negative strand RNA Viruses

RNA virus with DNA complete in lifecycle.

RNA -> cDNA by rtPCR - RT origin in retroviruses

phylogenetic analysis of conserved regions of the retrovirus polymerase gene An AS sequence alignment was constructed of residues in domains 1 to 4 and part of domain 5 of reverse transcriptase. an unrooted neighbor-joining phylogenetic tree was constrcuted by using the PHYLIP package

form:

slightly variable - slightly differences

• sperical, pleomorphic, 80-100nm

Envelope:

• lipid bilayer

• env protein (SU and TM)

Capsid

• CA protein, isometric or conical

• formes inner particle where RNA is. normally + strand, 2 copies

• single stranded (+) RNA, dimeric

• 5’ cap strucutre, 3’ poly A tail - protection

inside the capsid many proteins, which are important for lifecycle

• Alpharetrovirus, avian leukosis virus; type C morphology

• Betraretrovirus, mouse mammary tumor virus (MMTV); type B morphology

• gammaretrovirus, murine leukemia virus MLV

• deltaretrovirus, bovine leukemia virus (BLV)

• lentivirus, human immunodefiency virus 1 (HIV-1)

• spumavirus, simian foamy virus, formlerly calles HFV

virus needs to transfer the proteins from one cell to the other one, different types of viral particles

complexe ones have accessory genes

structural proteins: matrix proteins, capsid, nucleocapsid

esessential for replication: protease, RT, integrase (for + strand; RNA viruses = long polyprotein cleaved)

very simple ones: glycoprotein: entry

long term no repeat requences -> critical for replication

accessory genes: regulate gene expression, assembly and replication -> more complex viruses express more of these.

important of Karakul sheep to iceland from germany in the 1930s also brought several pathogens including maedi-visna virus (a lentivirus) and scrapie (prion)

• maedi causes a slowly progressive intestinal intestial pneumonia of adult sheep

• visna is a slowly progressive encephalomyelitis caused by the same virus

• mainly pneunomia

• feline immunodefiency virus was first isolated by pederson et al 1986, from a domestic cat

• clinical presentation is characterized by chronic opportunistic infection and neurological disease

• FIV has been viewed as a potential small animal model for HIV due to similarities in genome organisation, replication and cellular tropism

• overall seroprevalence of 11.04% worldwide

• acquired immunodeficiency syndrome is a term which applies to the most advanced stages of HIV infection. it is defined by the occurence of any of more than 20 opportunistic infections of HIV related cancers

• according to estimates by WHO and UNAIDS, 36.7mio people were living with HIV globally at the end of 2016. that same year, some 1.8 mio became newly infected and 1 mio died of HIV related causes.

• left without treatment, the majority of people infected with HIV will develop signs of HIV related illness within 5-10y, although this can be shorter. 10-15y, but sometimes longer - infected people will die with lower age due to stroke, heart disease

• HIV can be transmitted through unprotected sexual intercourse (vaginal or anal) and oral sex with an infected person; transfusion of contaminted blood; and the sharing of contaminated needles, syringes, surgical equipment or other sharp instruments. it may also be transmitted between a mother and her infant during pregnancy, childbirth and breastfeeding.

• tissues from patients were cultured and then searched for viruses, including retroviruses (using a RT assay). one RT assay turnes positive after three weeks of culture, but this was associated with the death of the cells, not characteristic of the retroviruses of the day. the virus was named lymphadenopathy-associated virus LAV

• electron microscopy showed the presence of a lentivirus (a genus in the familiy retroviridae)

the phylogenetic relationships of primate immunodeficiency retroviruses are shown in black lines, including HIV1 and HIV2 in humans. the precurser of HIV1 is a virus found in the chimpanzees, SIVcpz which is in turn the product of a recombinant between SIVrcm and SIVgsn

infects especially Tcells

HIV binds to surface of DC and than naturally infect T cells

female genital tract -> lymph -> draining lymph node:

-> non draining lymph node

-> spleen

-> gastrointestinal tract

-> Thymus

-> CNS -> once it is here can spreas everywhere. can infect other cell types

mucosal CD4+ tcell dont recover properly

1. fusion of HIV to the host cell surface

2. HIV RNA, RT, integrase and other viral proteins enter the host cell

   crucial step -> RNA converted to cDNA

3. viral DNA is formed by RT

4. viral DNA is transported across the nucleus and integrates into the host DNA

5. new viral RNA is used as genomic RNA and to make viral proteins

6. new viral RNA and proteins move to the cell surface, and a new immatrue HIV formes

7. virus is released. viral protease cleaces new polyproteins to create mature infectious virus.

