The transdominant endogenous retrovirus enJS56A1 associates with and blocks intracellular trafficking of Jaagsiekte sheep retrovirus Gag

The sheep genome harbors approximately 20 endogenous retroviruses (enJSRVs) highly related to the exogenous Jaagsiekte sheep retrovirus (JSRV). One of the enJSRV loci, enJS56A1, acts as a unique restriction factor by blocking JSRV in a transdominant fashion at a late stage of the retroviral cycle. To better understand the molecular basis of this restriction (termed JLR, for JSRV late restriction), we functionally characterized JSRV and enJS56A1 Gag proteins. We identified the putative JSRV Gag membrane binding and late domains and determined their lack of involvement in JLR. In addition, by using enJS56A1 truncation mutants, we established that the entire Gag protein is necessary to restrict JSRV exit. By using differentially tagged viruses, we observed, by confocal microscopy, colocalization between JSRV and enJS56A1 Gag proteins. By coimmunoprecipitation and molecular complementation analyses, we also revealed intracellular association and likely coassembly between JSRV and enJS56A1 Gag proteins. Interestingly, JSRV and enJS56A1 Gag proteins showed distinct intracellular targeting: JSRV exhibited pericentrosomal accumulation of Gag staining, while enJS56A1 Gag did not accumulate in this region. Furthermore, the number of cells displaying pericentrosomal JSRV Gag was drastically reduced in the presence of enJS56A1. We identified amino acid residue R21 in JSRV Gag as the primary determinant of centrosome targeting. We concluded that JLR is dependent on a Gag-Gag interaction between enJS56A1 and JSRV leading to altered cellular localization of the latter.     

Molecular cloning and functional analysis of three type D endogenous retroviruses of sheep reveal a different cell tropism from that of the highly related exogenous jaagsiekte sheep retrovirus

Integrated into the sheep genome are 15 to 20 copies of type D endogenous loci that are highly related to two exogenous oncogenic viruses, jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV). The exogenous viruses cause infectious neoplasms of the respiratory tract in small ruminants. In this study, we molecularly cloned three intact type D endogenous retroviruses of sheep (enJS56A1, enJS5F16, and enJS59A1; collectively called enJRSVs) and analyzed their genomic structures, their phylogenies with respect to their exogenous counterparts, their capacity to form viral particles, and the expression specificities of their long terminal repeats (LTRs). In addition, the pattern of expression of enJSRVs in vivo was studied by in situ hybridization. All of the three enJSRV proviruses had open reading frames for at least one of the structural genes. In particular, enJS56A1 had open reading frames for all structural genes, but it could not assemble viral particles when highly expressed in human 293T cells. We localized the defect for viral assembly in the first two-thirds of the gag gene by making a series of chimeras between enJS56A1 and the exogenous infectious molecular clone JSRV(21). Phylogenetic analysis distinguished five ovine type D retroviruses: enJSRV groups A and B, ENTV, and two exogenous JSRV groups (African versus United Kingdom/North America isolates). Transient transfection assays indicated that the LTRs of the three enJSRVs were not preferentially active in differentiated lung epithelial cells. This suggests that the pulmonary tropic JSRV developed from a type D retrovirus that did not have lung specificity. Consistent with this, in situ hybridization of a panel of normal ovine tissues revealed high expression of enJSRV mRNA in the luminal epithelium and glandular epithelium of the uterus; lower expression was localized in the lamina propria of the gut and in the bronchiolar epithelium of the lungs.     

