Retroviruses

Historically, retroviruses were divided into groups based on their morphology in negatively-stained E.M. pictures:

• A-type: Also known as 'intracisternal particles'. Non-enveloped, (non-infectious???) immature particles only seen inside cells, believed to result from endogenous retrovirus-like genetic elements.

• B-type: Enveloped, extracellular particles with a condensed, acentric core and prominent envelope spikes, e.g. MMTV.

• C-type: As B-type, but with a central core and barely visible spikes - e.g. most mammalian and avian retroviruses (MLV, ALV, HTLV, HIV).

• D-type: Usually slightly larger (to 120nm) and spikes less prominent, e.g. MPMV.

By and large, molecular genetic studies have borne out these morphologic differences, but have also largely replaced them - most comparisons now made on the basis of sequence conservation.

Retrovirus Structure:

All the above proteins are essential for replication; some retroviruses also encode additional essential and non-essential proteins.

Retroviruses have enveloped particles, somewhat variable in size/shape but ~100nm diameter. The envelope carries a virus-encoded glycoprotein, which forms spikes in the membrane. There are certain structural/functional similarities between the envelope glycoprotein and the influenza haemagglutinin (N.B: NO SEQUENCE SIMILARITIES). The mature protein is cleaved into 2 polypeptides:

• the outer envelope glycoprotein (SU), the major antigen of the virus, responsible for receptor binding, linked by disulphide bonds to:
• the trans-membrane glycoprotein (TM), holds the SU protein in the envelope, responsible for membrane fusion

Inside the membrane is the matrix (MA) protein, rather amorphous. This largely obscures the capsid (CA), which is believed to be icosahedral. CA is the most abundant protein in the particle (~33% total weight). Inside the capsid is the core = RNA genome+NC protein+RT+IN. This is usually a conical, electron-dense structure clearly visible in -ve stained E.M. pictures (matrix and capsid appear amorphous).
Turner B.G., Summers M.F. (1999) Structural Biology of HIV. J.Mol.Biol. 285: 1-32.

Genome:

1. They are the only viruses which are truly diploid.
2. They are the only RNA viruses whose genome is produced by cellular transcriptional machinery (without any participation by a virus-encoded polymerase).
3. They are the only viruses whose genome requires a specific cellular RNA (tRNA) for replication.
4. They are the only (+)sense RNA viruses whose genome does not serve directly as mRNA immediately after infection.

Some retroviruses have additional genes:

Sequence features of retrovirus genomes:

Replication:

• Ecotropic: Infect only mouse cells.
• Xenotropic: Infect only non-mouse cells (e.g. rat, hamster).
• Amphotropic: Infect both mouse and non-mouse cells.

Interference between an exogenous virus and an endogenous virus of the same receptor specificity results in 'interference groups' of viruses (e.g. ALVs). In recent years, a number of different retrovirus receptor molecules have been identified: Sommerfelt M.A. (1999) Retrovirus receptors. J.Gen.Virol. 80:3049-3064. See also: Restriction factors: a defense against retroviral infection Bieniasz P.D. (2003) Trends in Microbiology 11: 286-291.

It is probable that receptor binding results in conformational changes in the glycoprotein spike, revealing the (previously masked) fusion domain in the TM protein and resulting in fusion of the virus envelope with the cell membrane. Penetration and uncoating are poorly understood, but it is now known that uncoating is only partial, resulting eventually in a core (nucleocapsid) particle within the cytoplasm. Reverse transcription occurs inside the ordered structure of this core particle - with the reactants (RT + RNA + nucleotides) free in solution, reverse transcription is initiated but cannot be completed, and aborts soon after.

Reverse Transcription:

Reverse Transcription: The Movie

The d/s DNA product formed by this reaction is known as the provirus (c.f. 'prophage') and differs from the vRNA in being longer by one U3,R,U5 sequence. As a result, there is a direct repeat of this sequence present at each end of the provirus genome, and these are known as the long terminal repeats (LTRs). Three forms of provirus DNA are found in all infected cells:

It is not clear how these are related to one another, but the circles probably form by intracellular ligation. The linear and 2-LTR circle forms are infectious (unlike the (+)sense vRNA!). Reverse transcription occurs in the cytoplasm, after which the provirus DNA migrates into the nucleus.

Integration:

Catalysed by the IN polypeptide (part of the RTase complex). Integration is a highly specific reaction with respect to the provirus, but random with respect to host cell DNA. Formerly, it was thought that the 2-LTR circle was the substrate for integration, but it is now believed that the linear form (probably the direct product of reverse transcription) is the actual substrate used.

