The alphaviruses: gene expression, replication, and evolution

The alphaviruses are a genus of 26 enveloped viruses that cause disease in humans and domestic animals. Mosquitoes or other hematophagous arthropods serve as vectors for these viruses. The complete sequences of the +/- 11.7-kb plus-strand RNA genomes of eight alphaviruses have been determined, and partial sequences are known for several others; this has made possible evolutionary comparisons between different alphaviruses as well as comparisons of this group of viruses with other animal and plant viruses. Full-length cDNA clones from which infectious RNA can be recovered have been constructed for four alphaviruses; these clones have facilitated many molecular genetic studies as well as the development of these viruses as expression vectors. From these and studies involving biochemical approaches, many details of the replication cycle of the alphaviruses are known. The interactions of the viruses with host cells and host organisms have been exclusively studied, and the molecular basis of virulence and recovery from viral infection have been addressed in a large number of recent papers. The structure of the viruses has been determined to about 2.5 nm, making them the best-characterized enveloped virus to date. Because of the wealth of data that has appeared, these viruses represent a well-characterized system that tell us much about the evolution of RNA viruses, their replication, and their interactions with their hosts. This review summarizes our current knowledge of this group of viruses

Investigating the Roles of NEDD4.2s and Nef in the Release and Replication of HIV-1: A Dissertation

Replication of HIV-1 requires the assembly and release of mature and infectious viral particles. In order to accomplish this goal, HIV-1 has evolved multiple methods to interact with the host cell. HIV-1 recruits the host cell ESCRT machinery to facilitate the release of nascent viral particles from the host cell membrane. Recruitment of these cellular factors is dependent on the presence of short motifs in Gag referred to as Late-domains. Deletion or mutation of these domains results in substantial decrease in the release of infectious virions. However, previously published work has indicated that over-expression of the E3 ubiquitin ligase, NEDD4.2s is able to robustly rescue release of otherwise budding-defective HIV-1 particles. This rescue is specific to the NEDD4.2s isoform as related E3 ubiquitin ligases display no ability to rescue particle release. In addition, rescue of particle release is dependent on the presence of the partial C2 domain and a catalytically active HECT domain of NEDD4.2s. Here I provide evidence supporting the hypothesis that a partial C2 domain of NEDD4.2s constitutes a Gag interacting module capable of targeting the HECT domains of other E3 ubiquitin ligases to HIV-1 Gag. Also, by generating chimeras between HECT domains shown to form poly-ubiquitin chains linked through either K48 or K63 of ubiquitin, I demonstrate that the ability of NEDD4.2s to catalyze the formation of K63-polyubiquitin chains is required for its stimulation of HIV-1 L-domain mutant particle release. In addition, I present findings from on-going research into the role of the HIV-1 accessory protein Nef during viral replication using the culture T-cell line, MOLT3. My current findings indicate that downregulation of CD4 from the host cell membrane does not solely account for the dramatic dependence of HIV-1 replication on Nef expression in this system. In addition, I present evidence indicating that Nef proteins from diverse HIV-1 Groups and strains are capable of enhancing HIV-1 replication in this system. Analysis of a range of mutations in Nef known to impact interaction with cellular proteins suggest that the observed replication enhancement requires Nef targeting to the host cell membrane and may also require the ability to interact with select Src-kinases. Lastly, we find that the ability of Nef to enhance replication in this system is separate from any increase in viral particle infectivity, in agreement with current literature

Functional Analysis of Potential Phosphorylation Sites in the HIV-1 p6 Gag Domain

The human immunodeficiency virus type 1 (HIV-1) interacts with multiple host components and usurps cellular regulatory mechanisms, such as phosphorylation to help direct different events of its replication cycle. Protein phosphorylation is a posttranslational modification widely used by the cell to convey specific messages in response to specific stimuli and allows the coordination of a myriad of cellular processes in a timely and specific manner. The C-terminal p6 domain of the HIV-1 Gag protein, besides carrying the PTAP L-domain required to mediate virus release is subject to phosphorylation. Although p6 has been identified to exist as the major phosphoprotein in HIV-1 particles, an in-depth study of the functional role of p6 phosphorylation has not been conducted to date. Thus, these observations prompted us to perform a comprehensive analysis of the consequences of potential p6 phosphorylation in the HIV-1 replication cycle

Interaction of nonstructural protein NS3 of African horsesickness virus with viral and cellular proteins

