RETROVIRUSES Fahareen-Binta-Mosharraf MNS

Retroviruses Probably the most studied group of viruses in molecular biology Enveloped, positive-strand RNA viruses Unique morphology and replication Only virus that exists extracellular Retroviruses are double stranded RNA enveloped viruses mainly characterized by the ability to “reverse-transcribe” their genome from RNA to DNA

Retrovirus “ alternate reverse transcription” from RNA to DNA with transcription from DNA to RNA.

History Rous sarcoma virus: solid tumors in chicken Other cancer causing retroviruses from other animal species (oncogenes) 1981: first human retrovirus: Human T-lymphotropic virus (HTLV-1) 1983: Human immunodeficiency virus (HIV)

Retrovirus Virion Structure Virions measure nm in diameter and contain a dimeric genome of identical positive RNA strands complexes with the nucleocapsid (NC) proteins. The genome is enclosed in a protein capsid (CA) that also contains enzymatic proteins, namely the reverse transcriptase (RT), the integrase (IN) and proteases (PR), required for viral infection. The matrix proteins (MA) form a layer outside the capsid core that interacts with the envelope, a lipid bilayer derived from the host cellular membrane, which surrounds the viral core particle.

Anchored on this bilayer, are the viral envelope glycoproteins (Env) responsible for recognizing specific receptors on the host cell and initiating the infection process. Envelope proteins are formed by two subunits, the transmembrane (TM) that anchors the protein into the lipid membrane and the surface (SU) which binds to the cellular receptors

Retrovirus Genome Organization Based on the genome structure, retroviruses are classified into simple (e.g. MLV, murine leukemia virus) or complex retroviruses (e.g. HIV) All retroviral genomes contain three genes—gag, pol, and env— and are flanked by long-terminal repeats a.gag → nucleocapsid proteins b.pol → enzymes (reverse transcriptase, integrase, and protease) c.env → envelope glycoproteins complex retroviruses, such as HIV, have accessory genes including vif, vpr, vpu, nef, tat and rev that regulate viral gene expression, assembly of infectious particles and modulate viral replication in infected cells.

a. The gag sequence encodes the three main structural proteins: MA, CA, NC. The pro sequence, encodes proteases (PR) responsible for cleaving Gag and Gag-Pol during particles assembly, budding and maturation. b. The pol sequence encodes the enzymes RT and IN. The RT protein possesses three distinct activities: (a) RNA-dependent DNA polymerase activity, responsible for transcribing the two RNA genomes into a single cDNA; (b) RNAse H activity; and (c) DNA-dependent DNA polymerase activity IN is responsible for integrating the proviral DNA into the host cell genome.

c.The env sequence encodes for both SU and TM subunits of the envelope glycoprotein. Major envelope glycoprotein consists of two associated proteins: an attachment protein (HIV gp120) and transmembrane fusion protein (HIV gp41) both cleaved from gp160 precursor

The integrated provirus genome has 5’ and 3’ long terminal repeats (LTR) which each consist of three regions: (a) the U3 region, which functions as a promoter and contains transcriptional enhancer elements and a TATA box; (b) the R region, which is where transcription begins; and (c) the U5 region, which is involved in reverse transcription and carries a tRNA primer-binding site. Other important sequence elements of the provirus are the packaging signal (ψ, psi) and the polypurine tract (ppt), which serves as the site of initiation of positive-strand DNA synthesis during reverse transcription

Complex Retrovirus Additional Genome Organization The main feature of complex retroviral genomes distinguishing them from those of simple retroviruses is the presence of a set of accessory genes whose products are involved in the regulation of transcription, RNA transport, gene expression, and assembly. the Rev and Tat proteins as well as the accessory proteins Vpu, Vif, Vpr, and Nef. Rev is an RNA-binding protein that promotes late phase gene expression. Tat is an RNA-binding protein that enhances transcription. The Nef protein inhibits T-cell activation. Vpu enhances the release of the virus from the cell surface to the cytoplasm during entry. The Vif protein is necessary for replication due to its ability to down regulate the host’s antiviral response

