REVISION ESTRUCTURAL DE LA VIA POR EL METODO AASHTO

Multiple allelic variants of mouse APOBEC3 (mAPOBEC3) exist which differ in their ability to restrict murine retroviruses. The two main alleles are represented by inbred strains BALB/c and C57BL/6, and will be referred to as such (APOBEC3BALB/C and APOBEC3C57BL/6). These two alleles differ in protein sequence, splicing pattern, and expression level, all of which may contribute to differences in viral resistance (see Table 1.2).

There are also fifteen polymorphic amino acids between the two APOBEC3 alleles (Figure 1.6). Genetic analysis from dozens of inbred and wild mouse species identified twenty amino acids that are under positive selection, eleven of which are in this polymorphic group

(Sanville, 2010). Several of these residues are found within two clusters in the active cytidine deaminase domain, and are likely involved in long-term genetic conflict with pathogen targets. These amino acids are directly involved in viral restriction, with the APOBEC3C57BL/6-encoded residues creating a more restrictive enzyme. Genetic analysis also discovered a polymorphism in a splice branch selection site, and an inserted sequence in the more virus restrictive APOBEC3C57BL/6 that acts as an enhancer (Okeoma, 2009). This insertion is a long terminal repeat from a xenotropic mouse gammaretrovirus, and drives elevated mRNA expression levels (Sanville, 2010). Additional post-transcriptional regulation influences translation of APOBEC3BALB/c, as the inclusion of the fifth exon leads to decreased protein production (Li, 2012). This leads to significantly more APOBEC3 protein produced in animals carrying the APOBEC3C57BL/6 allele, further contributing to increased viral restriction. Data included herein support this differential expression as the primary influence on the difference in restriction (see Chapter 2.4.5).

Table 1.2. Murine APOBEC3 Alleles

APOBEC3C57BL/6 APOBEC3BALB/c

Exons 8 9

Polymorphic Residues 15 15

Prototype Strains C57BL/6, NZB/N, RF/J BALB/c, C3H/He, AKR, NIH

Swiss

mRNA Expression High Low

Murine Retrovirus Restriction

High Low

The full length Apobec3 gene encodes nine potential exons. Sequence variations between the two alleles, specifically a difference in number of TCCT repeats in intron 4 and a single nucleotide difference in exon 5, determine the inclusion or exclusion of exon 5. APOBEC3C57BL/6 excludes this exon, which translates into a slightly shorter protein of 49 kDa compared to the 52 kDa APOBEC3BALB/c (Li, 2012). Mice carrying the allele for APOBEC3BALB/c

produce primarily the full-length transcript, but also produce a small proportion of transcripts lacking exon 5. Transcripts have also been found that lack both exon 2 and 5, but are very rare (Sanville, 2010; Okeoma, 2009; Takeda, 2008).

Figure 1.6 Murine APOBEC3 Alleles. Protein sequence alignment of the two allelic variants of mouse APOBEC3. Alleles are labeled by the representative strain encoding each. APOBECBALB retains a fifth exon whereas APOBECC57BL/6 does not due to a putative change in splice acceptor site (Sanville, 2010). Polymorphic amino acids do not directly affect residues critical for catalysis of cytidine deamination, but several amino acid differences in the substrate recognition loop potentially alter the affinity for cytidines in different DNA sequence contexts (Kohli, 2009). Figure credit: Rahul Kohli

Mouse APOBEC3 shows a strong inhibitory phenotype against HIV in vitro. This inhibition is resistant to Vif, and is associated with heavy G-to-A mutagenesis of the viral genome, at a rate of 1 mutation per 100 bp (Bishop, 2004; Hakata and Landau, 2006). These mutations display a pattern of 5’-TT/CC-3’, giving mouse APOBEC3 a distinctive signature compared to human APOBEC3 preferred targets; human APOBEC3G prefers to deaminate at 5’-CCC’-3’; APOBEC3F prefers 5’-CTC-3’, and APOBEC3B prefers 5’-C/GTC-3’ (Bishop, 2004). The beteretrovirus mouse mammary tumor virus is inhibited by both human and murine versions of the protein, and served as the first model to demonstrate the antiviral activity of APOBEC3 in vivo, using APOBEC3-deficient knockout animals (Okeoma, 2007). Although MMTV is not completely restricted by APOBEC3, mice lacking the gene have increased viral loads, faster viral replication, and decreased time to mammary tumor formation. However, this restriction occurs in the absence of G-to-A mutagenesis in vivo, contrary to that seen in an HIV vector restricted by murine APOBEC3 in vitro (Okeoma, 2007). Mouse mammary tumor virus is also differentially restricted by different APOBEC3 alleles, with an identical phenotype compared to Friend MLV (Okeoma, 2009a). Mice expressing APOBEC3C57BL/6 are thus more resistant to several classes of murine retroviruses compared to those expressing APOBEC3BALB/c. This resistance appears to be directly correlated to levels of APOBEC3 expression, as inducing expression in vivo increases restriction in both APOBEC3C57BL/6- and APOBEC3BALB/c- expressing mice (Okeoma, 2009b).

