Bacteriophage is a virus which attacks bacteria and replicates using bacterial replication mechanisms. Bacteriophages are the most abundant viruses in the biosphere, and they can have either DNA or RNA genomes. This is the difference between retrovirus and bacteriophage. References: 1. Coffin, John M. “The Place of Retroviruses in Biology.”.

Maizels, Katherine A. Smith, in, 2011 3.1.1 Retroviral infections and suppression of CD8 + effector functionRetroviral infections in both mice and humans can be influenced by Treg populations ( Li et al., 2008; Rouse et al., 2006). In mice, infection with the chronic Friend retrovirus (FV) ( Iwashiro et al., 2001) or the LP-BM5 murine leukemia virus mixture (which causes mouse AIDS) stimulated expansion of CD4 + Treg cells co-expressing CD25 or CD38, cell surface markers associated with regulatory cells ( Antunes et al., 2008; Beilharz et al., 2004; Robertson et al., 2006; Zelinskyy et al., 2006). Life cycle of retrovirus.

Retrovirus infection involves the following sequential steps: (1) Entry into host cells, (2) Reverse transcription of the RNA genome and generation of the double-strand proviral DNA, (3) Integration of the proviral DNA into the host genome, (4) Transcription of the viral genes from the 5′-LTR, splicing, and translation, and (5) Production of viral particles. Integration of proviral DNA occurs only in dividing cells, and the integration site (only one per cell) is essentially random. See text for details. 1 Entry into Host CellsFirst, retroviruses interact with surface molecules of host cells.

The products of env, present at the virion surface, contact with the host membrane proteins (virus receptors). The ecotropic virus, which is infectious only to rodents, uses a transporter for basic amino acids, whereas amphotropic virus, which is infectious to a wide range of species, uses a sodium-dependent phosphate symporter as viral receptors.

After interaction of retroviruses and viral receptors, the virus and host cell membranes fuse together, and the virion core is delivered into the cytoplasm of the host cells ( Fig. 2 Reverse Transcription. As soon as the virion core enters the cytoplasm, reverse transcriptase included in the core initiates reverse transcription and produces the minus- strand DNA. For this process, tRNA annealed to the primer-binding site of the viral RNA genome is used as a primer for reverse transcription ( Fig.

This transcription stops at the 5′-end of the viral RNA. However, because the RNaseH activity of reverse transcriptase degrades the RNA strand of the DNA:RNA hybrid ( Fig. 3B), the resultant single- strand DNA containing the sequence complementary to R can hybridize to the 3′-R of the RNA genome ( Fig. This jump to the 3′-end then enables further reverse transcription to the 5′-end ( Fig. Next, the plus strand of DNA is produced, using the minus-strand DNA as a template by reverse transcriptase.

In this process, the polypurine tract, which is resistant to RNaseH, is used as a primer ( Fig. The plus-strand DNA synthesis proceeds to the annealed tRNA, which has the complementary sequence to the primer-binding site ( Fig. After degradation of tRNA by RNaseH ( Fig. 3G), the resultant single-strand region containing the primer-binding site hybridizes to the 3′-end of the minus-strand DNA, which contains the sequence complementary to the primer-binding site ( Fig.

This jump enables the synthesis of the complete double-strand proviral DNA ( Fig. Because of the two jumps, the U3 and U5 are duplicated at the 5′- and 3′-ends, respectively. Formation of the double-strand proviral DNA from RNA genome. (A) Reverse transcriptase included in the core initiates reverse transcription and produces the minus-strand DNA using tRNA as a primer, which is annealed to the primer-binding site (PB) of the viral RNA genome.

This transcription stops at the 5′-end of the viral RNA. (B) RNaseH activity of reverse transcriptase degrades the RNA strand of the DNA:RNA hybrid.

(C) The resultant single-strand DNA containing the sequence complementary to R can hybridize to the 3′-R of the RNA genome. (D) The jump to the 3′-end enables further reverse transcription to the 5′-end of the RNA genome. (E) RNaseH activity of reverse transcriptase degrades the RNA strand of the DNA:RNA hybrid. The polypurine tract (PP), located just upstream of U3, is resistant to RNaseH and serves as a primer for the plus-strand DNA synthesis.