M-PMV capsid transport is mediated by Env/Gag interactions at the pericentriolar recycling endosome

Cytoplasmic transport of Gag molecules to the site of budding is an important but poorly understand process in retroviral assembly. Our previous studies of Mason-Pfizer monkey virus showed that, for this retrovirus, Gag is assembled into capsids at a pericentriolar region and that Env is necessary for efficient transport out of the site. An Env requirement for cytoplasmic transport implicates vesicular trafficking in this process even though the capsids remain cytoplasmic and do not bud into intracellular compartments in the cells studied to date. We show here that the secretory pathway of the cell is not directly involved in Gag transport since the latter was not inhibited by BFA, nor did Gag colocalize with markers of the ER, Golgi, or TGN. Instead, colocalization was observed between Gag and endocytosed transferrin and with Rab11, suggesting that pericentriolar recycling endosomes play a critical role in this process. Mutants of Rab11 that inhibit efflux of transferrin from the recycling endosome also inhibited Gag transport. Our studies show that Env colocalizes with Gag at the pericentriolar assembly site, and provide evidence that Env must travel through this compartment in order to initiate export of the capsids from the site of assembly. Thus, for the first time, endocytic trafficking of a retroviral Env glycoprotein is linked to the efficient cytoplasmic transport of Gag.     

AP-3 directs the intracellular trafficking of HIV-1 Gag and plays a key role in particle assembly

Gag proteins direct the process of retroviral particle assembly and form the major protein constituents of the viral core. The matrix region of the HIV-1 Gag polyprotein plays a critical role in the transport of Gag to the plasma membrane assembly site. Recent evidence indicates that Gag trafficking to late endosomal compartments, including multivesicular bodies, occurs prior to viral particle budding from the plasma membrane. Here we demonstrate that the matrix region of HIV-1 Gag interacts directly with the delta subunit of the AP-3 complex, and that this interaction plays an important functional role in particle assembly. Disruption of this interaction eliminated Gag trafficking to multivesicular bodies and diminished HIV particle formation. These studies illuminate an early step in retroviral particle assembly and provide evidence that the trafficking of Gag to late endosomes is part of a productive particle assembly pathway.     

Intracellular versus cell surface assembly of retroviral pseudotypes is determined by the cellular localization of the viral glycoprotein, its capacity to interact with Gag, and the expression of the Nef protein

Retroviral Gag and Env glycoproteins (GPs) are expressed from distinct cellular areas and need to encounter to interact and assemble infectious particles. Retroviral particles may also incorporate GPs derived from other enveloped viruses via active or passive mechanisms, a process known as "pseudotyping." To further understand the mechanisms of pseudotyping, we have investigated the capacity of murine leukemia virus (MLV) or lentivirus core particles to recruit GPs derived from different virus families: the G protein of vesicular stomatitis virus (VSV-G), the hemagglutinin from an influenza virus, the E1E2 glycoproteins of hepatitis C virus (HCV-E1E2), and the retroviral Env glycoproteins of MLV and RD114 cat endogenous virus. The parameters that influenced the incorporation of viral GPs onto retroviral core particles were (i) the intrinsic cell localization properties of both viral GP and retroviral core proteins, (ii) the ability of the viral GP to interact with the retroviral core, and (iii) the expression of the lentiviral Nef protein. Whereas the hemagglutinin and VSV-G glycoproteins were recruited by MLV and lentivirus core proteins at the cell surface, the HCV and MLV GPs were most likely recruited in late endosomes. In addition, whereas these glycoproteins could be passively incorporated on either retrovirus type, the MLV GP was also actively recruited by MLV core proteins, which, through interactions with the cytoplasmic tail of the latter GP, induced its localization to late endosomal vesicles. Finally, the expression of Nef proteins specifically enhanced the incorporation of the retroviral GPs by increasing their localization in late endosomes.     

