Retroviruses Flashcards
RNA viruses that replicate through a DNA intermediate, which efficiently integrates into the genome of permissive cells forming a provirus
Retroviruses
A hallmark of the retrovirus life cycle is the process of
Reverse transcription
The viral RNA genome acts as template for the synthesis of viral DNA by the virus encoded polymerase during
Reverse transcription
Other viruses such as Hepatitis B virus and Cauliflower Mosaic virus utilize
Reverse transcription
The study of Retroviruses has taken on added intensity since the discovery of pathogenic human retroviruses such as
HIV-1 and 2 and Human T-lymphotrophic virus (HTLV-1)
Cause tumors in animals
Oncoretroviruses
Cause a variety of immunological and neurological diseases with a long latency
Lentiviruses
Cause vacuolation of some mammalian cells in culture
Spumaviruses
Surrounded by a lipid envelope derived from the infected cell upon budding of the virus
-Measure approximately 100 nm in diameter
Mature HIV-1 Virion
The virions have spikes emanating from the
Envelope
The spikes are composed of virus envelope proteins, which bind to receptors on permissive cells to initiate
Infection
The core of mature virions is conical in structure in the case of
HIV-1
The core of mature virions contains a round core in the center of the particle in the case of the
Murine leukemia virus
There are additional structures that have been observed for other types of retroviruses. But what is common to them all is that the infectious mature virions contain a condensed core of
Electron dense material
The genome within the core consists of two usually identical single-stranded, positive strand
RNA molecules
The RNA is capped and polyadenylated indicating that it is transcribed by cellular
RNA polymerase II
Most of the cis-acting elements that must be covalently linked to the genome for efficient replication to occur are located at the ends of the
Genome
The trans-acting sequences encoding the viral proteins are located between the
cis-acting elements
The retroviral genome is always organized in the same way as
5’ gag pol env 3”
At each end of the proviral genome (integrated double-stranded DNA) are
Long Terminal Repeats (LTRs)
The long terminal repeats can be funcitonally divided into which 3 parts?
U3, R, and U5
Begins in the 5’ LTR of the provirus at the U3-R border
Transcription initiation
It should be noted that the viral promoter is harbored in
U3
Occurs in the 3’ LTR at the R-U5 border
Polyadenylation
Thus, the single-stranded RNA genome found in virions is shorter than the viral doublestranded
DNA genome
The sequence that is lost during RNA transcription is replaced during
Reverse transcription
They are typically cell surface proteins with the different groups employing different proteins, as such they are important determinants of host range
Retrovirus receptors
A notable retrovirus receptor
-The receptor for HIV
CD4
Found on helper T cells and macrophages helping to account for the HIV host range
CD4 receptor
In contrast to other retrovirus receptors isolated to date, requires a co-receptor
HIV infection
The co-receptors are members of the chemokine receptor family with the most prominent being
CXCR4 and CCR5
Infection is initiated when the envelope glycoprotein SU (surface envelope protein) binds to the corresponding receptor(s) on the permissive
Target Cell
This initiates conformational changes resulting in direct membrane fusion between the membranes of the
Virus and cell
This fusion is mediated by the hydrophobic domain of
TM
After entry into the cell, the process of reverse transcription ensues during which the single-stranded virion RNA is converted into double-stranded DNA by the virus encoded
Reverse transcriptase
In order to generate the LTRs, RT makes two
-Unusual for a DNA polymerase
Strand transfers
Can integrate into non-dividing cells indicating that there is an active transport mechanism for the preintegration complex
HIV-1
On the other hand, other retroviruses integrate during
Mitosis
Integration occurs relatively randomly throughout the genome with a preference for open chromatin such as
Actively transcribed genes
Mediated by the virus encoded integrase (IN) and requires cis-acting attachment sites at the ends of the viral genome
Retroviral integration
During the process of integration, two nucleotides are lost at each end of the unintegrated dsDNA genome and the cellular target sequence is