African horsesickness virus (AHSV) is a dsRNA virus that belongs to the Orbivirus genus within the Reoviridae family. Each of the ten viral dsRNA segments encodes one virus-specific protein. During its life cycle AHSV replicates both in an insect vector and in a mammalian host, but while it has no detrimental effect on insect cells the virus is highly pathogenic to mammalian cells. It is postulated that this relates to different viral release mechanisms. Currently the main candidate for mediating viral release in both insects and mammals is the viral nonstructural protein NS3. In bluetongue virus (BTV), the prototype virus of the Orbivirus genus, it has been shown that NS3 interacts with both the viral outer capsid protein VP2 and a cellular exocytosis protein. For AHSV, we investigated whether the same mechanism was involved in viral release. This study aimed to identify and map possible protein-protein interaction between AHSV NS3 and VP2, and AHSV NS3 and unknown insect cellular proteins. For investigating the NS3-VP2 interactions a eukaryotic expression system (yeast twohybrid), a column binding assay utilising bacterially expressed NS3 and recombinant baculovirus expressed VP2 as well as a membrane flotation assay utilising recombinant baculovirus expressed VP2 and NS3-GFP, were used. A number of problems were encountered and no conclusive results were obtained. For investigating viral-cellular protein interactions the yeast two-hybrid system was also used, utilising NS3 as bait to screen proteins expressed from a Drosophila cDNA library. Results showed an interaction between the N-terminal region of AHSV NS3 and ubiquitin, an essential protein for the trafficking and degradation of membrane proteins from the endoplasmic reticulum. It also acts as a sorting signal in both the secretory pathway and in endosomes, where it targets proteins into multivesicular bodies in the lumen of vacuoles/lysosomes. It has been shown that ubiquitin could play a role in the pinching off of budding vesicles. An AHSV infected cell could therefore potentially use ubiquitin in its vesicular budding pathway, therefore giving the opportunity for viruses to use this to release them from the cell. The Hsp70 was another protein identified that interacts with AHSV NS3. This protein plays a role in folding reactions, protein translocation across membranes of organelles and protein assembly. It has been reported in other studies done that both ubiquitin and Hsp70 play roles in regulating the bioavailability of viral proteins, which could explain the different levels of NS3, high in insect cells and low in mammalian cells, which indirectly control the viral exit pathway used, budding versus lytic release. These results lay the foundation for explaining the potential role of NS3 in the AHSV life cycle in insect cells.Dissertation (MS)--University of Pretoria, 2007.Geneticsunrestricte

Interactions Between APOBEC3 and Murine Retroviruses: Mechanisms of Restriction and Drug Resistance

APOBEC3 proteins are important for antiretroviral defense in mammals. The activity of these factors has been well characterized in vitro, identifying cytidine deamination as an active source of viral restriction leading to hypermutation of viral DNA synthesized during reverse transcription. These mutations can result in viral lethality via disruption of critical genes, but in some cases is insufficient to completely obstruct viral replication. This sublethal level of mutagenesis could aid in viral evolution. A cytidine deaminase-independent mechanism of restriction has also been identified, as catalytically inactive proteins are still able to inhibit infection in vitro. Murine retroviruses do not exhibit characteristics of hypermutation by mouse APOBEC3 in vivo. However, human APOBEC3G protein expressed in transgenic mice maintains antiviral restriction and actively deaminates viral genomes. The mechanism by which endogenous APOBEC3 proteins function is unclear. The mouse provides a system amenable to studying the interaction of APOBEC3 and retroviral targets in vivo. Virions packaging endogenous protein were isolated from mice for analysis of APOBEC3 without a need for protein overexpression. Biochemical and molecular studies are possible using endogenous protein and viral nucleic acids. Additionally, the effect of APOBEC3-mediated viral mutagenesis and subsequent drug resistance can be modeled in this system. Human APOBEC3G transgenic mice infected with murine retroviruses and treated with an antiretroviral drug allows examination of natural levels of viral replication, APOBEC3 induced hypermutation, and potential viral escape. Studies described herein explore mechanisms of APOBEC3-mediated restriction and drug resistance in vivo. We show that endogenous APOBEC3 protein is efficiently packaged into viral cores, and this protein maintains catalytic activity against artificial substrates. We recovered low levels of G-to-A mutations from natural reverse transcription products, although approximately five to ten fold lower than that thought to be necessary for efficient viral restriction. We show that inhibition of reverse transcription is the main mechanism of restriction in vivo, and can be targeted through virion-packaged or cell-associated protein. Transgenically-expressed human APOBEC3G is instead able to heavily deaminate viral DNA, although frequently to sublethal levels. We assessed the effect of both murine APOBEC3 and APOBEC3G on viral replication in the presence and absence of an antiretroviral drug, and examined viruses for drug resistance mutations. APOBEC3G has a clear effect on the rate of viral mutagenesis in vivo, with the potential to induce drug resistance mutations

