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Retroviruses & Human Immunodeficiency Virus (HIV)


Unique Features of Retroviruses Key Features A. Human viruses are associated with tumors, leukemias, and immunodeficiencies. B. Common genetic organization and strategy - major differences lie in regulatory complexity. C. Genome consists of 2 copies of (+) stranded RNA. D. RNA converted to DNA (reverse transcription) by a bizarre mechanism followed by chromosomal integration. E. Integrated DNA co-linear with viral DNA.


Three Subfamilies of Human retroviruses Oncovirinae (HTLV-1, HTLV-2, and HTLV-5) include members that can immortalize and transform target cells Lentivirinae (HIV-1 and HIV-2) include members that are slow viruses and associated with neurologic and immunosuppressive disease Spumavirinae (Human foamy virus) not associated with human disease


Oncovirinae Transformation •Oncovirinae can immortalize and transform cells. They are fast acting and cause sarcomas and leukemias through incorporated cellular genes (protooncogenes) which cause dis-regulation of cell growth. •They can transform cells in vitro. At least 35 different protooncogenes have been identified which include growth hormones, growth hormone receptors, protein kinases, GTP-binding proteins and nuclear DNA binding proteins.


Leukemiaviruses A sub-class of oncoviruses that cause cancer by up-regulating cell growth •Some oncoviruses, such as HTLV, cannot transform cells in vitro • they produce cancer (in vivo) after a long latency period and promote unregulated growth indirectly rather than through the presence of a viral encoded oncogene. •T-cell leukemias are usually monoclonal.


HIV Kills cells rather than immortalizing them •HIV is a slow cytocidal virus with exquisite tropism for CD4+ expressing cells and macrophages. •It is the loss of CD4+ cells which destroys helper and delayed type hypersensitivity functions of the immune response.


Reverse Transcribing Viruses - HIV Fields Virology 4th edition, 2002, Chapter 59, Lippincott, Williams and Wilkins, 2002 Fig. 59-1

Figure 59-1 Primate lentiviruses have a distinct morphology and can induce syncytia during productive infections. A: Electron micrograph showing a single HIV-1 particle in the process of budding from an infected cultured human PBMC, and several mature virions containing the characteristic conical/bullet-shaped nucleoid (*100,000). (Photomicrograph kindly provided by Dr. Jan Orenstein.)


Retrovirus virion structure

Figure 59-17 Schematic representation of a mature HIV-1 virion.

Figure 60-2 Organization of a mature human immunodeficiency virus (HIV)-1 virion. Envelope glycoproteins project from the outer lipid membrane. Two copies of the HIV genome are contained within the core

Fields Virology 4th edition, 2002, Chapters 59 & 60, Lippincott, Williams and Wilkins, 2002


HIV-1 Replication Cycle Reverse Transcription

Assembly

Integrase

Attachment CD4

Integration

Reverse Transcriptase

Uncoating

Budding

CCR5

Maturation Protease

CXCR4 Beth D. Jamieson, Ph.D.


Comparison of oncogenic and non-oncogenic viruses Fields Virology 4th edition, 2002, Chapter 10, Lippincott, Williams and Wilkins, 2002 Fig. 10-2

Figure 10-2 Genomic structure of avian leukosis virus (ALV) and two transducing retroviruses. In addition to the long terminal repeat (LTR) sequences that provide transcriptional regulatory elements, the normal genome of ALV contains three major coding regions including gag, pol, and env. Gag encodes structural proteins of the virus, pol encodes enzymes involved in reverse transcription and integration, and env encodes the virion surface glycoproteins. In Rous sarcoma virus, the cellular src sequences are added to an otherwise intact retroviral genome. In contrast, in the MC29 virus, the addition of myc sequence is at the expense of the entire pol gene and parts of both gag and env.


Genetic organization of HTLV and HIV

Fields Virology 4th edition, 2002, Chapter 59, Lippincott, Williams and Wilkins, 2002 Fig. 59-4

Figure 59-4 Genomic organization of simple and complex retroviruses. The genes of Moloney murine leukemia virus (MuLV), HTLV-I, HIV-1 and HIV-2 are depicted as they are arranged in their respective proviral DNAs. The sizes of the different proviral DNAs are shown in proportion to the 9.7-kb HIV provirus


RNA and DNA intermediates in the retrovirus life cycle Fields Virology 4th edition, 2002, Chapter 57, Lippincott, Williams and Wilkins, 2002 Fig. 5706

Figure 57-6 Structures of the termini of the viral RNA and DNA genomes at various stages of the viral life cycle. Sequence blocks in RNA are indicated by lower case, and those in DNA by upper case. The structure of the RNA genome in the virion particle is indicated at the top. Reverse transcription of the RNA soon after infection involves the duplication and translocation of u5 and u3 sequence blocks, and it results in the formation of a doublestranded DNA molecule containing two terminal LTRs. The integration of the DNA genome occurs at the terminal sequences, establishing a provirus that is collinear with the preintegrative DNA. The forward transcription of the provirus is initiated at the U3/R border in the provirus; the resulting RNAs are cleaved and polyadenylated at the r/u5 border, recreating a viral RNA genome (bottom) identical to the infecting RNA.