The ends of the LTRs consist of inverted repeats of 4-6 bp. These are brought together to form a cleavage site for IN and are cleaved to form a staggered cut. This molecule is then inserted into the host cell DNA. The net result of the integration process is that:

1. The integrated provirus contains 1 or 2 less bases at the end of each LTR
2. The ends of the integrated LTRs always have the same sequence: 5' - TG...CA - 3'
3. 4-6 bp of host cell DNA flanking the integrated provirus are duplicated.

Gene expression:

LTR Structure:

The U3 region of the LTR contains the promoter elements responsible for the initiation of transcription:

In recent years, various LTRs have been intensively studied and dissected by molecular techniques, including:

• nucleotide sequencing (and comparison with cellular promoter elements with known functions)
• nuclease protection studies:
- S1 protection to determine precise transcription start sites - DNAse I protection to determine DNA-binding protein sites
• in vitro transcription studies (reconstitute 'the nucleus' in vitro).

Splicing:

Splicing is regulated by the cellular apparatus which interacts with cis-acting sequences present in the mRNA. The proteins encoded by gag, pol and pro (see below) genes are expressed from a full length genomic RNA (= vRNA). The env protein is expressed from a spliced mRNA. In more complex retro's, e.g. HTLV, Lentiviruses, there are multiply spliced mRNAs are produced. Pattern of splicing in HIV is very complex!
Expression of the protease gene: pro overlaps gag and/or pol, but is still expressed from the same full-length mRNA. Different viruses have a variety of post-transcriptional strategies to do this:

Assembly:

• B- & D-type viruses: Capsid/nucleocapsid assembly occurs in the cytoplasm, later bud out through the cell membrane, acquiring the envelope during this process.
• C-type viruses: Assembly occurs at the cell surface. Thickened patches begin to form in the membrane (env proteins on outer surface, gag proteins inside).

The genome is packaged as the particle buds out through the membrane. With both types, maturation occurs after the particle has budded, by cleavage events catalysed by the protease. Considerable structural changes occur during this process, resulting in the smooth gag shell of the immature particle being completely rearranged and leading to the condensation of the core visible in mature particles. N.B. - some types of retrovirus, notably Lentiviruses, are capable of infecting cells by direct cell-to-cell contact, without the formation of infectious extracellular particles.

Freed EO. (2004) HIV-1 and the host cell: an intimate association. Trends Microbiol. 12: 170-177.

A negatively stained electron micrograph of HIV (C-type) particles.

Retrovirus Genetics:

• High mutation rate - reverse transcription is an error-prone process.
• Recombination - occurs during recombination, promoted by the combination of 2 strands of RNA into 1 d/s DNA provirus.
• Interactions with the host cell - insertional mutagenesis, transduction.

Retrotransposons - endogenous retrovirus-like genetic elements:

Much of the human genome consists of interspersed repetitive DNA sequences. The origin these sequences seems to have been retrotransposition in the germ line, generated by:

• transcription of DNA templates by RNA polymerase
• reverse transcription into DNA by reverse transcriptase
• insertion into new genomic locations, increasing the number of genomic copies of the sequence

~11% of the mammalian genome is composed of retrovirus-like retrotransposons: "transposable elements in which transposition involves a process of reverse transcription with an RNA intermediate similar to that of a retrovirus". Compare this with only ~2.5% of the human genome which encodes unique (non-repeated) genes!
Alu and L1 are the major families of human interspersed repeated DNA, amounting to 10-15% of the genome. Another type of repetitive DNA element consists of retrovirus-like elements (RLEs), or human endogenous retroviruses (HERVs), representing about 7% of the human genome. Their structure closely resembles that of retroviruses, carrying internal sequences with homology to gag, pol, and sometimes env open reading frames flanked by long terminal repeats. Similar sequences occur in all organisms, from yeast to vertebrates. Bannert N, Kurth R. Retroelements and the human genome: new perspectives on an old relation. Proc Natl Acad Sci USA. 101: 14572-14579, 2004.

Phoenix from the ashes: The 5 million year old virus

Where do all these retroviruses come from?

So what have retroviruses ever done for us?

Mi, S. et al.(2000) Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis Nature 403: 785-9.

Pathogenesis:

Retroviral pathogenesis has concentrated on oncogenesis & more recently, AIDS, but retroviruses also cause a variety of haematopoetic and neurological conditions, including:

• Paralysis
• Wasting
• Ataxia
• Arthritis
• Dementia
• Neuropathy

It was recently reported that an ancient retrotransposon insertion is the cause of Fukuyama-type muscular dystrophy, one of the commonest autosomal recessive disorders in Japan (Kobayashi,K. et al, (1998) Nature 394: 388-392). To date this is the only known instance of insertional mutagenesis of the human genome caused by this type of element, but other examples look certain to be discovered in future.

Retroviruses are under active development as vectors for gene therapy.

Pretend to be a Retrovirus!

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