection and the role of cell surface receptors
Details of the events that lead to integration of the viral genome into the host cell genome
How HIV replicates itself
The principles of how the most effective anti-HIV drugs work
The problems associated with anti-HIV drug therapy and the solutions currently available
Derek Mann and Mike Ward