Expression of the jaagsiekte sheep retrovirus envelope glycoprotein is sufficient to induce lung tumors in sheep

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA). The expression of the JSRV envelope (Env) alone is sufficient to transform a variety of cell lines in vitro and induce lung cancer in immunodeficient mice. In order to determine the role of the JSRV Env in OPA tumorigenesis in sheep, we derived a JSRV replication-defective virus (JS-RD) which expresses env under the control of its own long terminal repeat (LTR). JS-RD was produced by transiently transfecting 293T cells with a two plasmid system, involving (i) a packaging plasmid, with the putative JSRV packaging signal deleted, expressing the structural and enzymatic proteins Gag, Pro, and Pol, and (ii) a plasmid which expresses env in trans for JS-RD particles and provides the genomes necessary to deliver JSRV env upon infection. During the optimization of the JS-RD system we determined that both R-U5 (in the viral 5' LTR) and the env region are important for JSRV particle production. Two independent experimental transmission studies were carried out with newborn lambs. Four of five lambs inoculated with JS-RD showed OPA lesions in the lungs at various times between 4 and 12 months postinoculation. Abundant expression of JSRV Env was detected in tumor cells of JS-RD-infected animals and PCR assays confirmed the presence of the deleted JS-RD genome. These data strongly suggest that the JSRV Env functions as a dominant oncoprotein in the natural immunocompetent host and that JSRV can induce OPA in the absence of viral spread.     

GGA and Arf proteins modulate retrovirus assembly and release

The Gag protein is the major structural determinant of retrovirus assembly. Although a number of cellular factors have been reported to facilitate retrovirus release, little is known about the cellular machinery that directs Gag to the site of virus assembly. Here, we report roles for the Golgi-localized gamma-ear containing Arf-binding (GGA) and ADP ribosylation factor (Arf) proteins in retrovirus particle assembly and release. Whereas siRNA-mediated depletion of GGA2 and GGA3 led to a significant increase in particle release in a late domain-dependent manner, GGA overexpression severely reduced retrovirus particle production by impairing Gag trafficking to the membrane. GGA overexpression inhibited retroviral assembly and release by disrupting Arf protein activity. Furthermore, disruption of endogenous Arf activity inhibited particle production by decreasing Gag-membrane binding. These findings identify the GGA proteins as modulators of HIV-1 release and the Arf proteins as critical cellular cofactors in retroviral Gag trafficking to the plasma membrane.     

A paradigm for virus-host coevolution: sequential counter-adaptations between endogenous and exogenous retroviruses

Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections of the host germline transmitted vertically from generation to generation. It is hypothesized that some ERVs are used by the host as restriction factors to block the infection of pathogenic retroviruses. Indeed, some ERVs efficiently interfere with the replication of related exogenous retroviruses. However, data suggesting that these mechanisms have influenced the coevolution of endogenous and/or exogenous retroviruses and their hosts have been more difficult to obtain. Sheep are an interesting model system to study retrovirus-host coevolution because of the coexistence in this animal species of two exogenous (i.e., horizontally transmitted) oncogenic retroviruses, Jaagsiekte sheep retrovirus and Enzootic nasal tumor virus, with highly related and biologically active endogenous retroviruses (enJSRVs). Here, we isolated and characterized the evolutionary history and molecular virology of 27 enJSRV proviruses. enJSRVs have been integrating in the host genome for the last 5-7 million y. Two enJSRV proviruses (enJS56A1 and enJSRV-20), which entered the host genome within the last 3 million y (before and during speciation within the genus Ovis), acquired in two temporally distinct events a defective Gag polyprotein resulting in a transdominant phenotype able to block late replication steps of related exogenous retroviruses. Both transdominant proviruses became fixed in the host genome before or around sheep domestication (approximately 9,000 y ago). Interestingly, a provirus escaping the transdominant enJSRVs has emerged very recently, most likely within the last 200 y. Thus, we determined sequentially distinct events during evolution that are indicative of an evolutionary antagonism between endogenous and exogenous retroviruses. This study strongly suggests that endogenization and selection of ERVs acting as restriction factors is a mechanism used by the host to fight retroviral infections.     