Duplicated
The retroviral genome is transcribed by cellular
RNA polymerase II
The viral transcripts then undergo RNA processing and
Transport
For all retroviruses at least two mRNAs are produced: 1) a full-length transcript which is translated to yield the
Gag and Gag-Pol polyproteins
For all retroviruses at least two mRNAs are produced: 2) a singly spliced mRNA, which encodes the
Env polyprotein
However, this represents the simplest splicing pattern as exemplified by
Moloney murine leukemia virus
Has a more complex splicing pattern because in addition to the expression of Gag, Pol, and Env, it encodes a number of other proteins translated from alternately processed transcripts
HIV-1
The gag and pol genes are usually in different translational reading frames, yet both are translated from the same unspliced
Viral mRNA
Most of the time just the Gag polyprotein is translated, but a percentage of the time (e.g. for HIV-1 about 5%) a larger Gag-Pol polyprotein is synthesized owing to
Translational frameshifting
One notable exception to translational frameshifting is in the case of MLV in which there is a stop codon between the
Two genes in the same reading frame
Again RNA secondary structure seems to play an important role in suppressing the stop codon in the case of
MLV
Once translated the Gag and Gag-Pol polyproteins are transported through the cytosol to the inner cell membrane where assembly takes place for most
Retroviruses
The polyprotein is proteolytically processed by the virus encoded
Protease
Translated from a singly spliced viral mRNA
Envelope glycoproteins
The Env polyprotein matures in the
Secretory pathway
It has a signal peptide for cotranslational insertion into the endoplasmic reticulum (ER), and there is a hydrophobic domain, which anchors it to the membrane
Env polyprotein
In the ER processing begins including significant glycosylation followed by transport to the
Golgi complex
Here, a cellular protease further cleaves Env polyprotein into
SU and TM
This distinguishes it from Gag and Gag-Pol processing, which is carried out by the
Viral protease
Remain associated via noncovalent interactions
SU and TM
For most retroviruses, assembly and budding occur at the
Cell surface
The Gag and Gag-Pol polyproteins have been targeted to the cell surface through the
Cytosol
Once all of the virus components are localized to the cell membrane, we see
Assembly into virions
Budding then ensues with subsequent viral maturation when the virus protease cleaves the
Gag and Gag-pol polyproteins
This produces the mature and infectious
Virion
Regulation of processing is controlled because protease is only active as a dimer, so it only becomes active
Post-assembly
Most retroviral vectors used for gene therapy trial have been derived from the simple
Murine Leukemia Virus (MLV)
A common strategy used for making the system safer is to separate the
Cis-acting and trans-acting sequences
The gene(s) that you want to transfer is placed into a transducing vector, which contains the
Cis-acting sequences
However, the transducing vector has deletion of some or all of the
Trans-acting sequences
A packaging cell is needed in order to propagate the
Vector virus
This cell expresses the genes encoding the viral protein using non-retroviral signals to direct their
Expression
The transducing vector, in the form of plasmid DNA, is introduced into the packaging cell, where the vector virus RNA is transcribed and packaged into
Vector virions
The vector virus buds from the packaging cell and can be used to target the cells of
Interest
Since the vector virus does not encode the viral proteins, the vector can integrate into the genome of the target cell but can not produce
Progeny virus
Recent trials that exemplify both the potential benefit and the risk of gene therapy are those involving the treatment of
Severe Combined Immunodeficiency (SCID)–X1
SCIDs are a series of genetic disorders in which T-cell differentiation is arrested resulting in severely impaired
Immunity
Ten different molecular defects have now been identified, all of which lead to early death in the absence of
Therapy
Transplantation of MHC compatible hematopoietic stem cells can restore T-cell development and rescue the lives of
SCID patients
An X-linked disease
SCID-X1
Contains early progenitor cells that can repopulate the lymphoid lineage
CD34+ population
