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P3N-PIPO, the only dedicated movement protein (MP) of potyviruses, directs cylindrical inclusion (CI) protein from the cytoplasm to the plasmodesma (PD), where CI forms conical structures for intercellular movement. To better understand potyviral cell-to-cell movement, we further characterized P3N-PIPO using Turnip mosaic virus (TuMV) as a model virus. We found that P3N-PIPO interacts with P3 via the shared P3N domain and that TuMV mutants lacking the P3N domain of either P3N-PIPO or P3 are defective in cell-to-cell movement. Moreover, we found that the PIPO domain of P3N-PIPO is sufficient to direct CI to the PD, whereas the P3N domain is necessary for localization of P3N-PIPO to 6K2-labeled vesicles or aggregates. Finally, we discovered that the interaction between P3 and P3N-PIPO is essential for the recruitment of CI to cytoplasmic 6K2-containing structures and the association of 6K2-containing structures with PD-located CI inclusions. These data suggest that both P3N and PIPO domains are indispensable for potyviral cell-to-cell movement and that the 6K2 vesicles in proximity to PDs resulting from multipartite interactions among 6K2, P3, P3N-PIPO, and CI may also play an essential role in this process. IMPORTANCE Potyviruses include numerous economically important viruses that represent approximately 30% of known plant viruses. However, there is still limited information about the mechanism of potyviral cell-to-cell movement. Here, we show that P3N-PIPO interacts with and recruits CI to the PD via the PIPO domain and interacts with P3 via the shared P3N domain. We further report that the interaction of P3N-PIPO and P3 is associated with 6K2 vesicles and brings the 6K2 vesicles into proximity with PD-located CI structures. These results support the notion that the replication and cell-to-cell movement of potyviruses are processes coupled by anchoring viral replication complexes at the entrance of PDs, which greatly increase our knowledge of the intercellular movement of potyviruses.

Plant viruses usually encode one or more movement proteins (MP) to accomplish their intercellular movement. A group of positive-strand RNA plant viruses requires three viral proteins (TGBp1, TGBp2, and TGBp3) that are encoded by an evolutionarily conserved genetic module of three partially overlapping open reading frames (ORFs), termed the triple gene block (TGB). However, how these three viral movement proteins function cooperatively in viral intercellular movement is still elusive. Using a novel in vivo double-stranded RNA (dsRNA) labeling system, we showed that the dsRNAs generated by potato virus X (PVX) RNA-dependent RNA polymerase (RdRp) are colocalized with viral RdRp, which are further tightly covered by “chain mail”-like TGBp2 aggregates and localizes alongside TGBp3 aggregates. We also discovered that TGBp2 interacts with the C-terminal domain of PVX RdRp, and this interaction is required for the localization of TGBp3 and itself to the RdRp/dsRNA bodies. Moreover, we reveal that the central and C-terminal hydrophilic domains of TGBp2 are required to interact with viral RdRp. Finally, we demonstrate that knockout of the entire TGBp2 or the domain involved in interacting with viral RdRp attenuates both PVX replication and movement. Collectively, these findings suggest that TGBp2 plays dual functional roles in PVX replication and intercellular movement. IMPORTANCE Many plant viruses contain three partially overlapping open reading frames (ORFs), termed the triple gene block (TGB), for intercellular movement. However, how the corresponding three proteins coordinate their functions remains obscure. In the present study, we provided multiple lines of evidence supporting the notion that PVX TGBp2 functions as the molecular adaptor bridging the interaction between the RdRp/dsRNA body and TGBp3 by forming “chain mail”-like structures in the RdRp/dsRNA body, which can also enhance viral replication. Taken together, our results provide new insights into the replication and movement of PVX and possibly also other TGB-containing plant viruses.