Comparison of the two lumpy skin disease virus vaccines, Neethling and Herbivac, and construction of a recombinant Herbivac-Rift Valley fever virus vaccine

There are two broad aims to this project. The first aim is to compare and characterise two lumpy skin disease virus (LSDV) vaccines namely the vaccine based on attenuated Neethling LSDV (nLSDV) and Herbivac®LS (Herbivac). The second aim is to construct a recombinant LSDV expressing Rift Valley fever virus (RVFV) genes. An LSDV vaccine is critical for sustainable control of lumpy skin disease (LSD). There are four commercially available live attenuated vaccines for LSDV, nLSDV, Herbivac, Lumpyvax and the Kenyan strain sheeppox virus (KS-1). In this study Herbivac was characterised by comparing it to its parent, nLSDV. Growth curves of the two viral strains were conducted in cell culture as well as in embryonated hens’ eggs. No notable difference in the growth rate of the two strains could be detected when the viruses were grown in cell culture, however a notable difference was detected when the viruses were grown on the chick allantoic membranes (CAMs) of embryonated hens’ eggs. When grown on CAMs a faster growth rate was observed for nLSDV compared to Herbivac. nLSDV also killed the embryos at 4 d.p.i where Herbivac did not. The two strains were then further characterised through histological analysis of CAMs after infection with each of the viruses. Overall, higher levels of hyperplasia and hypertrophy were observed in CAMs infected with either nLSDV or Herbivac compared to uninfected CAMs. Herbivac-infected CAMs resulted in thicker chorionic membranes and larger pocks compared to nLSDV. RVFV and LSDV both contribute to the disease burden among cattle in Africa and the Arabian Peninsula. The main aim of this study was to construct a recombinant Herbivac which expresses immunogenic proteins of Rift Valley fever virus (Herbivac-RVFV). Herbivac-RVFV was designed to express specific RVFV genes selected for their antigenic properties. The genes selected are also representative of the genes from recent viral outbreaks in the horn of Africa. The selection of outbreak relevant RVFV genes involved phylogenetic analysis of all full length M-segment and NC gene sequences available on Genbank. Phylogenetic trees were constructed for M-segments and NC genes and groups identified which were highly representative of sequences from recent outbreaks of the virus. Consensus sequences were derived from these groups and included in the transfer vector. The phylogenetic analysis also revealed that the sequences of current RVFV vaccines are phylogenetically distant from viruses isolated from current outbreaks, although high levels of sequence conservation was maintained across all viral strains. This is the first study in which the RVFV genes coding for proteins that will induce a protective immune response (Gn and Gc, as well as the nucleocapsid (NC) gene) were selected so as to be representative of current outbreak strains of the virus. These genes were inserted between LSDV ORFs 49 and 50, a novel insertion site. The transfer vector also contained an eGFP marker gene and an ECO-GPT selection gene, located outside of the LSDV flanking sequences. This meant a two-step isolation procedure, first to isolate the recombinant containing the entire transfer vector with eGFP and ECO-GPT, and then to isolate a recombinant with only the RVFV genes and not eGFP and ECO-GPT. Transient expression of RVFV proteins in cells infected with Herbivac and then transfected with the transfer vector was confirmed via western blotting and immunofluorescence. Here the proteins Gn, Gc and NC were shown to be expressed. In the present study, a single crossover Herbivac-RVFV recombinant was isolated through multiple passaging of cell lysates, originally obtained from Herbivac-infected FBT cells transfected with the transfer vector, in the presence of mycophenolic-acid selection medium

Doctor of Philosophy

dissertationThe cellular protein ALIX and other ESCRT proteins facilitate topologically equivalent membrane abscission events, including viral envelope separation from host membranes, biogenesis of multi-vesicular bodies, and midbody scission at the late stage of cytokinesis. Late domain motifs displayed by retroviral Gag polyproteins are responsible for recruiting ESCRT proteins. The three best-characterized classes of late domains are: the "P(S/T)AP" late domains that bind TSG101 of the ESCRT-I complex, the "PPXY" late domains that bind NEDD4 family ubiquitin E3 ligases, and the "YPXnL" late domains that bind ALIX. ALIX also binds the ESCRT-III protein CHMP4, which recruits other ESCRT-III subunits and VPS4 complexes to carry out membrane fission. My work in this dissertation is centered on how ALIX is recruited by various retroviruses and how ALIX function is regulated in viral budding. We first determined crystal structures of ALIXBro1, ALIXV and ALIXBro1-V. Second, in order to understand how the viral Gag proteins hijack ALIX, we determined the structure of ALIXBro1-V in complex with HIV and EIAV YPXnL late motifs. Third, we used surface plasmon resonance (SPR) to map a new type of ALIX-binding elements from certain SIV strains, which do not contain the canonical YPXnL late domains and still package ALIX in the virions. Furthermore, the new ALIX-binding motifs were crystallized with ALIXBro1-V. All these late-domain ligands adopt different conformations of backbones to interact with the equivalent interface on the ALIX V domain. Based on sequence analysis, nearly every known primate lentiviruses contains an ALIX-binding site, suggesting that the ability to recruit ALIX provide a strong selective advantage for viruses. Fourth, we discovered that the fulllength ALIX is autoinhibited by its C-terminal proline-rich region (PRR), which blocks the interaction of viral late domains based on the results of isothermal titration calorimetry (ITC), SPR and small-angle X-ray scattering (SAXS). The mutation that opens the closed conformation of the V domain partitioned ALIX into membrane-containing fractions and enhanced virus budding. These observations suggest that the function of ALIX is highly regulated, and ALIX activation requires dissociation of the autoinhibitory PRR, opening of the V domain, and probably protein dimerization

