Effects of cell cycle status on early events in retroviral replication

The study of retroviruses over the last century has revealed a wide variety of disease-producing mechanisms, as well as apparently harmless interactions with animal hosts. Despite their potential pathogenic properties, the intrinsic features of retroviruses have been harnessed to create gene transfer vectors that may be useful for the treatment of disease. Retroviruses, as all viruses, have evolved to infect specific cells within the host, and such specificities are relevant to both pathogenesis and retrovirus-based vector design. The majority of cells of an animal host are not progressing rapidly through the cell cycle, and such a cellular environment appears to be suboptimal for replication of all retroviruses. Retrovirus-based vectors can therefore be restricted in many important target cells, such as post-mitotic differentiated cells or stem cells that may divide only infrequently. Despite intense interest, our understanding of how cell cycle status influences retroviral infection is still quite limited. In this review, we focus on the importance of the cell cycle as it relates to the early steps in retroviral replication. Retroviruses have been categorized based on their abilities to complete these early steps in non-cycling cells. However, all retroviruses are subject to a variety of cell cycle restrictions. Here, we discuss such restrictions, and how they may block retroviral replication, be tolerated, or overcome.     

The road to chromatin - nuclear entry of retroviruses

Human immunodeficiency virus 1 (HIV-1) and other retroviruses synthesize a DNA copy of their genome after entry into the host cell. Integration of this DNA into the host cell's genome is an essential step in the viral replication cycle. The viral DNA is synthesized in the cytoplasm and is associated with viral and cellular proteins in a large nucleoprotein complex. Before integration into the host genome can occur, this complex must be transported to the nucleus and must cross the nuclear envelope. This Review summarizes our current knowledge of how this journey is accomplished.     

Cytoplasmic recruitment of INI1 and PML on incoming HIV preintegration complexes: interference with early steps of viral replication.

During the early phase of the retroviral life cycle, only a fraction of internalized virions end up integrating their genome into the chromosome, even though the resulting proviruses are almost systematically expressed. Here, we reveal that incoming retroviral preintegration complexes trigger the exportin-mediated cytoplasmic export of the SWI/SNF component INI1 and of the nuclear body constituent PML. We further show that the HIV genome associates with these proteins before nuclear migration. In the presence of arsenic, PML is sequestered in the nucleus, and the efficiency of HIV-mediated transduction is markedly increased. These results unveil a so far unsuspected cellular response that interferes with the early steps of HIV replication.     

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.     

Characterization of Resistance to Rhabdovirus and Retrovirus Infection in a Human Myeloid Cell Line

Viruses interact with various permissive and restrictive factors in host cells throughout their replication cycle. Cell lines that are non-permissive to viral infection have been particularly useful in discovering host cell proteins involved in viral life cycles. Here we describe the characterization of a human myeloid leukemia cell line, KG-1, that is resistant to infection by retroviruses and a Rhabdovirus. We show that KG-1 cells are resistant to infection by Vesicular Stomatits Virus as well as VSV Glycoprotein (VSVG) pseudotyped retroviruses due to a defect in binding. Moreover our results indicate that entry by xenotropic retroviral envelope glycoprotein RD114 is impaired in KG-1 cells. Finally we characterize a post- entry block in the early phase of the retroviral life cycle in KG-1 cells that renders the cell line refractory to infection. This cell line will have utility in discovering proteins involved in infection by VSV and HIV-1.     

DNA-tumor virus entry—From plasma membrane to the nucleus

DNA-tumor viruses comprise enveloped and non-enveloped agents that cause malignancies in a large variety of cell types and tissues by interfering with cell cycle control and immortalization. Those DNA-tumor viruses that replicate in the nucleus use cellular mechanisms to transport their genome and newly synthesized viral proteins into the nucleus. This requires cytoplasmic transport and nuclear import of their genome. Agents that employ this strategy include adenoviruses, hepadnaviruses, herpesviruses, and likely also papillomaviruses, and polyomaviruses, but not poxviruses which replicate in the cytoplasm. Here, we discuss how DNA-tumor viruses enter cells, take advantage of cytoplasmic transport, and import their DNA genome through the nuclear pore complex into the nucleus. Remarkably, nuclear import of incoming genomes does not necessarily follow the same pathways used by the structural proteins of the viruses during the replication and assembly phases of the viral life cycle. Understanding the mechanisms of DNA nuclear import can identify new pathways of cell regulation and anti-viral therapies.     

Retroviral infection of hES cells produces random-like integration patterns

Retroviral integration provides us with a powerful tool to realize prolonged gene expressions that are often critical to gene therapy. However, the perturbation of gene regulations in host cells by viral genome integration can lead to detrimental effects, yielding cancer. The oncogenic potential of retroviruses is linked to the preference of retroviruses to integrate into genomic regions that are enriched in gene regulatory elements. To better navigate the double-edged sword of retroviral integration we need to understand how retroviruses select their favored genomic loci during infections. In this study I showed that in addition to host proteins that tether retroviral pre-integration complexes to specific genomic regions, the epigenetic architecture of host genome might strongly affect retroviral integration patterns. Specifically, retroviruses showed their characteristic integration preference in differentiated somatic cells. In contrast, retroviral infections of hES cells, which are known to display decondensed chromatin, produced random-like integration patterns lacking of strong preference for regulatory-element-rich genomic regions. Better identification of the cellular and viral factors that determine retroviral integration patterns will facilitate the design of retroviral vectors for safer use in gene therapy.     