We have previously reported several lines of evidence that support a role for cellular DNA repair systems in completion of the retroviral DNA integration process. Failure to repair an intermediate in the process of integrating viral DNA into host DNA appears to trigger growth arrest or death of a large percentage of infected cells. Cellular proteins involved in the nonhomologous end joining (NHEJ) pathway (DNA-PK CS ) and the damage-signaling kinases (ATM and ATR) have been implicated in this process. However, some studies have suggested that NHEJ proteins may not be required for the completion of lentiviral DNA integration. Here we provide additional evidence that NHEJ proteins are required for stable transduction by human immunodeficiency type 1 (HIV-1)-based vectors. Our analyses with two different reporters show that the number of stably transduced DNA-PK CS -deficient scid fibroblasts was reduced by 80 to 90% compared to the number of control cells. Furthermore, transduction efficiency can be restored to wild-type levels in scid cells that are complemented with a functional DNA-PK CS gene. The efficiency of stable transduction by an HIV-1-based vector is also reduced upon infection of Xrcc4 and ligase IV-deficient cells, implying a role for these components of the NHEJ repair pathway. Finally, we show that cells deficient in ligase IV are killed by infection with an integrase-competent but not an integrase-deficient HIV-1 vector. Results presented in this study lend further support to a general role for the NHEJ DNA repair pathway in completion of the retroviral DNA integration process.

Mutations in the IN domain of retroviral DNA may affect multiple steps of the virus life cycle, suggesting that the IN protein may have other functions in addition to its integration function. We previously reported that the human immunodeficiency virus type 1 IN protein is required for efficient viral DNA synthesis and that this function requires specific interaction with other viral components but not enzyme (integration) activity. In this report, we characterized the structure and function of the Moloney murine leukemia virus (MLV) IN protein in viral DNA synthesis. Using an MLV vector containing green fluorescent protein as a sensitive reporter for virus infection, we found that mutations in either the catalytic triad (D184A) or the HHCC motif (H61A) reduced infectivity by approximately 1,000-fold. Mutations that deleted the entire IN (ΔIN) or 34 C-terminal amino acid residues (Δ34) were more severely defective, with infectivity levels consistently reduced by 10,000-fold. Immunoblot analysis indicated that these mutants were similar to wild-type MLV with respect to virion production and proteolytic processing of the Gag and Pol precursor proteins. Using semiquantitative PCR to analyze viral cDNA synthesis in infected cells, we found the Δ34 and ΔIN mutants to be markedly impaired while the D184A and H61A mutants synthesized cDNA at levels similar to the wild type. The DNA synthesis defect was rescued by complementing the Δ34 and ΔIN mutants in trans with either wild-type IN or the D184A mutant IN, provided as a Gag-IN fusion protein. However, the DNA synthesis defect of ΔIN mutant virions could not be complemented with the Δ34 IN mutant. Taken together, these analyses strongly suggested that the MLV IN protein itself is required for efficient viral DNA synthesis and that this function may be conserved among other retroviruses.

Integrase (IN) is the only retroviral enzyme necessary for the integration of retroviral cDNA into the host cell’s chromosomes. The structure and function of IN is highly conserved. The human immunodeficiency virus type 2 (HIV-2) IN has been shown to efficiently support 3′ processing and strand transfer of HIV-1 DNA substrate in vitro. To determine whether HIV-2 IN protein (IN 2 ) could substitute for HIV-1 IN function in vivo, we used HIV-1 Vpr to deliver the IN 2 into IN mutant HIV-1 virions by expression in trans as a Vpr-IN fusion protein. Trans -complementation with IN 2 markedly increased the infectivity of IN-minus HIV-1. Compared with the homologous trans -IN protein, infectivity was increased to a level of 16%. Since IN has been found to play a role in reverse transcription (Wu et al., J. Virol. 73:2126–2135, 1999), cells infected with IN 2 -complemented HIV-1 were analyzed for DNA products of reverse transcription. DNA levels of approximately 18% of that of wild type were detected. The homologous trans -IN protein restored the synthesis of viral cDNA to approximately 86% of that of wild-type virus. By complementing integration-defective HIV-1 IN mutant viruses, which were not impaired in cDNA synthesis, the trans -IN 2 protein was shown to support integration up to a level of 55% compared with that of the homologous trans -IN protein. The delivery of heterologous IN protein into HIV-1 particles in trans offers a novel approach to understand IN protein function in vivo.

