Retro-viruses and HIV Pathology Learning Objectives

Discuss the life/replication cycle of retroviruses, including receptor and coreceptors interaction with viral surface proteins and entry to release steps of viral replication and correlate with disease progression and antiretroviral targets. ( MK PC )

. Viral Entry: 1. For HIV-1, Env gp120 binds to CD4+ receptor on T lymphocytes or monocytes/macrophages. The HIV also needs chemokine receptor interaction to infect the cell. As such, the CP120 will bind to CDCR4 or CCR5. The transmembrane glycoprotein gp41 is responsible for fusion of viral and cellular membranes. CXCR4 is found mostly on T lymphocytes while CCR5 is found mostly on macrophages and monocytes. HIV-1 that interacts with CXCR4 is called X4 (t lymphocyte interaction) while HIV-1 that interacts with CCR5 is called R5 and is mostly linked to macrophage infection. X4 infections are specific for T cells while R5 infections are specific for both T cells and monocytes/ macrophages. b. Viral uncoating; 2. Release of genetic material in the cytoplasm. Partial uncoating and then reverse transcriptase begins c. Reverse transcription: Viral genome converted to double stranded DNA by virion associated RT. This will result in a linear DNA molecule which circularizes and form preintegration complex. RT happens in the cytoplasm d. Migration to nucleus: Double stranded DNA migrates to the nucleus. e. Integration: dsDNA enters into the nucleus, then integrates into the host chromosome at random sites. The viral genome (provirus) becomes part of cellular genome and replicated as long as infected cells divide. f. Transcription: LTR sequences that have integrated have the necessary promoter, enhancer and other signals required for transcription of viral genes by host RNA polymerase2. Full mRNA transcripts are produced that mostly lead to encoding for envelope glycoprotein. HIV can also encode a series of spliced mRNAs that encode viral regulatory proteins that are involved in transcriptional and post transcriptional events. g. Translation: structural proteins translated as polyproteins and processed to individual mature proteins by viral encoded protease. h. Viral assembly and release: Assembly takes place after protein synthesis. After assembly of the virus it will bud through the plasma membrane.

. Describe the structure and genomic complexity of retroviruses and functions of retroviral structural, regulatory (Tat and Rev) and accessory (Vif, Vpr, Vpu and Nef) proteins and correlate with course of infection, immunity, diagnosis and treatment. ( MK PC )

a. Retroviruses contain two copies of +ssRNA. The RNA genome is coated with nucleocapsid protein and RNA protein complex enclosed in capsid protein. The matrix protein will cover the capsid. The whole complex is wrapped by a lipid bilayer cell membrane containing two viral specific envelope glycoproteins. The two glycoproteins are SU (surface) and transmembrane ™. For HIV, the surface glycoprotein is call gp 120 while the transmembrane protein is GP41. b. Retroviral genes: The order of genes for a typical retrovirus is gag-pol-env. The three structural genes are flanked by long terminal repeats which act as a promoter for all HIV genes. a. Gag- gene encodes for structural proteins of the virus and sometimes the protease b. Pol: encodes reverse transcriptase, integrase and sometimes protease. c. Env (envelope) gene encodes the two glycoproteins found in lipid bilayer envelope. In HIV-1, the glycoprotein gp160 is processed to GP 120 and Gp41. GP120 binds to the CD4 receptor and co receptors (CCR5, CXCR4) on T lymphocytes and monocytes/macrophages for entering into the host cell. d. HIV-1 encodes two regulatory proteins TAT and Rex, Rev and four accessory proteins (Vif, Vpr, Vpu, Nef). HIV encodes all of them except for Vpu. Tat acts on the LTR sequences known as TAR to increase the rate of viral transcription and may also stabilize mRNA. REV is a post transcriptional activator and acts on specific sequences known as rev responsive element, within the envelope gene to increase the transport of unspliced and singly spliced RNA's to the cytoplasm for the synthesis of structural proteins. e. Accessory proteins Nef, Vif, Vpr and Vpu play a role in disease progression. i. Nef: Downregulated CD4 expression, required for viral pathogenesis ii. Vif: important for cellular infectivity iii. Vpr: Upregulates virus expression, prevents cell proliferation. Arrest cells in G2/ M phase iv. Vpu: important for virus release and/or assembly.