Murine leukemia viruses are also inhibited by murine APOBEC3. This observation was first made before the discovery of APOBEC3, as a genetic factor influencing recovery from F- MLV (see Chapter 1.6.1). A single autosomal gene named Rfv3 (Recovery from Friend virus 3) has long been known to influence neutralizing antibody responses and help control MLV viremia, but the molecular identity was not known until after the discovery of APOBEC3 (Chesebro and Wehrly, 1979). Mice are categorized as either resistant or susceptible to Friend virus, and this pattern matches the expression of allelic variants, with those expressing APOBEC3BALB/c characterized as susceptible (Santiago, 2008; Takeda, 2008). For MLV, APOBEC3 acts as an

intrinsic immune factor, but also affects the adaptive immune system. One theory suggests that restriction by APOBEC3 reduces virus-induced immune dysfunction, thereby promoting neutralizing antibody responses (Santiago, 2010).

APOBEC3’s role in viral restriction extends to other murine retroviruses as well, including Moloney murine leukemia virus. Bone marrow-derived dendritic cells isolated from APOBEC3-/- mice support higher levels of infection than those isolated from mice expressing APOBEC3. This higher level of infection also leads to more rapid development of leukemia (Low, 2009). Another gammaretrovirus, the endogenous AKV virus found specifically in AKR mice, is also restricted by both alleles of APOBEC3 in vitro, under identical expression conditions. APOBEC3 proteins expressed via transfection also caused low-level deamination of AKV genomes, at a rate of approximately 2 mutations per kB (0.2%) (Langlois, 2009).

Evidence suggests that mouse APOBEC3 may have had mutagenic activity in the past, as some clades of endogenous murine leukemia viruses show significant G-to-A mutations consistent with the mouse APOBEC3 editing pattern (Jern, 2007). These mutations may have helped inactivate the viruses as they integrated into the mouse germline. Retrotransposons, particularly the IAPE element, are also potently restricted by mouse APOBEC3 ex vivo, and show low-level mutagenesis (0.1-0.5%) (Esnault, 2008). Mouse APOBEC3 does not deaminate present day exogenous murine leukemia viruses, although the protein is packaged into budding virions and causes inhibition of reverse transcription (Browne and Littman, 2008). Based on the ability for mouse APOBEC3 to restrict and mutate HIV in vitro, it was hypothesized that murine retroviruses have developed mechanisms to resist the mutagenic effects of their species-specific APOBEC3 (Rulli, 2008).

Human APOBEC3G is an incredibly potent antiviral factor of HIVΔVif, but is unable to restrict wild-type HIV. Murine APOBEC3 shows an intermediate phenotype in restricting murine retroviruses such as MMTV and MLV. This inability to fully restrict these viruses suggests a potential Vif-like factor. However, MLV does not encode for any accessory proteins, and the accessory proteins of MMTV have other defined roles in the viral lifecycle. However, MLV does

encode for an elongated form of Gag, known as glycosylated Gag (gGag) (see Chapter 1.5). Without this factor, MLV replicates inefficiently in cells (and mice) expressing mouse APOBEC3, but replicates to wild-type levels in APOBEC3-deficient cells (Kolokithas, 2010). This restriction can be either virion- or cell-associated, and gGag does not prevent packaging of the protein into the virion. However, viruses expressing gGag have more stable viral cores, which may protect the viral reverse transcription complex from APOBEC3 (Stavrou, 2013). Reverse transcription within these viruses proceeds unimpeded, while reverse transcription of gGag-mutant viruses is reduced. The mechanism by which gGag improves capsid stability and prevents APOBEC3 access is unknown.