The reverse transcription of retroviral RNA to DNA Pbs=primer or tRNA binding site ppt=polypurine tract

Fields Virology 4th edition, 2002, Chapter 57, Lippincott, Williams and Wilkins, 2002 Fig. 57-7

Figure 57-7 The reverse transcription of the retroviral genome. Thin lines represent RNA; thick lines represent DNA. See text for details.


Integration of proviral DNA into the chromosome

Fields Virology 4th edition, 2002, Chapter 57, Lippincott, Williams and Wilkins, 2002 Fig. 57-11

Figure 57-11 Steps in the integration of the viral DNA. The full-length linear DNA (top) is processed by the viral integrase by the endonucleolytic removal of dinucleotides at the 3´ termini. The resulting DNA is then used in a strand transfer reaction in which the 3´ OH ends make staggered breaks in the two strands of the target site DNA. The resulting gapped intermediate is presumably repaired by host enzymes.


How non-oncogene containing viruses transform 57-18 Genetic alterations in target gene expression induced by retroviral insertional mutagenesis. Various changes in normal gene expression that have been observed upon insertion of retroviral DNA are diagrammed. A target gene containing four exons is used in these examples (top). Promoter insertion: Insertion of the provirus in the same transcriptional orientation in the first intron is shown to result in the formation of a new mRNA initiated in the 3´ LTR and extending into the downstream exons. Enhancer insertion: Insertion upstream of the gene, in this case in reverse orientation, is shown enhancing the expression from the natural promoter. Poly(A) site insertion: Insertion at the 3´ end of the gene in the forward orientation is shown providing a poly(A) addition signal and thereby increasing the levels of a prematurely truncated mRNA. Leader insertion: Insertion of the provirus in the same transcriptional orientation is shown to result in the formation of an RNA initiating in the 5´ LTR, extending through the provirus and into downstream exons. Splicing results in the Figure retention of only the viral leader on the chimeric mRNA. Inactivation: Insertion is shown causing premature end formation of the mRNA, resulting in the formation of an inactive fragment. Fields Virology 4th edition, 2002, Chapter 57, Lippincott, Williams and Wilkins, 2002 Fig. 57-11


The genes of Retroviruses with clinical consequences Table 65-2

=genes common to all retroviruses

From Murray et. al., Medical Microbiology 5th edition, 2005, Chapter 65, published by Mosby Philadelphia,,


The role of specialized genes in HIV The role of some of the specialized genes of lentiviruses (those other than gag-pol-env) are designed to transactivate genes (tax, tat) regulate mRNA splicing (rex,rev), alter cellular activation signals (nef)

Figure 65-5

From Murray et. al., Medical Microbiology 5th edition, 2005, Chapter 65, published by Mosby Philadelphia,,


Entry of HIV into cells requires cellular CD4 and chemokine receptors

Fields Virology 4th edition, 2002, Chapter 60, Lippincott, Williams and Wilkins, 2002 Fig. 60-3

Figure 60-3 Binding of the HIV envelope glycoprotein gp120 to the CD4 molecule on the cell surface enables a subsequent interaction with a coreceptor molecule. A conformational change occurs in the HIV envelope, and the fusion process is initiated. Envelope epitopes that are exposed during this conformational change may be so transient that they are not recognized by the immune system.


HIV – the disease •HIV primarily infects CD4 T cells and cells of the macrophage lineage •Monocytes •Macrophages(including alveolar macrophages) •Dendritic cells (skin) •Microglial cells (CNS) •Virus causes lytic infection of CD4 T cells and a persistent low-level productive infection of macrophages •Virus alters T-cell and macrophage cell functions


Natural Course of HIV-1 Infection


HIV pathogenesis

Figure 65-8

From Murray et. al., Medical Microbiology 5th edition, 2005, Chapter 65, published by Mosby Philadelphia,,


HIV pathogenesis Table 65-3

From Murray et. al., Medical Microbiology 5th edition, 2005, Chapter 65, published by Mosby Philadelphia,,


AIDS epidemiology Box 65-3.

From Murray et. al., Medical Microbiology 5th edition, 2005, Chapter 65, published by Mosby Philadelphia.


AIDS-associated secondary infections Table 65-5.

From Murray et. al., Medical Microbiology 5th edition, 2005, Chapter 65, published by Mosby Philadelphia.


Anti-Retroviral Therapy •Inhibitors of Reverse Transcriptase •Protease Inhibitors (1996)


The use of antiviral drugs leads to resistant virus

Fields Virology 4th edition, 2002, Chapter 15, Lippincott, Williams and Wilkins, 2002


Anti-Retroviral Therapy •Inhibitors of Reverse Transriptase •Protease Inhibitors (1996) •HAART: Highly Active Anti-Retroviral Therapy A combination of three or more of the above classes of drugs

Beth D. Jamieson, Ph.D.


Evidence for Viral Reservoirs

Plasma Viral RNA

Primary Infection Viral Rebound Viral Setpoint Cessation Of HAART

HAART

50 copies Infection


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