The human immunodeficiency virus type 1 (HIV-1) integrase protein (IN) is essential for integration of the viral DNA into host cell chromosomes. Since IN is expressed and assembled into virions as part of the 160-kDa Gag-Pol precursor polyprotein and catalyzes integration of the provirus in infected cells as a mature 32-kDa protein, mutations in IN are pleiotropic and may affect virus replication at different stages of the virus life cycle in addition to integration. Several different phenotypes have been observed for IN mutant viruses, including defects in virion morphology, protein composition, reverse transcription, nuclear import, and integration. Because the effects of mutations in the IN domain of Gag-Pol can not always be distinguished from those of mutations in the mature IN protein, there remains a significant gap in our understanding of IN function in vivo. To directly analyze the function of the mature IN protein itself, in the context of a replicating virus but independently from that of Gag-Pol, we used an approach developed in our laboratory for incorporating proteins into HIV virions by their expression in trans as fusion partners of either Vpr or Vpx. By providing IN in trans as a Vpr-IN fusion protein, our analysis revealed, for the first time, that the mature IN protein is essential for the efficient initiation of reverse transcription in infected cells and that this function does not require the IN protein to be enzymatically (integration) active. Our findings of a direct physical interaction between IN and reverse transcriptase and the failure of heterologous HIV-2 IN protein to efficiently support reverse transcription indicate that this novel function occurs through specific interactions with other viral components of the reverse transcription initiation complex. Studies involving complementation between integration- and DNA synthesis-defective IN mutants further support this conclusion and reveal that the highly conserved HHCC motif of IN is important for both activities. These findings provide important new insights into IN function and reverse transcription in the context of the nucleoprotein reverse transcription complex within the infected cell. Moreover, they validate a novel approach that obviates the need to mutate Gag-Pol in order to study the role of its individual mature components at the virus replication level.

The human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vpr and Vpx proteins are packaged into virions through virus type-specific interactions with the Gag polyprotein precursor. To examine whether HIV-1 Vpr (Vpr1) and HIV-2 Vpx (Vpx2) could be used to target foreign proteins to the HIV particle, their open reading frames were fused in frame with genes encoding the bacterial staphylococcal nuclease (SN), an enzymatically inactive mutant of SN (SN*), and chloramphenicol acetyltransferase (CAT). Transient expression in a T7-based vaccinia virus system demonstrated the synthesis of appropriately sized Vpr1-SN/SN* and Vpx2-SN/SN* fusion proteins which, when coexpressed with their cognate p55Gag protein, were efficiently incorporated into virus-like particles. Packaging of the fusion proteins was dependent on virus type-specific determinants, as previously seen with wild-type Vpr and Vpx proteins. Particle-associated Vpr1-SN and Vpx2-SN fusion proteins were enzymatically active, as determined by in vitro digestion of lambda phage DNA. To determine whether functional Vpr1 and Vpx2 fusion proteins could be targeted to HIV particles, the gene fusions were cloned into an HIV-2 long terminal repeat/Rev response element-regulated expression vector and cotransfected with wild-type HIV-1 and HIV-2 proviruses. Western blot (immunoblot) analysis of sucrose gradient-purified virions revealed that both Vpr1 and Vpx2 fusion proteins were efficiently packaged regardless of whether SN, SN*, or CAT was used as the C-terminal fusion partner. Moreover, the fusion proteins remained enzymatically active and were packaged in the presence of wild-type Vpr and Vpx proteins. Interestingly, virions also contained smaller proteins that reacted with antibodies specific for the accessory proteins as well as SN and CAT fusion partners. Since similar proteins were absent from Gag-derived virus-like particles and from virions propagated in the presence of an HIV protease inhibitor, they must represent cleavage products produced by the viral protease. Taken together, these results demonstrate that Vpr and Vpx can be used to target functional proteins, including potentially deleterious enzymes, to the human or simian immunodeficiency virus particle. These properties may be exploitable for studies of HIV particle assembly and maturation and for the development of novel antiviral strategies.

The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer comprised of two structurally distinct subunits (p51 and p66). Since p51 and p66 are derived from the same coding region, subunit-specific structure-function studies of RT have been conducted exclusively by in vitro biochemical approaches. To study RT subunit function in the context of infectious virus, we constructed an LTR-vpr-p51-IRES-p66 expression cassette in which the HIV-1 vpr gene was fused in frame with p51, followed by an internal ribosome entry site (IRES) sequence and the p66 coding region. By coexpression with RT-deficient proviral DNA, we demonstrated that the p66 subunit is specifically and selectively packaged into virions as a Vpr-p51/p66 complex. Our analysis showed that cleavage by the viral protease liberates Vpr and generates functional heterodimeric RT (p51/p66) that supports HIV-1 reverse transcription and virus infection. By exploiting this novel trans -complementation approach, we demonstrated, for the first time with infectious virions, that the YMDD aspartates of p66 are both required and sufficient for RT polymerase function. Mutational analyses of the p51 YMDD aspartates indicated that they play an important structural role in p51 folding and subunit interactions that are required for the formation of an active RT heterodimer within infected cells. Understanding the role of the individual RT subunits in RNA- and DNA-dependent DNA synthesis is integral to our understanding of RT function. Our findings will lead to important new insights into the role of the p51 and p66 subunits in HIV-1 reverse transcription.