HIV- integrated in the host cell genome - why hard to get rid of

• receptor binding and membrane fusion

• internalizing and uncoating

• RT of the RNA genome to form double stranded linear DNA

• nuclear entry of the DNA

• integration of the linear DNA to form the provirus

• transcription od the provirus to form viral RNA

• splicing and nuclear export of the RNA

• Translation of the RNA to form precursor proteins

• assembly of the virion and packaging of the viral RNA genome

• budding and release of the virions

• proteolytic processing of the precursors and maturation of the virions

• RT is an enzyme that synthesizes DNA using RNA as a template

• RT was discovered in 1970, seperatly by howard temin and david baltimore

• RT is an RNA directed DNA polymerase which was discovered in:

  • rauscher mouse leukemia virus (R-MLV) (baltimore)

  • rous sarcoma virus (temin)

• rt is an enzyme used to generate complementary DNA from RNA template

• RT has 3 biochmical activities:

  • RNA-dependent DNA polymerase activity

  • ribunuclease H

  • DNA-dependent DNA polymerase activity

• ss-RNA-> ds

  • -> experimental applications - molecular clonign, RNA sequencing, PCR, genome analysis

  • -> integration into host genome (retroviruses)

• HIV-1 RT is a multifunctional enzyme that is responsible for copying the ss RNA genome into dsDNA

• HIV1 RT is a heterodimer consisting of p66 and p51 subunits. both subunits are derived from a gag-pol polyprotein, which is cleaved by the viral protease

• RT contains a DNApolymerase that can copy either an RNA or DNA template and a ribonuclease H (RNaseH) activity that cleaves the RNA strans in RNA:DNA hybrids

• in addition to degrading the RNA genome after it has been copied to DNA, the RNase H cleavages define the ends of the ds genome that are the substrates for integration into the host genome

• in vivo studies demonstrate that inactivation of RNase H results in non-infectious virus partivles

• a distingushing feature of retrovirus replication is the insertion of a DNA copy of the viral genome into the host cell chromosomes ater RT

• the integrated viral DNA (provirus) serves as a template for the synthesis of viral RNAs and is maintained as part of the host cell genome for the lifetime of the infected cell

• retroviral mutants deficient in the ability to integrate generally fail to establish a productive infection

• the integration of viral DNA is catalyzed by IN, a 32kD protein generated by PR-mediated cleavage of the C-terminal portion of the hiv Gag-Pol polyprotein

• AIDS-related mortality has decreased by more than 80% since the introduction of combination therapy in the mid 1990s

• 30 antiretroviral drugs and druf combinations in 6 different classes are approved for the treatment of HIV1 infection, with more in clinical development.

• rates of spontaneuos mutation critically determine the genetic diversity and evolution of RNA viruses

• the high level of genetic diversity of the HIV1 virus grant in the ability to escape the immune system, to rapidly evolve drug resistance and to circumvent vaccination strategies

• HIV1 RT contributes only 2% of mutations, whereas 98% result from editing by host cytidine deaminases of the A3 family

• the HIV1 mutation rate tends to be higher in patients showing normal disease progression than in those undergoing rapid progressuin, emphasizing the negative impact on viral fitness of hypermutation by host cytidine deaminases

• in addition to the CD4+ memory Tcell subsets found in the peripheral blood, the lymphoid tissue, gut-associated lymphoid tissue, and the central nervous system are significant anatomic focuses of latent HIV infection. additional tissues, such as the lungs an skin may also contain cells harboring latent HIV

• additionally unknown potential reservoirs remain to be defined

• each of these tissues contains unique celltyoes that contribute to the persistance of HIV during effective ART