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Replication
and
Persistence
of

Primate
Lentiviruses





Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität
Erlangen-Nürnberg





zur
Erlangung des Doktorgrades Dr. rer. nat.





vorgelegt von


Andrea Kirmaier
aus Memmingen








1

Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der
Friedrich-Alexander Universität Erlangen-Nürnberg

































Tag der mündlichen Prüfung: 18.03.2011


Vorsitzende/r der Promotionskommission: Prof. Dr. Rainer Fink
Erstberichterstatter: Prof. Dr. Lars Nitschke
Zweitberichterstatter: Prof. Dr. Welkin E. Johnson


2 I SUMMARY
Cross-species transmission of simian immunodeficiency viruses (SIV) has created
both human immunodeficiency viruses, HIV-1 and HIV-2, the causative agents of
AIDS. The pandemic caused by HIV-1 has cost an estimated 25 million lives and
another 33 million are infected, with neither a cure nor a vaccine in sight. In central
Africa, humans are in ever-closer contact with monkey species that naturally harbor
SIV, so there is a constant danger of additional strains of HIV emerging in humans.
The determinants of cross-species transmission are still poorly understood, but host
species restriction factors are thought to potentially play an important role in de novo
transmission of viruses between species. In vitro, the restriction factor TRIM5α
inhibits retroviruses in a species-specific manner, but its influence on in vivo
pathogenesis was unknown, especially with respect to cross-species transmission.
The present study took advantage of the existence of the three functionally
distinct rhesus macaque TRIM5 alleles on restriction of related primate lentiviruses
was examined. First, restriction of a diverse panel of primate lentiviruses by rhesus
TFP CypAmacaque TRIM5 was confirmed in vitro: TRIM5 and TRIM5 both restricted all
Qof the macaque-unrelated SIV strains tested, whereas TRIM5 had no effect on any
of them. Then, the correlation of TRIM5 genotype and viral loads in two cohorts of
rhesus macaques experimentally infected with SIV from sooty mangabeys (SIVsm)
TFP CypAwas evaluated. Viral loads in TRIM5 or TRIM5 homozygous and
TFP CypATRIM5 /TRIM5 heterozygous macaques were over 1000-fold lower than those
Qfrom TRIM5 homozygous animals. In both cohorts, some monkeys with the
protective TRIM5 alleles developed high viral loads months into the infection and
viral clones from these animals showed signs of escape from restriction by TRIM5
through mutations in the viral capsid, which is the target of TRIM5. In vitro tests
confirmed these mutations to be adaptations to TRIM5.
The results indicate that allelic variation in the rhesus macaque TRIM5 gene
results in differences in susceptibility to infection and viral replication in the early
stages of cross-species transmission of SIVsm. Consistent with this observation,
SIVs that are pathogenic in rhesus macaques required adaptations in the viral capsid
protein to overcome suppression by two distinct types of TRIM5 allele.
3 II ZUSAMMENFASSUNG
Artenübergreifende Übertragung von Affen-Immunodefizienzviren (SIV) brachte
beide menschlichen Immunodefizienzviren, HIV-1 und HIV-2, hervor, welche AIDS
verursachen. Die von HIV-1 ausgelöste Pandemie kostete bisher geschätzte 25
Millionen Menschenleben und weitere 33 Millionen Menschen sind infiziert, wobei
weder ein Heilmittel noch ein Impfstoff in greifbarer Nähe sind. In Zentralafrika sind
Menschen in immer engerem Kontakt mit Affenarten, die SIV in sich tragen; dadurch
besteht die ständige Gefahr, dass neue HIV-Stämme in Menschen auftreten
könnten. Die bestimmenden Faktoren artenübergreifender Übertragung sind bisher
weitgehend unbekannt, aber wirtsspezifische Restriktionsfaktoren sollen
möglicherweise eine wichtige Rolle spielen bei der de novo-Übertragung von Viren
zwischen Arten. In vitro inhibiert der Restriktionsfaktor TRIM5α bestimmte
Retroviren, aber sein Einfluss auf Pathogenese in vivo war bislang unbekannt,
besonders im Hinblick auf artenübergreifende Übertragung.
Die vorliegende Studie zog einen Vorteil aus der Existenz dreier funktionell
verschiedener Allele von Rhesusaffen-TRIM5α auf die Restriktion verwandter
Lentiviren von Primaten untersucht. Zunächst konnte die einer Gruppe
von Primatenlentiviren durch Rhesusaffen-TRIM5α in vitro bestätigt werden:
TFP CypATRIM5 und TRIM5 restrigierten alle SIVs, die nicht von Rhesusaffen
