The limited coding capacity of picornavirus genomic RNAs necessitates utilization of host cell factors in the completion of an infectious cycle. One host protein that plays a role in both translation initiation and viral RNA synthesis is poly(rC) binding protein 2 (PCBP2). For picornavirus RNAs containing type I internal ribosome entry site (IRES) elements, PCBP2 binds the major stem-loop structure (stem-loop IV) in the IRES and is essential for translation initiation. Additionally, the binding of PCBP2 to the 5-terminal stem-loop structure (stem-loop I or cloverleaf) in concert with viral protein 3CD is required for initiation of RNA synthesis directed by poliovirus replication complexes. PCBP1, a highly homologous isoform of PCBP2, binds to poliovirus stem-loop I with an affinity similar to that of PCBP2; however, PCBP1 has reduced affinity for stem-loop IV. Using a dicistronic poliovirus RNA, we were able to functionally uncouple translation and RNA replication in PCBP-depleted extracts. Our results demonstrate that PCBP1 rescues RNA replication but is not able to rescue translation initiation. We have also generated mutated versions of PCBP2 containing sitedirected lesions in each of the three RNA-binding domains. Specific defects in RNA binding to either stem-loop I and/or stem-loop IV suggest that these domains may have differential functions in translation and RNA replication. These predictions were confirmed in functional assays that allow separation of RNA replication activities from translation. Our data have implications for differential picornavirus template utilization during viral translation and RNA replication and suggest that specific PCBP2 domains may have distinct roles in these activities.Following entry into a host cell, the single-stranded, positivesense genomic RNA of poliovirus (PV) must provide a nucleation point for the formation of numerous ribonucleoprotein (RNP) complexes. These complexes are capable of mediating the three major processes that the RNA must undergo to establish a successful infection of the host cell. The PV genomic RNA must be translated, replicated, and packaged for a productive virus infection to ensue. Given the small size (ϳ7,400 nucleotides [nt]) of the PV genome, the virus has evolved to utilize numerous proteins or other molecular machinery resident in the host cell to carry out its infectious cycle. One example of the molecular scavenging necessitated by the limited coding capacity of PV is utilization of a single cellular RNA-binding protein, poly(rC) binding protein 2 (PCBP2), for the formation of RNP complexes that form on two different PV 5Ј noncoding region (5Ј NCR) RNA secondary structures to mediate two of the three above-mentioned processes (16,24,25,50).PCBP2 (also known as hnRNP E2 and ␣CP-2) is a cellular RNA-binding protein that interacts with the 5Ј NCR of PV RNA (14,15,23). The protein contains three hnRNP K-homology (KH) RNA-binding domains and has a binding preference for poly(rC). In in vitro binding studies, the first and third KH domains ap...

During picornavirus infection, several cellular proteins are cleaved by virus-encoded proteinases. Such cleavage events are likely to be involved in the changing dynamics during the intracellular viral life cycle, from viral translation to host shutoff to RNA replication to virion assembly. For example, it has been proposed that there is an active switch from poliovirus translation to RNA replication mediated by changes in RNA-binding protein affinities. This switch could be a mechanism for controlling template selection for translation and negative-strand viral RNA synthesis, two processes that use the same positive-strand RNA as a template but proceed in opposing directions. The cellular protein poly(rC)-binding protein (PCBP) was identified as a primary candidate for regulating such a mechanism. Among the four different isoforms of PCBP in mammalian cells, PCBP2 is required for translation initiation on picornavirus genomes with type I internal ribosome entry site elements and also for RNA replication. Through its three K-homologous (KH) domains, PCPB2 forms functional protein-protein and RNA-protein complexes with components of the viral translation and replication machinery. We have found that the isoforms PCBP1 and -2 are cleaved during the mid-to-late phase of poliovirus infection. On the basis of in vitro cleavage assays, we determined that this cleavage event was mediated by the viral proteinases 3C/3CD. The primary cleavage occurs in the linker between the KH2 and KH3 domains, resulting in truncated PCBP2 lacking the KH3 domain. This cleaved protein, termed PCBP2-⌬KH3, is unable to function in translation but maintains its activity in viral RNA replication. We propose that through the loss of the KH3 domain, and therefore loss of its ability to function in translation, PCBP2 can mediate the switch from viral translation to RNA replication.