Viral Entry Binding of viral gp120/gp41 to CD4 and a chemokine coreceptor is required for HIV infection of cells. Preference for chemokine coreceptor genetically switches during disease from: Initial: CCR5—on macrophages, dendritic cells, and T cells Later: CXCR4—on T cells Receptor interaction allows for viral entry into cell in 2 ways 1. Receptor mediated endocytosis followed by virion release via a pH decrease release mechanism 2. Fusion at plasma membrane, capsid is released into cytosol

Reverse transcriptase carried in the virion synthesizes a complementary DNA (cDNA) from viral genome. 1 st is to synthesize DNA,forming an RNA-DNA hybrid 2 nd is to degrade RNA from DNA/RNA molecule because it does not degrade ssRNA. This activity is referred to as ribonuclease H activity Synthesizes a complementary DNA strand, forming double- stranded viral DNA. Reverse transcriptase causes a high mutation rate in newly formed viral DNA. Viral integrase catalyzes integration of viral DNA into host nuclear DNA, forming provirus. Provirus is part of host chromosome and vertically transmitted with host DNA and passed on to daughter cells. Formation of HIV provirus

Insertion sites are random Integrase binds the 2 ends of the viral dsDNA genome and brings them together Enzyme targets phosphodiester bonds for cleavage/insertion 2 hanging nucleotides are removed, loss of 2 nt from viral DNA is insignificant 4-6 nt apart of host ssDNA is matched and ligation site is fixed entirely DNA Genome Is Integrated Into Cellular Genome 4-6 NT NT hanging Host DNA Proviral DNA Integrase

The appropriate transcription factors are needed for expression of inserted genome to begin U3 region is the binding site for a number of cellular transcription factors a TATA box is present upstream (U3/R segments) allowing transcription initiation to begin by RNA Pol II Transcription begins at the junction of U3/R and proceeds through the whole genome Proviral DNA Will Be Expressed At Any Time In The Future

Host DNA-dependent RNA polymerase transcribes a full-length (+) RNA copy of the integrated genome and several shorter mRNA copies for individual proteins and polyproteins. At least 2 types of mRNAs are produced in retroviruses mRNAs gag and gag/pol proteins, env proteins are produced from this mRNA Genomic RNA for newly synthesized particles Only Viral messenger RNA (mRNA) and genome replication

(1) Viral genome and reverse transcriptase enter cell. (2) DNA copy synthesized by reverse transcriptase. (3) RNA degraded; second DNA strand synthesized. (8) Final viral assembly and budding take place. (5) With host cell activation, viral DNA is transcribed, yielding messenger RNAs and viral genome RNA. (6) Viral RNAs are translated, yielding viral enzymes (including protease) and structural proteins. Host cell nucleus Host cell genome Site of action of AZT and other reverse Transcriptase inhibitors Site of action of protease inhibitors RNA DNA (7) Viral membrane proteins are transported to host cell membrane. (4) DNA circularizes (unintegrated provirus) or integrase functions to incorporate DNA into host cell genome (integrated provirus). Site of action of antiretroviral drugs under development

Assembly After synthesis of viral proteins, nucleocapsids containing two copies of genome associate with glycoprotein modified plasma membrane and bud off. After budding, protease cleaves gag-pol polyprotein to produce mature nucleocapsid and functional integrase and reverse transcriptase enzymes.

Genetic variation 1.High rate of mutation in retroviruses results from numerous alterations introduced by reverse transcriptase 2.Generation of new HIV strains occurs during the course of infection of an individual, leading to changes in tissue tropism, antigenicity, and other properties of the virus.