Qstammten, während TRIM5 keinerlei Effekt hatte. Dann wurde der Zusammenhang
zwischen TRIM5-Genotyp und Viruslast in zwei Rhesusaffen-Kohorten untersucht, in
denen die Tiere experimentell mit SIV von Ruβmangaben (SIVsm) infiziert wurden.
TFP CypA TFP CypAViruslasten in TRIM5 - bzw. TRIM5 -homozygoten, oder TRIM5 /TRIM5 -
Qheterozygoten Affen, waren über tausendfach niedriger als in TRIM5 - homozygoten
Tieren. In beiden Kohorten entwickelten Affen mit den protektiven TRIM5-Allelen
mehrere Monate nach der Infektion hohe Viruslasten. Virusklone dieser Tiere wiesen
Zeichen von Resistenz gegen TRIM5 auf. Mutationen im viralen Kapsid, welches von
TRIM5 angegriffen wird, waren der Auslöser für die beobachtete Resistenz. In vitro-
Tests bestätigten, dass es sich bei den Mutationen um Adaptionen an TRIM5
handelte.
Die Ergebnisse zeigen auf, dass Allelvariationen im TRIM5-Gen von
Rhesusaffen zu unterschiedlicher Suszeptibilität für Infektion und Virusreplikation in
den Frühstadien von artübergreifender Übertragung von SIVsm führen. Mit dieser
4 Beobachtung übereinstimmend waren in pathogenen SIVs in Rhesusaffen
Adaptionen des viralen Capsidproteins notwendig, um Restriktion durch zwei
bestimmte TRIM5-Allele zu umgehen.
5 III INTRODUCTION
III.1 Retroviruses: from discovery to infection
The discovery of what are now known as retroviruses dates back over 100 years. In
1908, Danish scientists Vilhelm Ellerman and Olaf Bang described a transmissible
form of leukemia in chickens (39). Three years later, Peyton Rous discovered that
solid tumors could be induced in healthy chickens by transfer of tumor tissue from
diseased chickens (114). Both were observations of disease caused by cell-free,
thfilterable extracts from animals’ tissues. By the mid 20 century, advances in
scientific techniques had made it possible to identify the pathogenic agents as
viruses carrying a RNA genome; accordingly they were referred to as “RNA tumor
viruses”. In 1970, Howard Temin and David Baltimore independently discovered the
defining trait of this group of viruses: the reverse transcriptase enzyme which confers
the ability to transcribe their RNA genomes into a DNA intermediate (137), (10). The
DNA intermediate is then integrated into the host genome and serves as a template
for new viral RNA and proteins. This retrograde flux of genetic information gave the
RNA tumor viruses their current name “retrovirus”. Members of the family
Retroviridae are enveloped viruses carrying a dimeric, monopartite, single-stranded,
positive-sense, m7G5′ ppp5′ Gmp-capped, 3’-polyadenylated, polycistronic RNA
genome.
Today, a total number of over 120 retroviruses is known (29), categorized into
seven genera: alpha-, beta-, gamma-, delta-, epsilon-, lenti- and spumaviruses.
Retroviruses can be found in all vertebrates with the most studied representatives
being the ones isolated from mammals and birds (70). Exogenous lentiviruses exist
in five mammalian groups: primates, bovids, equids, felids and caprids/ovids (70).
The most commonly known retrovirus is likely the human immunodeficiency virus
type 1 (HIV-1), a primate lentiviruses that causes acquired
syndrome (AIDS) in its host (12, 41).
All primate lentiviruses (PLV) share a similar genetic arrangement and
morphology: the nine genes encoded in a lentiviral genome are arranged over all
three open reading frames (ORF) (figure 1A+B). Multiple splice sites allow for the
processing required in order to produce the nine different RNA messages. Two of
the nine genes, tat (trans-activator of transcription) and rev (regulator of virion), are
split up into two exons, each, whereas the remaining seven are single-exon genes.
Three genes encode more than one protein: The Gag polyprotein (group-specific
6 antigen) is cleaved into matrix (MA), capsid (CA), p2, nucleocapsid (NC), p1 and p6
proteins (figure 1C). Pol (polymerase) contains a protease (PR), a reverse
transcriptase (RT), an RNAse and integrase (IN). The glycoproteins gp120 and gp41
make up the Env (envelope) polyprotein. tat, rev, vif (viral infectivity factor), vpu (viral
protein u)/vpx (viral protein x), vpr (viral protein r), and nef (negative factor) are the
accessory genes. On either end, the viral RNA is flanked by repeat sequences,
which are important for IN-mediated integration of the viral DNA into the host
genome as well as for subsequent transcription.