Pericentriolar Targeting of the Mouse Mammary Tumor Virus GAG Protein

The Gag protein of the mouse mammary tumor virus (MMTV) is the chief determinant of subcellular targeting. Electron microscopy studies show that MMTV Gag forms capsids within the cytoplasm and assembles as immature particles with MMTV RNA and the Y box binding protein-1, required for centrosome maturation. Other betaretroviruses, such as Mason-Pfizer monkey retrovirus (M-PMV), assemble adjacent to the pericentriolar region because of a cytoplasmic targeting and retention signal in the Matrix protein. Previous studies suggest that the MMTV Matrix protein may also harbor a similar cytoplasmic targeting and retention signal. Herein, we show that a substantial fraction of MMTV Gag localizes to the pericentriolar region. This was observed in HEK293T, HeLa human cell lines and the mouse derived NMuMG mammary gland cells. Moreover, MMTV capsids were observed adjacent to centrioles when expressed from plasmids encoding either MMTV Gag alone, Gag-Pro-Pol or full-length virus. We found that the cytoplasmic targeting and retention signal in the MMTV Matrix protein was sufficient for pericentriolar targeting, whereas mutation of the glutamine to alanine at position 56 (D56/A) resulted in plasma membrane localization, similar to previous observations from mutational studies of M-PMV Gag. Furthermore, transmission electron microscopy studies showed that MMTV capsids accumulate around centrioles suggesting that, similar to M-PMV, the pericentriolar region may be a site for MMTV assembly. Together, the data imply that MMTV Gag targets the pericentriolar region as a result of the MMTV cytoplasmic targeting and retention signal, possibly aided by the Y box protein-1 required for the assembly of centrosomal microtubules.     

The clathrin adaptor complex AP-1 binds HIV-1 and MLV Gag and facilitates their budding

Retroviral assembly is driven by Gag, and nascent viral particles escape cells by recruiting the machinery that forms intralumenal vesicles of multivesicular bodies. In this study, we show that the clathrin adaptor complex AP-1 is involved in retroviral release. The absence of AP-1mu obtained by genetic knock-out or by RNA interference reduces budding of murine leukemia virus (MLV) and HIV-1, leading to a delay of viral propagation in cell culture. In contrast, overexpression of AP-1mu enhances release of HIV-1 Gag. We show that the AP-1 complex facilitates retroviral budding through a direct interaction between the matrix and AP-1mu. Less MLV Gag is found associated with late endosomes in cells lacking AP-1, and our results suggest that AP-1 and AP-3 could function on the same pathway that leads to Gag release. In addition, we find that AP-1 interacts with Tsg101 and Nedd4.1, two cellular proteins known to be involved in HIV-1 and MLV budding. We propose that AP-1 promotes Gag release by transporting it to intracellular sites of active budding, and/or by facilitating its interactions with other cellular partners.     

Nucleotide sequence of the jaagsiekte retrovirus, an exogenous and endogenous type D and B retrovirus of sheep and goats.

The complete genome of the jaagsiekte sheep retrovirus (JSRV), the suspected etiological agent of ovine pulmonary carcinoma, has been cloned from viral particles secreted in lung exudates of affected animals and sequenced. The genome is 7,462 nucleotides long and exhibits a genetic organization characteristic of the type B and D oncoviruses. Comparison of the amino acid sequences of JSRV proteins with those of other retrovirus proteins and phylogenetic studies suggest that JSRV diverged from its type B and D lineage after the type B mouse mammary tumor virus but before the type D oncoviruses captured the env gene of a reticuloendotheliosislike virus. Southern blot studies show that closely related sequences are present in sheep and goat normal genomic DNA, indicating that JSRV could be endogenous in ovine and caprine species.     

Tsg101: HIV-1's ticket to ride

Recent studies implicate the vacuolar protein-sorting pathway in the transport of the retroviral structural precursor (Gag) protein to its budding site on the plasma membrane of infected cells. This exploitation of the cellular endocytic trafficking machinery to release viral particles could lead to the identification of virus-specific modulators and provide opportunities to design new targeted anti-viral agents.     

What's going on post-budding?