(F) The plus-strand DNA is produced, using the minus-strand DNA as a template by reverse transcriptase. The plus-strand DNA synthesis proceeds to the annealed tRNA, which has the complementary sequence to the primer-binding site (PB). (G) The tRNA that was used as a primer is degraded by RNaseH. (H) The resultant single-strand region containing the primer-binding site (PB) hybridizes to the 3′-end of the minus-strand DNA, which contains the sequence complementary to the primer-binding site (PB).

This hybridization transiently makes a circular form since the U3–R–U5 portion of the plus strand remains hybridized to the U3–R–U5 portion of the minus strand. (I) The jump to the 3′-end of the minus-strand DNA enables the synthesis of the complete double-strand proviral DNA. Because of the two jumps, the U3 and U5 are duplicated at the 5′- and 3′-ends, respectively, generating two copies of a long terminal repeat (LTR). RNA and DNA are shown by thin and thick lines, respectively. 3 IntegrationThe double-strand proviral DNA next enters the nucleus ( Fig. This process requires the breakdown of the nuclear membrane and therefore occurs only in dividing cells.

After entry into the nucleus, the proviral DNA is integrated into the host chromosomal DNA through the att sites of the U5 and U3 regions. This process is controlled by integrase. Only a single copy of the proviral DNA is integrated.

The integration sites of the proviral DNA are essentially random. 4 Expression of Viral GenesThe U3 of the 5′-LTR functions as a promoter, which directs transcription from the U3–R border. Transcription proceeds to the 3′-LTR, and the RNA is cleaved and polyadenylated at the R–U5 border of the 3′-LTR ( Fig. For MoMLV, two distinct RNAs are generated from the viral genome ( Fig. One is the full- length form containing the region from the 5′-R to the 3′-R.

This RNA serves both as the viral genome to be packaged into virion particles and as the template for translation of gag and pol. The other one is the spliced form and serves as the template for translation of env. In the latter form, the gag and pol regions are removed as an intron.From the full-length form of RNA, both Gag and Gag–Pol fusion proteins are produced. Because the open reading frame of gag ends at the stop codon, translation of the full-length RNA mostly generates only Gag protein. However, occasionally termination at the stop codon is suppressed, and translation continues to the open reading frame of pol, thereby producing Gag–Pol fusion protein. This fusion protein is later processed by a protease encoded by pol.

5 Production of Viral ParticlesThe full-length RNA form is now packaged into virion particles ( Fig. The ψ sequence of the RNA genome is recognized as a packaging signal by Gag, and two copies of the viral RNA are packaged into one virus particle. In addition, reverse transcriptase and host tRNA, which are used for proviral DNA synthesis, are incorporated into virus particles. Finally, the virus particles are budded from the cell surface. Murphy, Richard G. Vile, in, 2016 3.2.4 Integration of Closed Circle Retroviral Cassettes Delivered by Adenoviral VectorsFollowing retroviral infection, reverse transcribed proviral DNA serves as a substrate for an integration reaction catalyzed by the retroviral INT protein, which, along with viral GAG proteins, forms the pre-integration complex. Tractor parking games download.

87 This complex brings the 5′ end of the 5′LTR (the U3 region) into close juxtaposition with the 3′ end of the 3′LTR (the U5 region). 87 The direct substrate for INT is most likely a linear, double-stranded molecule with blunt ends. 88 INT-mediated integration then occurs by a very precise mechanism in which the terminal-two base pairs of each LTR are lost prior to integration into the target cell genome. 87,89 However, closed circular molecules have also been detected in the nuclei of retrovirally infected cells, which contain 2LTRs joined covalently together at the so-called circle junction. 87,90,91 Although there is considerable evidence that MLV probably does not use a 2LTR circle as the principal integration intermediate, 87,89,92 it was hypothesized that it may still be possible for INT to use such a molecule as a template for integration if it were the only, or predominant, species delivered into the nucleus. 87 This hypothesis is supported by the existence of the 2LTR circles in MLV-infected cells 90 and evidence from the spleen necrosis virus (SNV) system that the LTR junction fragment can be an effective substrate for integration.