Figure 1: Genomic organization of two lineages of primate lentiviruses, (A) the SIVcpz/HIV-1
lineage and (B) the SIVsm/SIVmac/HIV-2 lineage. Coding regions for the nine genes are
distributed over all three ORFs. Both viral lineages share the structural gag, pol and env genes,
as well as the accessory genes vif, vpr, tat, rev and nef, with one exception: while the
SIVcpz/HIV-1 lineage encodes vpu, the SIVsm/SIVmac/HIV-2 as well as other SIVs carry vpx.
Though a common gene, vpr is located in different locations between the two lineages. The long
terminal repeats (LTR) are only present in retrotranscribed viral genomes. (C) Upon maturation,
the Gag polyprotein is cleaved into six separate proteins: MA, CA, and NC, the p2 and p1 spacer
proteins, and p6, which is a late protein essential for assembly and budding. CA consists of a N-
terminal and a C-terminal domain, divided by the major homology region (MHR).

A mature virion is an 80-100 nanometer (nm) wide sphere enveloped by a lipid bi-
layer that is derived from the host cell membrane (figure 2). The membrane contains
seven to twelve trimeric envelope spikes, with each trimer made up from three
7 molecules each of gp120 and of the transmembrane protein gp41. The inside of the
lipid bi-layer is lined with MA. Contained within this outer shell is the viral core, made
up of a CA lattice. The characteristic cone shape of the lentiviral core is achieved
through directed assembly of approximately 250 hexameric and twelve pentameric
CA units (106). Within the CA core, the two copies of the RNA genome are
associated with NC. The virally encoded proteins RT, RNAse, IN, PR, Nef, and Vpr
are also found in the core (21).


Figure 2: Schematic overview of a lentivirus. The lipid bi-layer (gray) contains seven to twelve
copies of the envelope trimer, which is made up of three gp120 and gp41 moieties (blue),
respectively. Matrix proteins (dark red circles) line the inside of the membrane. The viral core,
made up of capsid hexamers and pentamers (light blue), contains the RNA genome (dark
green), nucleocapsid (light green), and the protease (dark gray pacman), integrase (light gray
triangles) and reverse transcriptase/RNAse enzymes (gray horseshoe).