Explain the mechanisms of genetic variation in HIV and discuss the hurdles in the development of an HIV/AIDS vaccine. ( MK PC )

. HIV-1 has the most error prone reverse transcriptase compared to other retroviruses. The variability mostly comes from the envelope region. The viral tropism or cells targeted may change which could affect the immune systems ability to target the HIV virus. Example could be switching from CCR5 chemokine interactions early on to CXCR4 chemokine interactions later. Additional reasons for genetic variatns include immunologic pressure for change, alteration in cell tropism, replication efficiency. Genetic variation causes problems in vaccine development given that the immune cell may not be able to prevent the wide spread nature of HIV-1.

Explain the three mechanisms by which retroviruses transform cells to an oncogenic state.

. Presence of an oncogene. Required for acute transforming viruses. If the oncogene is expressed from the LTR promoter, there will be rapid and cute onset of malignant disease. Persistent transformation is possible only by those retroviruses that are not cytocidal. No human retroviruses are known to transform this way 2. Insertional mutagenesis. Integration of the retrovirus in the vicinity of particular genes may cause inappropriate expression of the gene, resulting in uncontrolled cell growth. These cellular oncogenes are called proto-oncogenes. Integration is random and rarely occurs near a cellular proto-oncogene and as such, no human cancers are known to be caused by this mechanism 3. Integration of the provirus in the leukemia cells from any one patient which takes place at a unique location on a particular chromosome. The chromosomal location of the provirus is never the same in any two patients, thus the cnacer is not the result of insertional mutagenesis. The transformation results from the continued expression of Tax gene that activates viral transcription and expression of cellular genes. This mechanism is found in HTLV-1, the causative agent of adult T cell lymphoma.

Explain the complex interaction of HIV with the immune system and explain why does the immune system fail to eliminate HIV infection. ( MK PC )

a. HIV infects macrophages first, then CD4 cells via CCR5 receptors. The immune response will response via a humoral antibody production against gp120 and gp41. Likewise, NK activation will try to eliminate the infection early on. However, the virus will reduce its excess production such that it can keep producing in cells without immune recognition (latent phase). After an extended period of time, the virus will accelerate its production and overwhelm the immune system killing most of the CD4 cells (x4 phase of CXCR4) and diminish the immune system making the individual susceptible to opportunistic infections. b. Reason the immune system fails to eliminate HIV: a. Cell to cell spread avoids virus recognition by antibodies b. High rate of mutation results in antigenic variation c. Virus escapes from antibody and CTL(CD8) recognition d. Immune system unable to keep the pace with rapidly mutating virus e. Suppression of MHC 1/MHC2 complex's and interference with cytokine function. f. Persistent infection has been established g. Integration of viral genome into host chromosome h. Impaired T and B cell function, development of opportunistic infections.

9. Describe the pathogenesis of human retroviruses, HTLV-1, II and correlate with diseases caused by HTLV.

a. HTLV-1 (ATL): Adult cell leukemia-highly malignant disease, survival is months however 1% of infected people progress to disease. HTLV associated myelopathy. CSF contains anti HTLV-1. Lymphocytix pleocytosis seen, protein levels elevated, hematologic malignancies, B cell chronic lymphocytic leukemia, immunosuppression. b. HTLV-II: Leukemia-Hairy cell leukemia of B cell origin found as T cell origin called T cell variant hairy cell leukemia. c. Diagnosis: Antibody to ELISA, cross reactivity with HTLV-1 and II. PCR which will specifically be able to differentiate between HTLV-I and II. d. Treatment: No specific treatment

Differentiate between onco-retroviruses and lentiviruses with respect to their course of infection and disease progression. ( MK PC )

a. Onco-retroviruses: Include HTLV-1/HTLV-2. Association with cancer development. HTLV-1 is linked to adult T cell leukemia and lymphoma (ATLL). HTLV-2 is linked to human leukemias including hairy cell leukemia. Oncoviruses do not kill the cell but rather transform it so that new virus is produced indefinitely. Further, they can manipulate cellular signaling mechanisms to enhance pro growth and malignancy potential. b. Lentiviruses: Slow disease causing viruses include HIV-1 and HIV-2. HIV-1 is the major cause of aids world wide. HIV infects and kills T4 cd helper cells and also infects other cells such as macrophages, dendritic cells, langerhan cells and brain cells. The virus is slow and can cause acute symptoms resembling mono but can persist in a latent phase for years until the adaptive immune system can no longer regulate viral replication and CD4 cells die off and AID's and opportunistic infections begin to appear.

