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Home , Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, Lentivirus

Where do come from and why do they do what they do?

John M. Coffin Tufts University

Tufts University Where did viruses come from? (Actually, I haven’t a clue.)

RNA world

“RNA cell”

Prokaryotes

Eukaryotes Proto cell Why do they do what they do? (Because they can.)

1. However they arose, they have evolved multiple times into a variety of niches. 2. 2 Phases – virion and intracellular. 3. Rely on cell for most or all replicative functions. 4. Virion allows cell-cell and host-host . 5. Their “job” is to direct cells to make more of themselves, not to cause disease (unless it promotes transmission). 6. Long term host- co evolution often leads to a well- adapted, relatively benign interaction. 7. Unlike most other viruses, have left behind a fossil record that lets us study this evolution. The Replication Cycle

Entry via fusion

Adsorbtion to specific receptor (CD4)

Maturation via protease The Retrovirus Family Tree

Virus HFV b e l1 , b e l2 MLV FeLV HERV-C WDSV o rf A , o rf B , o rf C HIV-1 t a t , re v HIV-2 EIAV VMV d u t MPMV s a g n e w e n v MMTV HERV-K IAP ASLV

BLV HTLV-1 t a x , re x HTLV-2

New At least 30 million years ago! Endogenous Retroviruses

1. Remnants of germ line infections by exogenous (infectious) retroviruses.

2. Became fixed in the host . Some confer protection against future infections by the same or similar viruses. A few others have salutary effects. 3. Inherited like normal genes. 4. Present in every vertebrate and many invertebrates. 5. Comprise 6-8% of the human . (More viruses than us). Endogenous Retroviruses

6. Provide a fossil record of pathogen-host interaction unavailable in any other system.

7. Can participate in evolutionary processes as well as inform us about them.

8. Involved in disease in some animals. Humans? Formation of Endogenous

Long terminal repeats (LTR’s) Identical sequence

Endogenous provirus Host-Virus Evolution

++ Host-Retrovirus Evolution Effects of Endogenous Proviruses (Good and Bad)

Syncytin Endogenous Retroviruses and Evolution Endogenous Proviruses as Evolutionary Markers

1. Represent unique and irreversible mutations: • Shared insertions indicate common ancestry.

2. Can be used to provide important evolutionary information: • Estimate time of integration. • Detect and quantitate various kinds of events • Detect various kinds of recombination events.

HERV-K (HML-2) Group

1. First entered genome more than 30 million years ago; successive waves of spread through our ancestors.

2. There are <100 full-length proviruses in each of us, as well as about 900 solo LTR’s. 3. Expressed as transcripts and particles in certain normal tissues and disease states.

3. Mining from the human genome sequence: • 91 full (or near full) length proviruses have been found • Most are fixed in our ; a few are polymorphic.

HERV-K (HML-2) proviruses

• Many have retained ORF

• Insertional polymorphisms

• Could be circulating at low rate (unknown)

• No provirus has been shown to be infectious as-is

Bhardwaj N, Coffin JM. Cell Host Microbe. 2014 Mar 12;15(3):255-9. Evolution of HERV-K (HML-2)

1. From the pattern of mutations in the LTRs of a single provirus, we can also estimate: • its age. • the phylogeny of its host species.

2. We can also detect various kinds of mutation and recombination events. Endogenous Proviruses and Identical Primate Phylogeny integration site 5 Millions of years 8 Human ago Chimpanzee 12 Bonobo 18 2 30 Gorilla > 45 Orangutan Gibbon Old World Stump-tailed macaque Rhesus macaque Baboon African green monkey New World Squirrel monkey Evolution of HERV-K LTRs

5’ LTR 3’ LTR Mutations accumulate •• •• •

Species arise •• •• • •• •• •

Mutations accumulate ••• • •• •• ••••• •••••

5’ LTRs

3’ LTRs HERV-K19q13

74 Human 5' Chimpanzee 5' Bonobo 5' 7 7 Gorilla 5' Orangutan 5' Gibbon 5' Human 3' Chimpanzee 3' 7 9 Bonobo 3' Gorilla 3' Orangutan 3' Gibbon 3' HERV-K10 5' HERV-K10 3' MP, = 5 subst. HERV-K20q