Two retroviruses packaged in one cell line can combined inhibit the replication of HIV-1 in TZM-bl cells

The cellular protein tetherin tethers the HIV-1 viral particles on the cellular membrane to inhibit the replication of HIV-1. However, the HIV-1 accessory protein Vpu counteracts the antiviral function of tetherin. In this study, two retroviral vector plasmids were constructed. One inhibited the vpu gene expression; the other one over-expressed the tetherin. Both retroviral vector plasmids could be packaged in the packaging cell line PT67 to obtain the corresponding retroviruses. The retroviral vector plasmids’ functions of tetherin over-expression or vpu-RNAi were detected at the cell level. Retroviral vector plasmids were transfected to PT67 cells at different ratios from 0T3V to 3T0V, and then mixed retroviruses were harvested. The antiviral functions of mixed retroviruses were detected in HIV-1 infected TZM-bl cells. The results showed that packaged mixed retroviruses could repress the replication of HIV-1 in TZM-bl cells.     

Retroviral DNA integration: HIV and the role of LEDGF/p75

To replicate, a retrovirus must integrate a DNA copy of its RNA genome into a chromosome of the host cell. Integration is not random in the host genome but favors particular regions, and preferences differ among retroviruses. Several mechanisms might play a part in this favored integration targeting: (i) open chromatin might be preferentially accessible for viral DNA integration; (ii) DNA replication during cell division might facilitate access of integration complexes to favored sites; and (iii) cellular proteins bound to the host chromosome might tether integration complexes to favored regions. This review summarizes recent advances in understanding the mechanisms of retroviral integration, focusing on LEDGF/p75--the first cellular protein shown to have a role in directing HIV DNA integration. Studies on LEDGF/p75 indicate that it directs HIV integration site selection by a tethering interaction, whereas the chromatin accessibility or cell cycle models are less well supported. Understanding viral integration will help improve the safety of retrovirus-based vectors used in gene therapy.     

Cleavage of vimentin by different retroviral proteases

Proteases (PRs) of retroviruses cleave viral polyproteins into their mature structural proteins and replication enzymes. Besides this essential role in the replication cycle of retroviruses, PRs also cleave a variety of host cell proteins. We have analyzed the in vitro cleavage of mouse vimentin by proteases of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), bovine leukemia virus (BLV), Mason-Pfizer monkey virus (M-PMV), myeloblastosis-associated virus (MAV), and two active-site mutants of MAV PR. Retroviral proteases display significant differences in specificity requirements. Here, we show a comparison of substrate specificities of several retroviral proteases on vimentin as a substrate. Vimentin was cleaved by all the proteases at different sites and with different rates. The results show that the physiologically important cellular protein vimentin can be degraded by different retroviral proteases.     

The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life cycles. This junction may determine the characteristic parvovirus tropism for proliferative and cancer cells, and its disturbance could critically contribute to persistence in host tissues.     

泡沫病毒Gag蛋白的研究进展

Gag蛋白在逆转录病毒复制周期的许多阶段中发挥重要作用。泡沫病毒基因组结构与其它逆转录病毒类似,但它们的基因组成分和生活周期存在明显差异,这在一定程度上是由其Gag蛋白的结构和功能所决定的。本文对18种不同株型的泡沫病毒gag基因序列进行进化树分析,探究不同来源的泡沫病毒的亲缘关系;并以典型的原型泡沫病毒为代表,阐述泡沫病毒Gag蛋白的结构和功能以及对病毒复制不同阶段的影响。     

Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region.

Replication-competent retroviruses can be modified to carry nonviral genes. Such gene transfer vectors help define regions of the retroviral genome that are required in cis for retroviral replication. Moloney murine leukemia virus has been used extensively in vector construction, and all of the internal protein-encoding regions can be removed and replaced with other genes while still allowing production of virions containing and transmitting the altered retroviral genome. However, inclusion of a portion of the gag region from Moloney murine leukemia virus markedly increases the titer of virus derived from these vectors. We determined that this effect was due to more efficient packaging of the vector RNA into particles and did not depend on protein synthesis from the gag region. We conclude that the retrovirus packaging signal extends into the gag region. We have found that retroviral vectors containing the complete packaging signal allow more efficient gene transfer into a variety of cell types. In addition, these results may help explain why many oncogenic retroviruses have retained gag sequences and often express transforming proteins that are gag-onc hybrids.     

Inhibition of retroviral pathogenesis by RNA interference

BACKGROUND: RNA interference (RNAi) is a newly discovered cellular defense system that is known to suppress replication of genomic parasites in model organisms. It has been widely conjectured that RNAi may also serve as an antiviral system in vertebrates. RESULTS: Retroviral infection could be initiated by electroporation of cloned Rous sarcoma virus (RSV) proviral DNA into the developing chick neural tube. Coelectroporation of proviral DNA and short double-stranded RNAs matching sequences of avain retroviruses, which were designed to induce RNAi against RSV, inhibited viral replication. Replication of RSV after electroporation resulted in disruption of embryonic development and early death, but this, too, could be suppressed by RNAi against the RSV genome. RNAi could also inhibit the growth of RSV and HIV in cell culture. Analysis of the step of the retroviral life cycle that is inhibited by RNAi revealed that it primarily prevented accumulation of the viral RNAs synthesized late during infection. RNA genomes introduced in viral particles early during infection were less sensitive. CONCLUSIONS: RNAi can block retroviral infection in vertebrates. The tissue electroporation method described here should allow RNAi to be used widely to study gene function and control of infection in vertebrate animals.