T-20 is a synthetic peptide that potently inhibits replication of human immunodeficiency virus type 1 by interfering with the transition of the transmembrane protein, gp41, to a fusion active state following interactions of the surface glycoprotein, gp120, with CD4 and coreceptor molecules displayed on the target cell surface. Although T-20 is postulated to interact with an N-terminal heptad repeat within gp41 in a trans -dominant manner, we show here that sensitivity to T-20 is strongly influenced by coreceptor specificity. When 14 T-20-naive primary isolates were analyzed for sensitivity to T-20, the mean 50% inhibitory concentration (IC 50 ) for isolates that utilize CCR5 for entry (R5 viruses) was 0.8 log 10 higher than the mean IC 50 for CXCR4 (X4) isolates ( P = 0.0055). Using NL4.3-based envelope chimeras that contain combinations of envelope sequences derived from R5 and X4 viruses, we found that determinants of coreceptor specificity contained within the gp120 V3 loop modulate this sensitivity to T-20. The IC 50 for all chimeric envelope viruses containing R5 V3 sequences was 0.6 to 0.8 log 10 higher than that for viruses containing X4 V3 sequences. In addition, we confirmed that the N-terminal heptad repeat of gp41 determines the baseline sensitivity to T-20 and that the IC 50 for viruses containing GIV at amino acid residues 36 to 38 was 1.0 log 10 lower than the IC 50 for viruses containing a G-to-D substitution. The results of this study show that gp120-coreceptor interactions and the gp41 N-terminal heptad repeat independently contribute to sensitivity to T-20. These results have important implications for the therapeutic uses of T-20 as well as for unraveling the complex mechanisms of virus fusion and entry.

Viral protein X (Vpx) is a human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus accessory protein that is packaged into virions in molar amounts equivalent to Gag proteins. To delineate the processes of virus assembly that mediate Vpx packaging, we used a recombinant vaccinia virus-T7 RNA polymerase system to facilitate Gag protein expression, particle assembly, and extracellular release. HIV genes were placed under control of the bacteriophage T7 promoter and transfected into HeLa cells expressing T7 RNA polymerase. Western immunoblot analysis detected p55gag and its cleavage products p39 and p27 in purified particles derived by expression of gag and gag-pol, respectively. In trans expression of vpx with either HIV-2 gag or gag-pol gave rise to virus-like particles that contained Vpx in amounts similar to that detected in HIV-2 virus produced from productively infected T cells. Using C-terminal deletion and truncation mutants of HIV-2 Gag, we mapped the p15 coding sequence for determinants of Vpx packaging. This analysis revealed a region (residues 439 to 497) downstream of the nucleocapsid protein (NC) required for incorporation of Vpx into virions. HIV-1/HIV-2 gag chimeras were constructed to further characterize the requirements for incorporation of Vpx into virions. Chimeric HIV-1/HIV-2 Gag particles consisting of HIV-1 p17 and p24 fused in frame at the C terminus with HIV-2 p15 effectively incorporate Vpx, while chimeric HIV-2/HIV-1 Gag particles consisting of HIV-2 p17 and p27 fused in frame at the C terminus with HIV-1 p15 do not. Expression of a 68-amino-acid sequence of HIV-2 containing residues 439 to 497 fused to the coding regions of HIV-1 p17 and p24 also produced virus-like particles capable of packaging Vpx in amounts similar to that of full-length HIV-2 Gag. Sucrose gradient analysis confirmed particle association of Vpx and Gag proteins. These results demonstrate that the HIV-2 Gag precursor (p55) regulates incorporation of Vpx into virions and indicates that the packaging signal is located within residues 439 to 497.