Analysis of retroviral assembly and maturation using cryo-electron tomography

Retroviruses are a family of membrane-enveloped RNA viruses that can retrotranscribe and integrate their genome in the host cell chromatin. During the active production of virions the structural polyprotein Gag assembles together with the genomic RNA to form immature particles, which bud out from the host cell. After budding the Gag protein undergoes proteolytic maturation and is cleaved into MA, CA, NC, p6 and two spacer peptides, SP1 and SP2. This leads to dramatic changes in the core morphology and the gain of infectivity. The immature retro-virions are known to have Gag organized into a round, incomplete hexameric lattice with a spacing of ~7.5nm. The core of mature virions is organized into a mixture of hexamers and pentamers which are organized along a lattice with a spacing of ~9.6nm. The shape of the core in the mature virions is genus-dependent, but can be cylindrical, conical or round. During my PhD I have studied the immature Gag assembly across four retroviral genera in order to understand the structural requirements for the assembly of the immature retroviral lattice, and to shed more light on the principles of HIV-1 maturation. The major conclusions of my studies are the following: The CA region of Gag is the most structurally conserved across genera. The presence of a domain upstream of CA is not critical for the assembly although it stabilizes the lattice. In order to maintain an immature lattice is important to have a Gag multimerization domain downstream of CA. The region between CA and NC, which is highly variable, is not critical for the assembly but it can stabilise the lattice and therefore affect the structural changes that occur during the maturation. The maturation in retroviruses is an extremely fast process. In order to investigate the structural changes occurring during the maturation in HIV-1 I analysed the products of partial Gag maturation, which were obtained through selective mutations of the cleavage sites in Gag. This confirmed that the order in which Gag cleavages occur is important for a correct processing. The immature Gag lattice is destabilized only if both sides of the CA-SP1 region are cleaved. Furthermore, it showed that the condensation of the RNP has an effect on the core morphology in the mature virion

Investigation of the sequences and structural elements required for HIV-2 infectivity and genome dimerisation

A unique feature of retroviruses is that two copies of the genomic RNA are packaged in each particle. The selective encapsidation of viral genomes is ensured by the binding of the Nucleocapsid to a specific motif on the RNA genome, the packaging signal (Psi). The Psi regions of many retroviruses overlap with sequences that promote the dimerisation of the genome, the dimerisation initiation site (DIS), and it has been suggested that the two mechanisms are closely linked. The aim of the research presented herein was to identify the sequences and structural elements required for the dimerisation of HIV-2 genomic RNA and to investigate the relationship between HIV-2 genome dimerisation and encapsidation, infectivity and particle morphogenesis. Mutations of two palindromic sequences, introduced in an infectious molecular clone of the HIV-2rod isolate, revealed that a palindrome within HIV-2 Psi was important for genome dimerisation. In contrast with previous studies, the palindrome termed DIS is not required for genome dimerisation and viral replication. Viruses bearing mutations within the Psi region failed to dimerise and to replicate in T-cells, a defect that could not be rescued by targeting more genomes to the cells. Psi-deleted viruses also displayed a defect in particle morphogenesis. A reduced packaging efficiency, combined with the presence of RNA monomers or unstable dimers in these virions, resulted in the production of fewer mature particles. However an increase in the number of particles containing two cores was observed. Further characterisation of the sequences and structural elements required for RNA dimerisation, packaging and viral replication showed that the formation of stem B is not critical for viral replication. However, a GGAG purine-rich motif at position 392-395 of the HIV-2rod genome is absolutely essential for genome dimerisation and viral infectivity, and a correlation was observed between dimer formation and viral replication