In general, the retrovirus particles become infectious on post-budding with cleavages of structural protein Gag by viral protease. Protease defective mutants bud particles normally, but the particles are non-infectious and called donuts-like particle because of their morphology. The viral genomes inside the donuts-like particles form very fragile dimer, which are far different from those in wild-type particles. The ordered particle maturation process is essential for infectivity of virus, but its mechanism largely remains unclear. We have constructed HIV-1 Gag cleavage site mutants to enable the steady state observation of virion maturation steps, and precisely study Gag processing, RNA dimerization, virion morphology and infectivity. As results, we found that these process progressed synchronously, but each transition point did not coincide completely. The mutual relationship between viral protein and RNA maturation is discussed for a further understanding of the retroviral life cycle.     

Protease-dependent uncoating of a complex retrovirus

Although retrovirus egress and budding have been partly unraveled, little is known about early stages of the replication cycle. In particular, retroviral uncoating, a process during which incoming retroviral cores are altered to allow the integration of the viral genome into host chromosomes, is poorly understood. To get insights into these early events of the retroviral cycle, we have used foamy complex retroviruses as a model. In this report, we show that a protease-defective foamy retrovirus is noninfectious, although it is still able to bud and enter target cells efficiently. Similarly, a retrovirus mutated in an essential viral protease-dependent cleavage site in the central part of Gag is noninfectious. Following entry, wild-type and mutant retroviruses are able to traffic along microtubules towards the microtubule-organizing center (MTOC). However, whereas nuclear import of Gag and of the viral genome was observed for the wild-type virus as early as 8 hours postinfection, incoming capsids and genome from mutant viruses remained at the MTOC. Interestingly, a specific viral protease-dependent Gag cleavage product was detected only for the wild-type retrovirus early after infection, demonstrating that cleavage of Gag by the viral protease at this stage of the virus life cycle is absolutely required for productive infection, an unprecedented observation among retroviruses.     

Retroviral genomic RNAs are transported to the plasma membrane by endosomal vesicles

The viral genomes of alpha- and gamma-retroviruses follow an outbound route through the cytoplasm before assembling with the budding particle at the plasma membrane. We show here that murine leukemia virus (MLV) RNAs are transported on lysosomes and transferrin-positive endosomes. Transport on transferrin-positive vesicles requires both Gag and Env polyproteins. In the presence of Env, Gag is rerouted from lysosomes to transferrin-positive endosomes, and virion production becomes highly sensitive to drugs poisoning vesicular and endosomal traffic. Vesicular transport of the RNA does not require prior endocytosis, indicating that it is recruited directly from the cytosol. Viral prebudding complexes containing Env, Gag, and retroviral RNAs are thus formed on endosomes, and subsequently routed to the plasma membrane. This may allow retroviruses to hijack the endosomal machinery as part of their biosynthetic pathway. More generally, tethering to vesicles may provide an efficient mechanism for directed RNA transport.     

Actin Associates with the Nucleocapsid Domain of the Human Immunodeficiency Virus Gag Polyprotein

Recently, it was shown that actin molecules are present in human immunodeficiency virus type 1 (HIV-1) particles. We have examined the basis for incorporation and the location of actin molecules within HIV-1 and murine retrovirus particles. Our results show that the retroviral Gag polyprotein is sufficient for actin uptake. Immunolabeling studies demonstrate that actin molecules localize to a specific radial position within the immature particle, clearly displaced from the matrix domain underneath the viral membrane but in proximity to the nucleocapsid (NC) domain of the Gag polyprotein. When virus or subviral Gag particles were disrupted with nonionic detergent, actin molecules remained associated with the disrupted particles. Actin molecules remained in a stable complex with the NC cleavage product (or an NC-RNA complex) after treatment of the disrupted HIV-1 particles with recombinant HIV-1 protease. In contrast, matrix and capsid molecules were released. The same result was obtained when mature HIV-1 particles were disrupted with detergent. Taken together, these results indicate that actin molecules are associated with the NC domain of the viral polyprotein.