The Importance of the Gag Polyprotein in the Propagation of Retrovirus Particles Introduction.

The virus is known as an anomaly in the biological world. Not even considered alive by many, viruses are some of the most successful specimens in biology. Made up of only a simplistic genome and a protein coat made mostly from host membrane proteins, viruses are able to adapt and evolve quickly to their environments. In the late 19th century, Dimitrii Ivanofsky and Martinus Beijerinck were the first to discover and classify viruses. In 1911, Peyton Rous classified Rous sarcoma virus (RSV) as the first known tumor virus. Because RSV has an RNA genome, it is also the first known RNA tumor virus. In the 1960s, research demonstrated that RSV encoded an oncogene that is not required for its replication. During this time, RSV was used in the theory of the provirus and reverse transcription. Reverse transcription is the process in which the retroviral RNA viral genome is converted into a double stranded DNA copy (Javier and Butel, 2008). The viral genome is integrated into the host genome by viral integrase enzymes. This integrated viral genome is known as the provirus. In RSV, the mRNA encoded by the provirus contains 4 open reading frames: 5- gag-pol-env-src-3. The full-length unspliced mRNA codes for a single polyprotein that is later cleaved into the Gag and Pol proteins (Withers and Beemon, 2010). In RSV, gag codes for the main structural components of the viral protein shell, known as the capsid, and a viral protease, while pol codes for the reverse transcriptase and integrase. The Gag polyprotein is cleaved into multiple protein complexes which include the matrix sequence (MA). MA plays an important role in membrane binding during the assembly of progeny virus particles (Parent et al., 1996). It is now known that the encapsidation of new virus particles by Gag is required for proper virus replication. While there are currently no anti-retroviral drugs that target this mechanism, a better understanding of it could lead to new ways to treat human retroviral infections (Parent, 2011). In retroviruses, the reverse transcribed proviral DNA is integrated into the cellular DNA via the viral integrase. This proviral DNA is transcribed into a number of RNAs, one of which is unspliced and acts as mRNA for the translation of the Gag and Pol polyproteins and as the RNA genome for progeny viruses. In eukaryotic cells, RNA has an inherently short lifespan in the cell, and unspliced RNAs in the nucleus have an especially short lifespan. Scientists were baffled when they found that the unspliced RNA of RSV was stable within the host cell. Arrigo and Beemon (1988) found that a frameshift mutation in RSV caused a 10-fold decrease in unspliced RNA levels, but did not decrease the level of env RNA Barker and Beemon (1991) found that mutations in gag that encoded premature stop codons experienced significantly lower unspliced RNA levels than in wild-type viruses. In 2006, further research designated the 400 nucleotides directly following the natural gag stop codon as the RSV stability element (RSE) (Withers and Beemon, 2010). Myr1E is an RSV mutant where the membrane-binding domain of the Src protein has been added to the N-terminus of the Gag polyprotein, which prevents it from undergoing nuclear trafficking (Parent et al., 2000). This mutant is often used in the lab in studies on the RSV Gag polyprotein. There are multiple compounds that Myr1E is often treated with in the lab during experiments on Gag. A nuclear localization sequence (NLS) is a type of nuclear signal, comprised of amino acids, that targets proteins for selective entry into the nucleus. The NLS that is normally used in experiments on RSV is derived from the frog, Xenopus (Robbins et al., 1991). The NLS is placed within certain amino acid residues in the RSV Gag protein. Leptomycin B (LMB) is a nuclear export inhibitor that inhibits export by targeting the CRM1 export receptor (Kudo et al., 1999). LMB is used to increase nuclear localization of certain proteins in cells that have been transfected with a viral provirus. Certain proteins are treated with a green fluorescent protein (GFP) so that levels of protein within the nucleus can be quantified by measuring fluorescence levels using confocal microscopy (Garbitt-Hirst et al., 2009).