Correlate the immunopathogenesis of HIV-1 infection, including transmission, initial replication in target cells, and viral spread with establishment of infection, clinical latency, viral reservoirs and disease progression. ( MK PC )

a. HIV-1 infection usually transmitted through sex, vertically or through blood. Characterized by an acute phase of intense viral replication and dissemination to lymphoid tissues (flu/mono like symptoms), activation of innate and adaptive immune response but an inability to contain the highly replicating virus, a chronic-persistent asymptomatic phase of continued viral replication and immune activation and finally an advanced phase of marked depletion of CD4 T lymphocytes and AIDs diagnosis. b. Infection: Sexual transmission takes place. The target of HIV-1 is CD4 molecules which initially interact with CCR5 on macrophages, langerhand cell and mucosal CD4 T cells. HIV productively replicated in the genital mucosa CD4 T lymphocytes and migrates via draining lymph nodes to GALT and replicated and depletes memory CD4 T cells. HIV will then disseminate to other secondary lymphoid tissue to establish stable viral reservoirs. This is when a majority of patients experience flu like symptoms. Without immune response, there will be an initial viral replication whereby the concentration of HIV could reach 10 million copies. Depletion of CD4 cells in the blood and significant depletion in the GALT. Early control of viral infected cells via CD8 cells. Antibodies generated against TM gp41 and Gp120. Antibodies detected 4-6 weeks after infection. Virus hits a set point where it can continue to replicate without initiating an immune respone. CD4 t cells will rebound an increase in the blood which is suggestive of the clinical latency period. Infected monocytes via CCR5 interaction can be the first infected. They can differentiate into perivascular macrophages and become the resident cells harboring HIV in the CNS. CXCR4 which is specific for T cells is the target for HIV-1 later in the disease progression and accounts for the rapid depression of circulating CD4 cells linked clinically to aids. c. Latency: Long asymptomatic period that has a set point where you will have viral replication but not enough to initiate an immune respone. Mutations, tropism (R5 to X4), activation of infected T cells by mitogens and DNA viruses, reduction in CD4 lymphocytes, immunodeficiency. d. Immunodeficiency: Primary defect in the reduction of CD4 T lymphocytes due to the direct killing of the target cells. Leads to a generalized failure of cell mediated immune responses and can also lead to the development of opportunistic infections. I. Destruction of CD4+ cells: In HIV infected patients, CD4:CD8 ratio is 1 to 0.2. Normal CD4/CD8 ratio is 2.0 2. Depletion of HIV-1 infected CD4+ T cells: No damage to the cells, if there is a very low or no virus production. Active virus production leads to cell death. Humoral or cell mediated response may lead to destruction of infected cells. CD4+ cells which also express gp120 and gp41 on their membrane can be killed by antibody plus complement lysis. Actual number of HIV infected T cells in aids patients is 0.01-1%. Lymph nodes contain 10 to 100 times more virus than circulating CD4 T cells. 3. Depletion of uninfected CD4+ T cells: Syncytia formation, destruction mediated by soluble gp120, interference with T cell maturation, inappropriate programmed cell death. 4. Disease progression of HIV: First cells to be infected are macrophages followed by langerhan cells and mucosa t lymphocytes. The virus replicates slowly and uses these cell types as reservoirs. The infected cells can interact with one another via cell-cell fusion to spread without being targeted by neutralizing antibodies. R5 (CCR5 on macrophages and some CD4 cells) dominates the early HIV infection while X4 (CXCR4) develops during late stages of the infection. The x4 virus interaction is more efficient at transmitting the virus to CD4 cells relative to R5 cells. Once the CD4 cells and macrophages drop too low, the disease will now be classified as AIDS and the patient will be susceptible to opportunistic infections. The viral load is directly related to disease progression. The higher the viral load the faster the disease progression. HIV persists in reservoir cells that are able to resist anti retroviral therapy. This formation of reservoir cells is the biggest hurdle in eradicating HIV. Resting CD4 T lymphocytes remain silently infected with HIV provirus.

Define the term retroviruses and name the different groups of retroviruses and compare and contrast retroviruses from other viruses. ( MK PC )

a. Retroviruses are enveloped, single stranded+ RNA viruses that contain an enzyme known as reverse transcriptase which converts the RNA genome to DNA genome after infecting the host cell. The viral genome can then enter into the genetic information of the host. Retroviruses are not transmittable through air, dust or normal conditions but require intimate contact including sexual, IV drug use and the transfer of blood or mother to child transmission. The route of transmission and the mechanism (use of a reverse transcriptase) is a distinguishing characteristic of + ssRNA retro viruses relative to other kinds of viruses