Transmission Blood, semen,vaginal secretions Sexual contact Exposure to contaminated blood and blood products From infected mother to her baby perinatally

Pathogenesis HIV primarily infects helper T (T H ) cells and myeloid-lineage cells, which express CD4 and a chemokine coreceptor. a.Lytic infection and latent infection are established in T cells. b.Persistent low-level productive infection is established in macrophages.

Consequences of target cell infection Persistently infected macrophages may act as the major reservoir and distribution vehicle for HIV in the body. Killing of infected CD4 T cells leads to decreased CD4 T cell count and eventually to other immune system abnormalities. Examples include: 1. Decreased proliferation of CD8 T cells (due to reduced interleukin [IL]-2 production) 2. Increased susceptibility to viruses 3. Decreased macrophage function 4. Reduced macrophage function increases susceptibility to fungi, mycobacterial recurrences, and bacterial infections. 5. Changes in the balance of cytokines

Pathogenesis

Stage 1 – Primary symptoms Short, flu-like illness - occurs one to six weeks after infection no symptoms at all Infected person can infect other people Stage 2 – Asymptomatic symptoms Lasts for an average of ten years This stage is free from symptoms There may be swollen glands The level of HIV in the blood drops to very low levels HIV antibodies are detectable in the blood

Stage 3 - Symptomatic The symptoms are mild The immune system deteriorates emergence of opportunistic infections and cancers Stage 4 - HIV  AIDS The immune system weakens The illnesses become more severe leading to an AIDS diagnosis

Mechanisms of HIV Persistence At any given time, most of the lymphocytes in the body are in a resting state. When a lymphocyte encounters a bacterial or viral protein that it is programmed to recognize, it becomes activated and begins to proliferate, generating effector cells that eliminate the invading microorganism. Most of these effector cells die, but some survive and return to a resting state as memory cells. These cells persist indefinitely, allowing effective responses to future challenges with the relevant microorganism. HIV-1 preferentially infects activated CD4 + T lymphocytes, and in the process the viral genome is stably integrated into the genome of the host cells. Generally this leads to the production of new virus particles and the death of the infected cell

However, a small subset of the activated CD4 + T cells that are infected survive long enough to revert back to a resting memory state. Because the expression of HIV-1 genes depends on host transcription factors induced in activated T cells, viral gene expression is automatically turned off when these cells return to a quiescent state. The result is a stably integrated but transcriptionally silent form of the HIV-1 genome in a memory T cell, a cell whose function it is to survive for years in a quiescent state. Upon subsequent reexposure to the relevant HIV antigen, the latently infected cell is reactivated and becomes competent for HIV-1 gene expression and virus production

Diagnosis Acute Primary Infection Once HIV enters the body, the virus infects a large number of CD4+ T cells and replicates rapidly. During this acute phase of infection, the blood has a high number of HIV copies (viral load) that spread throughout the body, seeding in various organs, particularly the lymphoid organs such as the thymus, spleen, and lymph nodes. During this phase, the virus may integrate and hide in the cell’s genetic material. Shielded from the immune system, the virus lies dormant for an extended period of time. In the acute phase of infection, up to 70 percent of HIV-infected people suffer flu-like symptoms. The Immune System Strikes Back Two to 4 weeks after exposure to the virus, the immune system fights back with killer T cells (CD8+ T cells) and B-cell-produced antibodies. At this point, HIV levels in the blood are dramatically reduced. At the same time, CD4+ T cell counts rebound, and for some people the number rises to its original level.

Clinical Latency During this phase, a person infected with HIV may remain free of HIV- related symptoms for several years despite the fact that HIV continues to replicate in the lymphoid organs where it initially seeded. Progression to AIDS The immune system eventually deteriorates to the point that the human body is unable to fight off other infections. The HIV viral load in the blood dramatically increases while the number of CD4+ T cells drops to dangerously low levels. An HIV-infected person is diagnosed with AIDS when he or she has one or more opportunistic infections, such as pneumonia or tuberculosis, and has fewer than 200 CD4+ T cells per cubic millimeter of blood.