Page |2 Genetic Evidence for a Connection between Rous Sarcoma Virus Gag Nuclear Trafficking and Genomic RNA Packaging The retroviral Gag polyprotein has many functions within the infected host cell. Most notably, it is responsible for encapsidation of the progeny virus particles. The authors of this article, however, suspect that Gag plays a major role in the packaging of gRNA. The authors have set out to prove that proper nuclear of trafficking of the Gag polyprotein is required for proper gRNA packaging and infectivity. Effect of inserted NLS on nuclear trafficking of Myr1E Gag. Earlier research had found that insertion of NLS into the matrix sequence lead to stronger nuclear trafficking, but it remained unknown whether or not the inserted NLS would dominate over the Myr1E mutation and allow proper nuclear trafficking. To measure the amount of Gag in the nucleus, cells transfected with Gag-GFP were examined via a confocal microscopy. The samples were then treated with LMB. Western-blot analysis of the samples was used to ensure that the fluorescence seen in the samples was from stably fused Gag-GFP, and not free GFP protein within the nucleus. It was found that insertion of the NLS had little effect on distribution of the protein within the cell under steady-state conditions, but there was a significant increase of Gag accumulation in the nucleus after incubation with LMB (Garbitt-Hirst et al., 2009). To make controls for the NLS insertions, alanines were substituted for basic residues in NLS. In Myr1E.KR/AA, upstream lysine and arginine codons were mutated to encode alanines, which reduced the degree of nuclear localization after LMB treatment. Myr1E. KR/AA.KKK/AAA also experienced less nuclear trafficking than Myr1E.NLS Gag-GFP (Garbitt-Hirst et al., 2009). It was found that there was a 49% increase of wild-type Gag-GFP nuclear localization after treatment with LMB. Myr1E Gag-GFP only experienced a 4% increase after treatment, indicating that it is much less sensitive to the drug than the wild-type protein. Myr1E.NLS Gag-GFP saw a 17% increase in nuclear localization upon treatment with LMB. The two mutants of My1E.NLS Gag-GFP, Myr1E. KR/AA.KKK/AAA and Myr1E.KR/AA, experienced and intermediate increase in localization of 10 and 9%, respectively (Garbitt-Hirst et al., 2009). These results make sense because the substitution of alanines in the two control mutants reduces the mutants sensitivity to LMB. The fact that the wild-type experienced a large increase of nuclear localization, where Myr1E experienced a very low rate of nuclear localization leads to the conclusion that Myr1E Gag may be exported from the nucleus prematurely, compared to the wild-type Gag. Restoration of gRNA packaging. Mean measurements of RT activity and an RNase protection assay of viral gRNA from transfected avian cells was used to determine if increasing Myr1E Gag nuclear trafficking would increase gRNA packaging. Myre1E progeny viruses contained approximately 40% of the wild-type level of gRNA, where Myre1E.NLS contained 87.7% of the viral gRNA compared to the wild-type virus. This indicated that the mutant was able to restore gRNA packaging to near wild-type efficiencies. The level of packaging for the two NLS mutants was significantly higher than Myr1E, and lower than that of the wild-type and Myr1E.NLS, although the value was not significant in this case. This shows that the introduction of the NLS into the Myr1E mutant, which significantly increased nuclear trafficking, also significantly increased genomic RNA packaging (Garbitt-Hirst et al., 2009). This provides evidence that proper Gag nuclear trafficking is pivotal for gRNA packaging. Budding efficiencies of Myr1E mutants. It was found that upon addition of an Src membrane-targeting domain to Myr1E mutants, the budding efficiency significantly increased. This makes sense because the Src membrane-targeting domain targets the Gag for export to the plasma membrane. This is provides a possible reason for the inability of Myr1E to package gRNA properly. The authors hypothesize that