We investigated whether a 5′LTR–3′LTR junction fragment, in a closed circular DNA molecule excised from an incoming plasmid by Cre recombinase and in the absence of the preferred, linear viral DNA molecules, could be recognized by the retroviral integration machinery ( Fig. A fused LTR junction fragment was thus cloned, containing the entire 3′LTR and just 28 bp of the U3 region of the 5′LTR ( Fig. This LTR junction together with the puromycin resistance gene was flanked by LoxP sites and was demonstrated to efficiently excise a circular proviral intermediate in vitro upon supply of Cre recombinase in trans. 93 Further studies in cell lines trans-complementing Gag/Pol gene functions, together with Cre recombinase generated long-term neomycin-resistant clones.

Genomic DNA extracted from stable clones was used to investigate the proviral integration structures by utilizing a panel of diagnostic PCR primers. The PCR demonstrated that integration following plasmid transfection, Cre excision, and puromycin selection for 1 month can produce a very specific molecular structure which is distinct from that produced by random plasmid integration.

PCR results demonstrated that the 5′ and 3′ LTRs, which are adjoining in the plasmid backbone, become separated by the intervening sequences of the retroviral vector genome (between the loxP sites). A molecule is thus generated in which the proviral genome is now bounded by the LTRs in a manner typical of INT-mediated integration ( Fig. Critically, the terminal two base pairs of both the 5′LTR (U3 region) and the 3′LTR (U5 region) were lost ( Fig. Hence these studies confirm that a circular retroviral genome with terminally fused LTR structures can indeed serve as a substrate for the retroviral machinery. Genomic integration of an adenovirally delivered retroviral circular provirus cassette.A LoxP flanked cassette containing a fused terminal LTR junction and transgenes of interest were inserted into an adenoviral vector. Upon infection of cells expressing cre recombinase, this cassette is efficiently excised as a closed circular molecule.

The fused LTR junction contained in this circular proviral molecule are subsequently recognized by retroviral integrase directing integration into the host chromosome. Sequencing of the integration junctions of the circular RV proviral cassette.(A) Schematic representation of the RV genome conformation in the non-covalently linked circular pre-integration complex and the subsequently cloned fused LTR junction. (B) A human cell line expressing the retroviral gag/pol genes and cre recombinase (TelCre) were infected with the Ad/RV hybrid vector expressing puromycin resistance.

Colonies were selected which had stably integrated the RV proviral cassettes and the genomic DNA extracted. The integration junctions were subsequently cloned by PCR amplification of re-ligated restriction digested fragments containing the integration site, 93 which were subsequently sequenced through the integration site.From this initial proof of concept, the LoxP cassette was subsequently assembled into an E1-deleted Ad vector. The Ad virus is used to deliver the LTR junction fragment into the nuclei of cells; the proviral-like intermediate can then be excised from the Ad genome by the Cre/lox system and forms a template for INT-mediated integration. This hybrid Ad/RV system thus has the high transient titer of Ad vectors, does not depend upon cell division for infection, and leads to long-term gene expression via integration of a proviral transgene cassette.

Delivery of the Lox-Puro-Junc-Lox cassette in an Ad vector, in the presence of Cre and GAG and POL allowed cloning of cells which are resistant to puromycin for long periods in culture. Without Cre, such clones were impossible to obtain. Moreover, these clones contain a molecular structure consistent with proviral integration by PCR and contain integration sites which, for the majority of the clones (7 of 9), are typical of INT-mediated, rather than random integration processes ( Fig. Codelivery of three separate Ad vectors, Ad-GAG/POL, AdCre, and Ad.LTR.Junc, was also able to produce long-term integrants. Therefore, we are currently optimizing the design and use of this novel hybrid vector system into a single, or double, Ad delivery system. Recent experiments have shown that Pol-expressed INT alone is sufficient to drive the integration of the Cre-excised proviral form in vitro without the need for additional Pol or Gag proteins.

An Ad vector was thus cloned incorporating the INT gene in the same cassette as the transgene cassette to enable a two-vector transduction strategy, which is currently under investigation in our laboratory. This novel hybrid vector system presents great potential in enabling the stable transduction of all cells primarily infected by the Ad vectors. Lowenstine, Kent G. Osborn, in, 2012 RetrovirusesSeveral different retrovirus infections have been described in nonhuman primates. Of the exogenous or pathogenic retroviruses, only infections with simian type D retroviruses (SRV) and simian immunodeficiency viruses (SIV) have been associated with respiratory tract lesions.