The PLVs use the Cluster of Differentiation 4 (CD4) cell-surface glycoprotein as main
receptor. CD4 is expressed on T 1- and T 2-type T helper cells, regulatory T cell h h
subpopulations, monocytes, macrophages and dendritic cells. Members of the C-C
and CXC chemokine receptor families (CCR, CXCR) serve as main co-receptors
(18, 27, 28, 35,Choe, 1998 #1706, 100). Both, CD4 and one of the co-receptors
must be present on a cell to make it susceptible to lentiviral infection. In the
HIV/SIVmac lineage, CCR5 and CXCR4 serve as co-receptors. CCR5 is mainly
expressed on monocytes, macrophages and dendritic cells, while CXCR4 is present
on many cell types and in many tissues. Infection of a susceptible cell is a multi-step
+process. First, the virus attaches to a CD4 cell via interactions of gp120 with CD4
8 on the cell surface (117). Binding to the co-receptor trigger conformational changes
in the envelope spike, exposing the fusion peptide within gp41, which is inserted into
the host cell membrane. The formation of the 6-helix-bundle, in which previously
extended helices within gp41 fold back onto each other, brings the viral and cellular
membranes into close proximity so they can fuse and the viral core is released into
the host cytoplasm. Once inside the cell, RT and RNAse begin to transcribe the
single-stranded viral RNA into a double-stranded DNA inside the CA core. The fully
retro-transcribed DNA then associates with several viral proteins to form the pre-
integration complex (PIC). Lentiviral cores use the microtubular cytoskeleton to travel
towards the nuclear membrane, where the core falls apart and the PIC is actively
transported across the intact nuclear membrane. This characteristic ability enables
lentiviruses to infect non-dividing cells. In dividing cells, Vpr, which is associated with
PICs, actually causes a G2 cell-cycle arrest of the infected cell. This is thought to
greatly increase viral multiplication, since the cellular transcription and translation
machinery is mostly available for the virus. Inside the nucleus, IN integrates the viral
DNA into the cellular genome so that it becomes a permanent and stable part of the
host genome. The viral LTRs then serve as a promoter recognized by the cellular
RNA polymerase II, which translates the DNA into m7G5′ ppp5′ Gmp-capped, 3’-
polyadenylated mRNAs, albeit at a low level (135). Since the cellular mRNA export
machinery only allows for fully spliced mRNAs to be exported to the cytoplasm, a
subset of viral genes is translated first. Binding of one of the early proteins, Tat, to a
Tat-responsive element in the LTR strongly enhances RNA polymerase processivity,
facilitating the accumulation of full-length transcripts of viral RNA. These contain
another cis-element towards the 3’ end, which is recognized by the accessory Rev
protein. Rev facilitates the nuclear export of any spliced or unspliced RNA containing
the Rev-responsive element. The spliced, partially spliced and unspliced mRNA
transcripts serve different purposes: the first two are being translated into the
accessory proteins and the Env polyprotein. The latter serves as template for the
Gag and Pol polyproteins, and is incorporated, together with the polyproteins and
some of the accessory proteins, into newly forming virions at the cell membrane.
After budding from the cell membrane in this immature state, virus maturation is
induced by proteolytic cleavage of the Gag and Pol and by structural
rearrangements. Only fully matured virions are capable of subsequent rounds of
infection.
9 III.2 Primate lentiviruses: HIV and SIV
The first primate lentivirus identified was HIV-1 in humans in 1983 (12), followed
shortly by the discovery of a SIV in rhesus macaques (Macaca mulatta, SIVmac) in a
captive colony in the US in 1985 (32). When numerous additional SIV species were
discovered in feral monkey species on the African continent, phylogenetic analyses
eventually revealed that both human immunodeficiency viruses, HIV-1 and the later
isolated HIV-2, were derived from naturally occurring SIVs. All HIV-1 strains
originated from different strains of SIVs of chimpanzees (Pan troglodytes, SIVcpz),
which must have been transmitted to humans on at least three occasions, giving rise
to the M, N and O groups and the various clades of HIV-1 group M viruses (66).
SIVcpz was also transmitted to gorillas (Gorilla gorilla), giving rise to SIVgor (136,
142). HIV-2, in contrast, is derived from SIVs of sooty mangabeys (Cercocebus atys,
SIVsm) (50), which were transmitted to humans at least seven times (23). The
transmission events leading to the emergence of HIV-1 and HIV-2 in humans are
ththought to have happened in the early to mid 20 century (151).


Figure 3: Worldwide prevalence of HIV/AIDS in the adult population by country in 2007. Color
code indicates low prevalence (< 0.1% of the population) in gray color up to high prevalence
(15.0 – 28.0% of the population) in dark red color. Adapted from: 09 AIDS epidemic update,
UNAIDS/WHO.

10

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