Page |3 Myr1E may not have sufficient time to interact with gRNA and package it correctly (Garbitt-Hirst et al., 2009). Analysis of Myr1E infectivity after addition of NLS. Relative to wild-type viruses, the infectivity of Myr1E is greatly decreased. The authors wanted to know if addition of the NLS domain to Myr1E would restore gRNA encapsidation to high enough levels to restore infectivity. Avian QT6 cells were transfected with Myr1E, Myr1E.NLS and wild-type proviral plasmids and pelleted through centrifugation. Virus particles were obtained from the pellet and equivalent amounts of virus particles were added to new cells that were passaged every 3 days for a 21 day period. Before each passage, the medium was collected and pelleted through a sucrose cushion and stored. Each sample was measured for reverse transcription activity and mean values were calculated for each time period (Garbitt-Hirst et al., 2009). Myr1E saw greatly decreased infectivity than the wild-type provirus. It was found that the wildtype virus was able to propagate, but both Myr1E and Myr1E.NLS were unable to propagate despite having near wild-type levels of gRNA packaging (Garbitt-Hirst et al., 2009). Previous findings have shown that Myr1E has a defect in RNA dimerization and is only able to package 1 copy of the viral RNA, rather than the required 2 copies (Parent et al., 2000). This explains the ability for Myr1E.NLS to restore nuclear trafficking to near wild-type levels, but was still unable to propagate. Implications and future experimentation. Addition of NLS to the Gag protein increased gRNA packaging to nearly wild-type levels, but did not restore infectivity in the mutant, which is caused by Myr1Es RNA dimer defect. This shows that Gag must have some interaction with the viral gRNA within the host nucleus that is required for proper gRNA packaging. While it is not known what this interaction is, the authors speculate that Gag undergoes a modification in the nucleus that allows gRNA encapsidation, or that Gag transports packaging proteins across the nuclear membrane into the cytoplasm (Garbitt-Hirst et al., 2009). The authors state that the mechanisms of the RSV Gag protein are not exclusive to RSV, or even retroviruses (Garbitt-Hirst et al., 2009). This provides wide implications for this research. While RSV does not directly affect human health, studying it can help us understand the mechanisms of retroviruses that do, such as HIV. This research has proven that proper nuclear trafficking of the Gag protein is required for propagation of the progeny virus particles. While the HIV-1 and other retroviruses utilize the Rev protein to allow the unspliced gRNA into the cytoplasm and RSV does not, this research shows that a disruption in nuclear trafficking can lead to non-propagation in retroviruses. This information gives scientists a potential mechanism to target in medications that would be used on many viral and retroviral infections.

The Structure and Function of the Rous Sarcoma virus RNA Stability Element It is understood that retroviruses are able to evade degradation of the unspliced genomic RNA, but scientists have been puzzled by how this is achieved. In RSV, this is achieved through a secondary mRNA structure now known as the Rous Sarcoma virus RNA stability element (RSE). While we now know that RSE acts to inhibit nonsense-mediated decay (NMD), the mechanisms by which it works are still largely unknown. The authors of this article have attempted to shine light on the mechanisms by which RSE works, and how this knowledge might affect the treatment of certain cancers and genetic diseases in the future.

Page |4 Determining functional sequence elements of the RSE. The RSE has a specific secondary structure that allows it to function correctly. The figure below shows the secondary structure of RSE. The colored nucleotides represent the effects of a mutation on that particular nucleotide. The effects range from no effect (black) to partial loss of function (blue, purple, and green) to complete loss of function (red). The authors hypothesize that mutations on the affected nucleotides results in a disruption of the RSEs secondary structure. It is believed that the loops in the RSEs structure may act as protein binding sites. Disruption of these binding sites could lead to loss of protein binding and loss of function of the RSE. The function of these mutated RSE nucleotides was quantified by measuring the amount of accumulated viral RNA in cells that were transfected with proviruses that contained mutated RSE sequences. It was observed that the majority of the mutations lead to a partial loss of function of RSE. A possible explanation for this is that it is speculated that the RSE can fold into multiple conformations, and each of these conformations plays a role in RNA protection. Further experimentation led to the discovery that only a specific 155 nucleotide sequence of the RSE is required for function. Even though the minimum nucleotide sequence for a functional RSE is 155 nucleotides, specific nucleotides are needed for minimal RSE function. Experimentation by the authors has revealed that a single mutant containing mutations at each red nucleotide on figure 1 experiences complete loss of RSE function (Withers and Beemon, 2010). Role of NMD in decay of RSV RNA. Nonsense mediated decay is a cellular mechanism used to identify and decay mRNAs with premature stop codons. Identification of premature stop codons is mediated by the exon-junction complex. During splicing, the spliceosome deposits multiple proteins 20-24 nucleotides upstream from an exon-exon junction. The exon-junction complex acts as a marker for where splicing occurs. Since the natural stop codon is found in the terminal exon of nearly every mRNA, a stop codon that is detected before an exon-junction complex signals the cell to degrade the mRNA and nascent protein (Le Hir et al., 2000). RSV RNA that contains a premature stop codon or a nonfunctional RSE has been found to be subject to NMD. This would be easily explained if the RNAs were spliced, but genomic viral RNA is not spliced, and therefore has no exon-exon junctions. It is currently thought that the distance between a termination codon and the poly-A tail can also be a factor in NMD (Withers and Beemon, 2010). Studies have found that in both human and yeast cells, mutations that result in a significantly longer 3 UTR result in NMD (Higgs et al., 1983; Zaret and Sherman, 1984). Experimentation in 2004 and again in 2008