In infections with SRV the lesions encountered are due to secondary opportunistic infections ( Lowenstine, 1993a). Similar infections may also be seen in SIV-infected animals; however, SIV also causes a primary retroviral pneumonia in macaques ( Baskerville et al., 1992; King, 1993a). Simian Immunodeficiency VirusLentiviruses of cercopithecine monkeys are indigenous to African monkeys of the genera Cercopithecus, Chlorocebus, Cercocebus, and Papio ( Mandrillus). The viruses are of very low pathogenicity in African species. In Asian macaques, however, these viruses cause devastating disease characterized by immune dysfunction, wasting, opportunistic infections, and primary retroviral pneumonia and encephalitis. Historically, many macaques were infected by accidental iatrogenic exposure by direct contact with African species and through exposure to other infected macaques ( Lowenstine et al., 1992; Aptrei et al., 2006).

In most vivaria, natural SIV infection of macaques is rare to nonexistent ( Daniel et al., 1984). However, macaques are commonly experimentally infected as models for human HIV infection. Primary retroviral pneumonia is a common finding in experimentally infected rhesus ( Baskin et al., 1989; Baskerville et al., 1992; King, 1993a). Clinical signs are nonspecific, including anorexia, weight loss, and inactivity. Grossly, the lungs fail to collapse and are diffusely or patchily discolored tan to cream or yellow, sometimes with pleural opacification. They are spongy to slightly firm on palpation with minimal free exudate on cut surface ( Figure 9.7).

Histologic examination reveals thickening of alveolar septa, marked exudation of foamy macrophages, lesser amounts of proteinaceous material, and large numbers of syncytial giant cells. The pneumonia is readily differentiated from measles giant cell pneumonia by the absence of inclusion bodies in the SIV pneumonia giant cells and the diffuse alveolar involvement as opposed to the measles orientation around small bronchioles (i.e., no necrotizing bronchiolitis).

In, 2017Diseases associated with retrovirus infections have been recognized for more than a century. Equine infectious anemia, Jaagsiekte (pulmonary adenomatosis) of sheep, and bovine leukosis were all described in the 19th century, long before their causative etiology was understood. Retroviruses in tissue filtrates from chickens with leukosis were investigated by physician Vilhem Ellerman and veterinarian Oluf Bang in Copenhagen in 1908. They were able to transmit leukemia by inoculating chickens with cell-free filtrates. Peyton Rous, a medical pathologist, succeeded in producing transplantable sarcomas in chickens by injecting chicken tumor-derived cell-free filtrates in 1910–1911 (the eponymously named Rous sarcoma virus).

Nearly 60 years after this discovery, Rous was awarded the Nobel Prize. Guo-li Ming, in, 2012 Viral-labeled Cell Toxicity and Physiological ChangesOne concern with retroviral infection of cells is that high-titer viruses may result in multicopy insertion into the host genome and lead to potentially toxic effects due to a high level of expression. For example, GFP has been reported to be deleterious when highly expressed ( Liu et al., 1999; Baens et al., 2006). The expression level of retroviral-labeled GFP under the ubi promoter is generally weaker than that of genetically encoded GFP in the Thy-1-GFP transgenic mouse lines ( Feng et al., 2000).

In the past decade, neurons in Thy-1-GFP mice have been thoroughly investigated with no apparent fluorescent protein-induced toxicity or alteration of neural activity. Several studies directly compared nonretroviral-labeled GFP − mature neurons and GFP + adult-born neurons older than 4 weeks of cell age in the same animal and found no discernible differences in electrophysiological properties ( van Praag et al., 2002; Ge et al., 2006; Overstreet-Wadiche and Westbrook, 2006 ). In addition, retroviral-labeled cells with GFP expression have been shown to survive up to 14 months, supporting GFP being relatively inert by retroviral mediated expression ( Zhao et al., 2006). Reitz Jr., Robert C.

Gallo, in, 2015 Origin and Classification of Human RetrovirusesCurrent knowledge places retroviral infection of humans as zoonoses that originated in primate-to-human species-jumping events. For HIV-1 and HIV-2, these events occurred in Central and West Africa, most likely at multiple times, with the more recent attaining major epidemic significance. Simian immunodeficiency virus of chimpanzees (SIV cpz) is the immediate precursor to HIV-1. 14 It now appears likely that similar species-jumping events occurred between certain types of monkeys and chimpanzees. 15Retroviruses have been classified by a number of different biologic features into at least seven genera. Oncogenic retroviruses occur in all classes of vertebrates. The first infectious agents that produced cancer in chickens were isolated by Ellerman and Bang (1908) 16 and by Peyton Rous (1910).