9 Human 5' 4 Gorilla 5' Orangutan 5' Chimpanzee 5' Bonobo 5' Human 3' 7 74 3 Chimpanzee 3' Bonobo 3' 7 Gorilla 3' 9 Orangutan 3' Baboon 3' Af Gr Monkey 3' HERV-K10 5'

HERV-K10 3' MP, = 5 subst. Polymorphism of HERV-K 11q22 Provirus

Donor Biaka Mbuti Maya Quecha Khmer Druze Ami 1 2 3 4 5 6 7 8 910 Adygei

X

Solo LTR

1. Frequent solo LTR formation.

Empty site 2. Far flung groups have empty sites, implying integration more than a few hundred thousand years ago.

Are Any HERVs Still Active? • Can we see differences in provirus content among individuals? Predicted bands Oligonucleotide Donor: probe 1 2 3 4 5 6 7 8 9 10 AseI Sites

* * HERVs Extracted from the Human Genome

• 91 “Full-length” (2-LTR) • Ca 900 solo LTRs • A few polymorphic • Only one is complete (but not infectious)

Subramanian et al. 2013

Rarer proviruses are more likely to be recently integrated, and perhaps retain infectivity.

Are there Recently Active Proviruses?

1. In humans, there is much less evidence for recent activity of endogenous proviruses than in mice.

2. Nevertheless, there have been a few new insertions of HERV-K’s within the last few hundred thousand years and some may still be capable of infection.

3. Such proviruses would be expected to be rare and perhaps confined to a small population.

4. They might be seen in the “thousand genomes” project, a databse of over 2500 human genomes from populations worldwide.

However, the way the “sequencing is done (alignment of short reads to a reference genome) causes rearranged sequences like proviruses, to be discarded. Fortunately, they are saved in a non- aligned “dumpster” file.

Newly Found Proviruses

90

80

70

60

50

40

Frequency Frequency 30

20

10

0 1q41 9q34 6q26 4p16a 12q12 19q12 4p16b 5q14.1 1p21.1 3q11.2 8q24.3 6p22.3 7q36.3 5q12.3 1p13.2 19p12e 19p12d 19p12b 10q26.3 5p15.32 20p12.1 11q12.2 13q31.3 6p21.32 15q22.2 Xq21.33 12q24.32 19q13.43 12q24.31 Provirus

2-LTR Proviruses (All the rest are solo LTRs) Structures of Newly Found 2-LTR Proviruses

Can Xq21,33 be the infectious provirus we are looking for? • Relatively recently inserted • All reading frames intact with no obvious muations • Rare in the population (ca 1.5%) and geographically limited (Africa)

We’ll see…

HML-2 expression in different diseases

• RNA, protein and/or particles seen in many types of disease: – Teratocarcinoma (pictured) – Melanoma – Breast cancer – Lymphoma – Multiple sclerosis – ALS – HIV-1 infection – Normal Placenta – Hypothesis: The expression pattern reflects the natural history of the ancestral virus (parent to child transmission) Scale Bar = 250nm.

Bieda et al. J Gen Virol. 2001; 82:591-6. Potential application of HML-2 expression

• Therapeutics designed to target HML-2 epitopes – HML-2 epitopes could present an unchanging marker for infection if specifically expressed on infected/ diseased cells – Vaccine to elicit anti-HML-2 Env and Gag responses in HIV-1 infection (Sacha et al., 2012; Shephard et al., 2014) – HML-2 Env monoclonal Ab to target breast cancer cells (Wang-Johanning et al., 2012) – Elicit anti-HML-2 Env T-cell responses to target ovarian cancer cells (Rycaj et al., 2015)