Page |5 found that mRNA, when bound to Poly-A binding protein (PABP), is able to evade NMD if PABP is in close enough proximity to the ribosome. This means that mRNAs having an unusually long 3 UTR are subject to NMD because PABP is not in close enough proximity to the ribosome during termination, and cannot protect the mRNA from decay (Withers and Beemon, 2010). Models of RSE function. The full viral mRNA is a polycistronic mRNA with two open reading frames encoding the Gag and Pol polyproteins. NMD targets mRNAs with multiple reading frames, seeing the initial stop codon as a premature stop codon. In the case of RSV, the gag stop codon needs protected from recognition by NMD proteins. When a ribosome prematurely initiates termination, several proteins are recruited to the ribosome for NMD. The proteins SMG1 and Upf1 are recruited to the ribosome and Upf2 and Upf3 are bound to the mRNA. Phosphorylation of Upf1 signals the beginning of NMD (Huntzinger et al., 2008). Because modifications to Upf1 signal NMD to start, it is believed that the RSE somehow inhibits Upf1. Studies have attempted to find which function of Upf1 is inhibited by RSE, but have only lead to speculations. It is also unsure as to whether RSE directly interacts with Upf1 or whether it recruits a secondary protein for inhibition (Withers and Beemon, 2010). Upf1 in RSV and HIV-1. Upf1 is highly regulated in cells undergoing NMD. Phosphorylation of Upf1 is believed to be a signal for the initiation of NMD. Upf1 is phosphorylated by an ATPase (Withers and Beemon, 2010). In RSV, overexpression of non-phosphorylated Upf1 prevents initiation of further rounds of translation. In HIV-1, however, it has been found that Gag production is higher than in wildtype cells. One explanation for this is that certain Upf1 mutants have been observed to show helicase activity (Withers and Beemon, 2010). Implications and further experimentation. While this study does reveal much about how retroviruses are able to evade mRNA degradation, there is still much research to be done. It is not clear exactly how RSE inhibits Upf1 and if it uses the same inhibition mechanism as other retroviruses, such as HIV-1. Recent studies have shown that Upf1 specifically targets long 3 UTR in HIV-1 for NMD. It remains unsure as to whether HIV-1 uses an RSE-like structure to inhibit this process or whether it uses something altogether different. If HIV does indeed use an RNA secondary structure similar to RSE, it opens the doors for a new way to target HIV infection. Scientists are also trying to find whether or not an RSE-like mechanism is used in cellular genes that possess a long 3 UTR. There are multiple human genetic diseases that have been attributed to mutations resulting in premature stop codons and an unusually long 3 UTR. It is estimated that roughly one third of genetic disorders and cancers are caused by mutations that result in premature stop codons. One such disease, Beta-Thalassemia is one of the first disorders to be attributed to premature stop codons. The mutation in Beta-Thalassemia causes NMD of the mRNA that codes for the Betaglobin chain on hemoglobin (Frischmeyer and Dietz, 1999). If a conserved RSE-like structure can be found in eukaryotes, the first step will have been made in finding treatment for this, and many other similar diseases. Bibliography Frischmeyer, P.A., and Dietz, H.C. (1999). Nonsense-Mediated mRNA Decay in Health and Disease. Human Molecular Genetics 8, 18931900. Garbitt-Hirst, R., Kenney, S.P., and Parent, L.J. (2009). Genetic Evidence for a Connection between Rous Sarcoma Virus Gag Nuclear Trafficking and Genomic RNA Packaging. J Virol 83, 67906797.

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