17 These workers were considerably ahead of their time, and the biologic systems to culture and study these viruses had not yet been described. Rous eventually won a Nobel Prize in 1966 for his work. The pioneering work of Ludwig Gross in the 1950s stimulated renewed interest by demonstrating that oncogenic viruses could produce tumors in mammals, 18 but for the next 3 decades, it remained orthodoxy for most scientists that human retroviruses did not exist. We now know that the pathogenic human retroviruses include lentiviruses (HIV-1 and HIV-2) and oncoviruses (HTLV-1 and HTLV-2).

Human endogenous retroviruses, present in the human germ line, are often replication defective and have not yet been shown to cause any disease.As a replication strategy, retroviruses use the reverse transcription of viral RNA into linear double-stranded DNA, with subsequent integration into the host genome. The characteristic enzyme used for this process, an RNA-dependent DNA polymerase that reverses the flow of genetic information, is known as reverse transcriptase. The discovery of this enzyme altered the “central dogma” of molecular biology—namely, that genetic information must necessarily flow from DNA to RNA 19,20—and helped initiate the modern era of molecular biology. This enzyme is error prone; with the massive turnover of virions in the infected host, these errors accumulate in the viral DNA, accounting for the relatively high mutability of HIV-1. The lifestyle of the retrovirus therefore involves two forms, a DNA provirus and RNA-containing infectious virion.The discovery of HTLV-1 and its etiologic association, first with adult T-cell leukemia, an aggressive T-cell lymphoma, 21-23 and later with a neurologic disease, tropical spastic paraparesis/HTLV-1–associated myelopathy (TSP/HAM), 24 were pivotal events in modern medicine (see Chapter 170).

Although there is relatively little variation among HTLV-1 isolates, HTLV-2, the second human retrovirus, is 50% identical to HTLV-1 at the genomic level. 4 Similarly, HIV-2, the fourth human retrovirus, was identified as a serologic variant of HIV-1, the third human retrovirus, and was isolated from patients in western Africa. 25,26 Some types of SIV are so closely related to HIV-2 that they may form an overlapping continuum with recent common ancestors.

HIV-2 is known to infect several monkey species, including the sooty mangabey, its natural host, and SIV has been known to be transmitted, albeit rarely, to laboratory workers. SIVs and SIV/HIV hybrids (SHIVs) have been used extensively to study animal models of immunodeficiency. Other species, including cats (feline leukemia virus FeLV and feline immunodeficiency virus FIV) and cattle (bovine leukemia virus BLV and bovine immunodeficiency virus BIV), harbor retroviruses analogous to those of humans and some African primates.

HIV-related retroviruses, known as lentiretroviruses, also include the ungulate viruses, maedi-visna virus of sheep, caprine arthritis-encephalitis virus, and equine infectious anemia virus.As RNA viruses, retroviruses have the survival advantage of great genetic diversity. As viruses with a DNA intermediate in their replication cycle, they also have the advantage of latency, as do many DNA viruses, but even more so because the DNA provirus is integrated into the chromosomal DNA of the infected cell. As a CD4 + T-cell and macrophage-tropic virus, HIV also has the advantage of reducing the effectiveness of the host immune response. Retroviruses are typically 100 nm in diameter and contain two single strands of RNA, which permits recombination between the strands ( Fig. The typical genome is approximately 10 kb or less in size and contains three major structural genes, namely, gag (group-specific antigen), pol (polymerase), and env (envelope).

HIV-1 also contains several additional genes; these “extra” genes were first described in HTLV-1. In both viruses, some of these extra genes are essential to viral replication, whereas others may modulate interactions of the virus with its host. Figure 171-2 outlines the genome composition of HIV-1 and HTLV-1. FIGURE 171-2. Genomic organization of human retroviruses.A comparison of the genomes of human T-cell lymphotropic virus type 1 (HTLV-1) and human immunodeficiency virus (HIV) is shown.