The translation of picornavirus genomic RNAs occurs by a cap-independent mechanism that requires the formation of specific ribonucleoprotein complexes involving host cell factors and highly structured regions of picornavirus 59 noncoding regions known as internal ribosome entry sites (IRES). Although a number of cellular proteins have been shown to be involved in picornavirus RNA translation, the precise role of these factors in picornavirus internal ribosome entry is not understood. In this report, we provide evidence for the existence of distinct mechanisms for the internal initiation of translation between type I and type II picornavirus IRES elements. In vitro translation reactions were conducted in HeLa cell cytoplasmic translation extracts that were depleted of the cellular protein, poly(rC) binding protein 2 (PCBP2). Upon depletion of PCBP2, these extracts possessed a significantly diminished capacity to translate reporter RNAs containing the type I IRES elements of poliovirus, coxsackievirus, or human rhinovirus linked to luciferase; however, the addition of recombinant PCBP2 could reconstitute translation. Furthermore, RNA electrophoretic mobility-shift analysis demonstrated specific interactions between PCBP2 and both type I and type II picornavirus IRES elements; however, the translation of reporter RNAs containing the type II IRES elements of encephalomyocarditis virus and foot-and-mouth disease virus was not PCBP2 dependent. These data demonstrate that PCBP2 is essential for the internal initiation of translation on picornavirus type I IRES elements but is dispensable for translation directed by the structurally distinct type II elements.

Human immunodeficiency virus type 1 (HIV-1) isolates vary in their ability to infect macrophages. Previous experiments have mapped viral determinants of macrophage infectivity to the V3 hypervariable region of the HIV-1 envelope glycoprotein. In our earlier studies, V1 and V2 sequences of HIV-1 were also shown to alter the ability of virus to spread in macrophage cultures, whereas no effect was seen in lymphocyte cultures. In the present study, determinants that allowed certain HIV-1 clones to infect and spread in macrophages were primarily mapped to the V2 region and were found to act by influencing early events of viral infection. By an assay of viral entry into macrophages, it was shown that viruses with the V2 region from the Ba-L strain of HIV-1 had >10-fold-higher entry efficiency than viruses with the V2 region derived from the NL4-3 strain. V1 region differences between these groups caused a twofold difference in entry. The known low expression of CD4 on macrophages appeared to be important in this process. In entry assays conducted with HeLa cell lines expressing various levels of CD4 and CCR5, low levels of CD4 influenced the efficiency of entry and fusion which were dependent on viral V1 and V2 envelope sequences. In contrast, no effect of V1 or V2 was seen in HeLa cells expressing high levels of CD4. Thus, the limited expression of CD4 on macrophages or other cell types could serve as a selective factor for V1 and V2 envelope sequences, and this selection could in turn influence many aspects of AIDS pathogenesis in vivo. Infection of cells by human immunodeficiency virus (HIV)begins with the sequential interaction of the viral envelope glycoproteins with cellular receptor molecules including CD4 plus one of several chemokine receptors. While more than a dozen chemokine receptors are known to mediate HIV entry, the two that are most commonly utilized by HIV isolates are CXCR4 and CCR5. CXCR4 HIV variants (T-cell tropic) typically arise late in the disease course and utilize CXCR4 coreceptors that are expressed on T cells (17), while CCR5 variants (macrophage tropic) are typically the transmitted form and enter cells using CCR5 coreceptors that are expressed on macrophages and T cells (2, 14, 16). The primary determinants necessary for CCR5 or CXCR4 coreceptor usage have been previously mapped to the V3 region of the envelope. However, this domain is not sufficient for high levels of virus infection in macrophages, as previous experiments have shown that both nonenvelope and envelope regions other than V3 can alter macrophage infection (3,23,25,31,45).Previously, sequences within the V1 and V2 regions of HIV envelope have been shown to influence various aspects of macrophage infection. For example, V2 can modulate the influence of V3 on macrophage tropism and also influences envelope interactions with soluble CD4 (25, 41). In addition, V1 and V2 changes may be required for the efficient production of virus in macrophages (40,46). In our previous studies, differences in V1 and V2 envelope sequences were...