HML-2 RNA Reported in HIV-1 plasma

30,000 copies/mL HIV RNA

Contreras-Galindo et al. Genome Res.2013;23(9):1505-13. HML-2 RNA is not detected in HIV-1 plasma

Uninfected HIV-1 infected

Bhardwaj N, Maldarelli F, Mellors J, Coffin JM. J Virol. 2014;88(19):11108-20. HML-2 RNA is upregulated in PBMCs

Bhardwaj N, Maldarelli F, Mellors J, Coffin JM. J Virol. 2014;88(19):11108-20. Possibility of HML-2 recombination

3. Recombination

* * * * * * * *

2. Co-packaged RNA “a” * gag-pro-pol *

* env “ab” “b” * gag-pro-pol-env

1. Complementation

4. Cell produces infectious virus Possibility of HML-2 recombination

• In-vitro recombinant HML-2 viruses are weakly infectious – Recombination between 3 provirus genomes could lead to functional virus (Dewannieux et al., 2006; Lee et al., 2007)

• HML-2 expression in HIV-1 patients may lead to expression of proviruses with functional ORFs – Need to profile which proviruses are expressed

Conclusions

1. Retroviruses have uniquely left behind a rich fossil record of their (relatively) recent (30+ million year) coevolution with their host animals.

2. All of the tens of thousands of known HERVs are incapable of replication and most are very old (>5 million years).

3. Nevertheless, there have been a few new insertions of HML-2’s within the last few hundred thousand years and some may still be capable of infection. (Maybe we’ve found one.)

4. HML-2 expression is upregulated in HIV-infected patient PBMCs (but no virus in blood!).

5. Are any of polymorphic proviruses associated with disease, such as breast cancer? (We’re working on it.)

Acknowledgements -Jenn Hughes -Julia Wildschutte -Ravi Subramanian -Neeru Bhardwaj -Zachary Williams -Meaghan Montesion

Tufts University

Acknowledgements

-Mary Kearney -Ann Wiegand -Jon Spindler -Wei Shao

HIV Drug Resistance Program National Cancer Institute at Frederick

Phylogeny of HML-2 proviruses gag

LTRHs

LTR5B

LTR5A

Subramanian RP, Wildschutte JH, Russo C, Coffin JM. Retrovirology. 2011;8:90. Profile HML-2 expression using RNASeq

• Use RNASeq to: – Identify proviruses that could recombine to make a functional virus – Determine how expression changes between individuals

• Advantages of total RNASeq: – Analyze expression in the context of other genes – No primer bias introduced – Long read lengths allow for unique read placement

Profile HML-2 Expression Using RNASeq

• Validate RNASeq on the teratocarcinoma cell line Tera-1 – Known to express HML-2 RNA and protein in cells – Produces non-infectious HML-2 virions – Perform RNASeq on passage-matched cell and virion samples

Scale Bar = 250nm. Bieda et al. J Gen Virol. 2001; 82:591-6. RNASeq Work Flow: Library Preparation

Extract RNA Library MiSeq Te ra - 1 v i r i o n R N A

Treat RNA Virion cDNA 300 base with DNase made with paired-end Nugen reads qPCR to Ovation v2 verify lack ~50 million of DNA Libraries reads total constructed QC RNA with with TruSeq Trim and NanoDrop total RNA align reads and BioA reagents

Extract Tera-1 cell RNA

Bieda et al. J Gen Virol. 2001; 82:591-6. Comparison of HML-2 Proviruses Detected in Tera-1 Cells and Virions

Tera-1 Cells

Tera-1 Virions

Bhardwaj N, Montesion M, Roy F, Coffin JM. Viruses. 2015;7(3):939-68. HML-2 proviruses expressed in Tera-1 cells

* Underrepresented in Bhardwajsimulation N, Montesion M, Roy F, Coffin JM. Viruses. 2015;7(3):939-68. Expression of 22q11.23 in Tera-1 Cells Expression of 22q11.21 in Tera-1 Cells Comparison of Transcript Levels and LTR Promoter Activity in Tera-1 Cells