Studies of HTLV genes and gene products laid the foundation for an understanding of their functional homologues subsequently found in HIV (e.g., tat and rev), although there is little sequence homology of these genes between HTLV and HIV. Env, envelope; gag, group-specific antigen; LTR, long terminal repeat; nef, negative regulatory factor; ORFs, open reading frames; pol, polymerase; pro, protease; rev, regulator of viral expression; rex, regulator of viral expression; tat, trans-activator; tax, trans-activator of transcription; vif, virion infectivity factor; vpr, viral protein R; vpu, viral protein U.

Retroviruses first bind to cellular receptors on the cell surface via the viral envelope (Env) glycoproteins incorporated into their membranes. These include the entry receptors that mediate virus–cell fusion and entry, either from the plasma membrane or from within an endosomal compartment ( Fig. The presence or absence of entry receptors on cells determines whether they are susceptible to infection. The entry receptors themselves, such as the chemokine coreceptors used by HIV, or additional virus-binding receptors such as the C-type lectin pattern recognition receptor (PRR) DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), also transduce intracellular signals on retrovirus binding and trigger responses that can be pro- or antiviral ( Collman et al., 2000; Gringhuis et al., 2009). Some retroviruses interact with additional PRRs on the cell surface, such as toll-like receptor 2 (TLR2) or TLR4, either via direct interaction with viral glycoproteins that present conserved structural features, termed pathogen-associated molecular patterns (PAMPs), or because the virions themselves bind to bacterial ligands that serve as PAMPs ( Del Corno et al., 2016; Nazli et al., 2013; Rassa et al., 2002; Wilks et al., 2015) ( Fig.

Steps at which different host genes restrict retrovirus infection. Viruses can enter via the plasma membrane after binding to a cell surface receptor or after internalization into an acidic compartment. Entry is determined by the binding specificity of the virus Env for receptor. Binding of virus to cell surface pattern recognition receptors (PRRs), such as TLR2, TLR4, or DC-SIGN can also trigger activation of antiviral signal transduction pathways.

There are also endosomal PRRs activated by viral RNA; this occurs when defective virions are endocytosed, when phagocytic cells ingest retrovirus-infected cells, or when molecules such as tetherin cause the internalization of virions trapped on the cell surface. During productive infection, virions enter the cell and the capsid, which forms a lattice-like structure, can be recognized by host cell factors such as TRIM5α. After or during capsid partial or total dissociation, factors such as virus-packaged or cell-intrinsic APOBEC3 proteins can mutate viral DNA by deaminating cytidine residues or block reverse transcription, whereas cell-intrinsic SAMHD1 can deplete nucleotide pools needed for reverse transcription. Reverse transcription products, including single-stranded viral DNA, DNA/RNA hybrid molecules, and double-stranded viral DNA can be recognized by cytosolic sensors such as cGAS, DDX41, and members of the absent in melanoma 2–like receptor family. Some sensors are also present in the nucleus, although there have not yet been reports that they can recognize retroviral DNA prior to integration.

For HIV-1, it has also been reported that early after infection, short polyA- transcripts are generated and recognized by RNA sensors such as DDX3, which in turn signal through the MAVS restriction factor (see Fig. After fusion of the virus–cell membrane, the retroviral capsid enters the cytoplasm.

The regular lattice-like structure of the capsid protein in retroviral virions can serve as a PAMP. For example, it has been reported that the HIV capsid is recognized by cellular restriction factors such as TRIM5α, leading to the induction of innate immune responses in nonhuman cells; recent studies suggest that TRIM5α may function as a cell-type specific PRR in humans Langerhans DCs ( Pertel et al., 2011; Ribeiro et al., 2016) ( Figs. 11.1 and 11.2).

Another antiviral restriction factor in mice, termed Fv1, also interacts with incoming capsid, but it is not known if this triggers innate immunity ( Sanz-Ramos and Stoye, 2013). Interestingly, not all species have functional TRIM5-like proteins. For example, the TRIM5α gene in cats is truncated, making it unable to interact with feline immunodeficiency (FIV) capsid ( Zielonka and Munk, 2011). Pathways activated by viral gene products, resulting in innate/intrinsic immune responses to retroviruses. During infection, viral RNA/DNA hybrids are recognized by DDX41 and the ALRs IFI16 (humans) or IFI203 (mice), which then activate STING), localized in the endosome. STING activation causes phosphorylation ofTBK1 and in turn IRF3, which enters the nucleus and induces transcription of type 1 IFNs and chemokines such as CXCL10.

Some ALRs, such as IFI16, have also been shown to sense nuclear DNA, although this has not been shown for retroviruses. It has also been demonstrated that truncated polyA- transcripts generated after integration of HIV provirus into the host chromosome are recognized by DDX3, which in turn activates the MAVS sensing pathway. Virion RNA that ends up in endosomal compartments, as shown in Fig. 11.1, can bind to TLR7 or TLR8 and trigger signaling through the MyD88 adaptor molecule, resulting in IκK phosphorylation and activation of the transcription factors IRF7 and NF-κB, which traffic to the nucleus and activate transcription of type 1 IFNs and cytokines, respectively. It has also been reported that the dsRNA PRR TLR3 may recognize retroviral RNA in endosomes.

TRIM5α recognition of HIV-1 capsid has also been reported to activate the NF-κB pathway, via phosphorylation of the cellular kinase TAK1. During the course of reverse transcription in the cytoplasm, several forms of nucleic acid are generated, all of which may be perceived as “foreign” by the cell. These include RNA/DNA hybrids, single-stranded and double-stranded viral DNA (ssDNA and dsDNA, respectively) ( Figs. 11.2 and 11.3). Moreover, all retroviruses have a structurally unusual replication intermediate consisting of a tRNA-bound DNA; the tRNA primer is removed by the RNAase H activity of reverse transcriptase during DNA synthesis ( Fig.

Reverse transcription in the cytoplasm likely initiates within the capsid structure and thus the capsid serves to protect the nucleic acids from recognition by host cytosolic factors that recognize foreign nucleic acid ( Lahaye et al., 2013; Stavrou et al., 2013). Eventually, the capsid structure dissociates, and the dsDNA enters the nucleus, where it could also be recognized by nuclear DNA sensors, although this has not been reported ( Fig. Once integrated into the genome, viral RNA is transcribed and resembles host mRNAs.

However, it has also been reported that at least for HIV-1, truncated viral transcripts that lack polyA tails are produced from integrated proviruses early in infection and that these are sensed by host RNA sensors ( Gringhuis et al., 2017). Nucleic acid structures generated during retrovirus replication that are recognized by host sensors. Light purple structures indicate RNA and dark purple DNA. The specific tRNA packaged in virions serves as the primer for reverse transcription. Different retroviruses use different tRNA as primers.

The RNaseH function of reverse transcription degrades viral RNA as the DNA is synthesized, although it leaves behind a small piece of RNA termed the polypyrimidine tract (ppt) to serve as a primer for plus strand DNA synthesis. The RNaseH activity also eventually degrades the tRNA primer.

After integration in the genome, viral genomic RNA and mRNAs are likely not recognized by host sensor. However, it has been suggested that HIV produces short polyA- transcripts until activation of the TEFb elongation factor by NF-κB occurs and that these are sensed by the DDX3 RNA helicase ( Barboric et al., 2001; Gringhuis et al., 2017; Ribeiro et al., 2016). DsDNA, double-stranded viral DNA; ssDNA, single-stranded viral DNA; RIG-I, retinoic acid-inducible gene I.Incomplete or defective retroviruses may also enter cells via endocytosis into endosomal compartments, where PRRs that recognize single-stranded RNA (ssRNA) such as TLR7/8 reside ( Beignon et al., 2005; Browne, 2011; Heil et al., 2004; Kane et al., 2011). Similarly, host factors such as bone-marrow stromal cell antigen 2/tetherin, which tethers virions to the cell surface, may lead to their endocytosis and subsequent degradation within compartments where these PRRs reside ( Fig. 11.1) ( Li et al., 2016) Thus, retroviral proteins, nucleic acid replication intermediates, and transcripts all potentially serve as recognition elements for host nucleic acid sensors. Goff, in, 2018 Applications for TherapyThe early steps of retroviral infection described here provide a number of potential opportunities for intervention by antiviral drugs. The uncoating of virion cores has been studied as a particularly attractive target (for recent review, see Thenin-Houssier and Valente, 2016).

Molecules that bind CA to block uncoating or conversely enhance premature uncoating could in principle strongly inhibit virus infection and most attractively would block infection before formation of the integrated provirus. Such compounds do indeed exist, including PF74 ( Shi et al., 2011), BI-1 and BI-2 ( Lamorte et al., 2013; Price et al., 2014; Fricke et al., 2014a), I-XW-053 ( Kortagere et al., 2012), Ebselen ( Thenin-Houssier et al., 2016), and PF-1385801 ( Blair et al., 2010); some have been entered into clinical trials.PF74 is the most extensively studied. PF74 binds in a pocket formed by the CA NTD and CTD interface, within the binding site for CPSF6 ( Bhattacharya et al., 2014).

The binding is thought to destabilize the capsid ( Shi et al., 2011). HIV-1 escape from inhibition by PF74 requires multiple mutations in the binding pocket of CA that collectively reduce drug binding ( Shi et al., 2015). These substitution mutations also reduced binding of the host protein CPSF6 and enhanced replication in the presence of the dominant-acting inhibitory protein fragment CPSF6-358. Many of the effects of PF47 on virus replication are plausibly mediated by its effects on host factor binding ( Saito et al., 2016). Some of the mutations are associated with considerable loss in fitness, but others compensate for that fitness cost. The selection for PF74 resistance in some cases was found to result in the appearance of mutants that were not merely resistant but dependent on the drug for replication ( Zhou et al., 2015). These mutants also showed altered dependence on the host factors CPSF6, the nucleoporin Nup153, and the importin TNP03.Other compounds under investigation, BI-1 and -2, also bind CA in the context of incoming capsids and block events postreverse transcription; escape mutations also arise in CA ( Lamorte et al., 2013; Price et al., 2014; Fricke et al., 2014a).

The binding sites here also lie within the binding site for CPSF6 and Nup153, and the compounds induce conformational changes that prevent host factor binding. These results are encouraging in the sense that escape from the inhibitors will be constrained by effects on host factor functions. While viral escape will surely arise in vivo, these compounds may ultimately have a significant role in combination therapies. Ebselen is particularly interesting and unusual in forming a covalent disulfide bond with CA ( Thenin-Houssier et al., 2016).The many host factors discussed here all raise the potential for antiviral therapies that act through modulation of the interaction between host and viral proteins or of the specific activity of the host factor. Targeting host factors in this way has always drawn much attention, as a possible way to avoid the viral escape from inhibitors through rapid mutation.

This is not likely to be a perfect solution to the problem—viruses can escape even those inhibitors that target a host protein (such as the CCR5 inhibitor maraviroc) by finding alternate routes of infection—but at a minimum these efforts will provide novel mechanisms to block virus replication.Inhibitors of several host factors have been considered or tested. Microtubule modulators such as taxol, vinblastine, colchicine, and nocodazole have been examined, but their antiviral effects are limited by issues of redundant pathways for virus trafficking ( Yoder et al., 2011), and cell toxicity. A number of drugs targeting the cyclophilins show some promise as antivirals. Cyclosporin, the prototypical inhibitor of CypA, can meaningfully inhibit HIV-1 in some settings, but its inhibition of T-cell signaling and the immune response is a dangerous unwanted consequence.

There exist nonimmunosuppressive derivatives, most notably SDZ NIM811, which binds CypA and prevent its interaction with HIV-1 Gag but does not block T-cell signaling ( Rosenwirth et al., 1994; Billich et al., 1995; Steinkasserer et al., 1995). Their effect is to block events after reverse transcription, either PIC uncoating or movement of the PIC to the nucleus. Studies suggest that these compounds could be promising antiretroviral drugs.

Lastly we note that any molecules that activate the innate immune response and IFN production have the potential to serve as antivirals. Indeed IFN itself is one of the oldest antivirals to have been tested and is used to treat other viral infections. Generally this approach is fraught with toxic side effects due to the induction of dozens, if not hundreds, of ISGs with unwanted consequences. Molecules that would activate expression of only select ISGs, rather than all of them, could be more successful antivirals. Thus, inducers of only TRIM5α, or MX2, or the APOBECs, should have more limited deleterious effects on the host. Even here, however, there may be unacceptable toxicities.

The APOBECs, for example, are potent mutagens, with some limited activity even on double-stranded DNA, and the potential for oncogenesis is probably too high to seriously contemplate their intentional induction as an antiviral therapy.